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Essential roles of the T7 Endonuclease (Gene 3) and the T7 Exonuclease (Gene 6) in recombination of Bacteriophage… Lee, Marion A. 1976

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THE ESSENTIAL ROLES OF THE T7 ENDONUCLEASE (GENE 3) AND THE T7 EXONUCLEASE (GENE 6) IN RECOMBINATION OF BACTERIOPHAGE DNA by Marion A. Lee B.Sc, University of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF MICROBIOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1976 0 Marion A. L e e , 1976 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 ABSTRACT The role of the T7-induced exonuclease (gene 6) i n recombi-nation was studied using both molecular and genetic techniques. In the molecular method the fate of parental DNA during parentr-to-progeny recombination was examined. A comparison of infections with T7 +, T7am6 (amber gene 6), or T7ts6 (temperature sensitive gene 6) under permissive and nonpermissive conditions was made. CsCl density gradient analysis of r e p l i c a t i v e DNA indicated that the T7 exonuclease i s necessary for recombination to occur, i . e . , i n the absence of the exonuclease the parental DNA r e p l i -cated continuously as a hybrid molecule and did not recombine. Analysis of denatured r e p l i c a t i v e DNA by CsCl density gradient centrifugation indicated that the exonuclease also may be needed for a l i m i t e d amount of covalent repair of recombinants. Further confirmation of the ess e n t i a l role which the exonuclease plays in recombination came from genetic analysis. The T7 exonuclease was shown to be necessary for intragenic and intergenic recombi-nation i n several areas of the T7 genetic map; genetic recombi-nation frequencies were found to be decreased from 3 to 18-fold under conditions nonpermissive for the exonuclease. The role of the T7-induced endonuclease (gene 3) i n molecular recombination was studied by examining the fate of parental DNA during parent-to-progeny recombination using a shear technique. The T7 endonuclease was found to be necessary for the dispersion of parental DNA i n the newly r e p l i c a t e d DNA. Concatemers syn-thesized by either T7 + or T7am3 (amber gene 3) phage containing the newly r e p l i c a t e d DNA were sheared to the size of mature phage DNA and also to quarter size molecules. In the presence of gene 3 protein, parental DNA and newly rep l i c a t e d DNA were i n t e r -spersed, i . e . , the 3 2 P - l a b e l from the sheared DNA was found to sediment at the density of recombined DNA. In the absence of gene 3 protein, the parental strand of each sheared DNA molecule was usually found i n t a c t , i . e . , the 3 2 P - l a b e l from the sheared DNA was found to sediment at the density of hybrid DNA. These results support the previous genetic data (52, 83) that the gene 3 protein i s esse n t i a l for T7 recombination. The r o l e of T7 recombination enzymes i n the formation of concatemers was studied by examining selected gene 3 and gene 6 mutants. Results of sucrose gradient analysis showed that DNA concatemers were formed when both the T7 exonuclease (gene 6) and the T7 endonuclease (gene 3) were absent. Further r e s u l t s showed that concatemers cannot be maintained i n the absence of the exonu-clease unless the endonuclease was eliminated. In a T7am6 i n f e c -t i o n DNA concatemers formed early were prematurely broken down and accumulated as fragments smaller than mature si z e phage DNA. In a T7am3am6 (amber i n both genes 3 and 6) i n f e c t i o n concatemers accumulated and were not matured. These results indicate that concatemers are formed by a process other than normal phage recombination. However, s e l e c t i v e defects i n the recombination system do i n t e r f e r e with the s t a b i l i t y of concatemers. i v TABLE OF CONTENTS Page Abstract i i Table of Contents i v L i s t of Figures v i Acknowledgement ix Chapter I - Introduction 1 Section 1. Recombination 1 A. Methods of Analysis 2 B. Molecular Models 5 C. Evidence Supporting the Branch Migration Model of Recombination 11 Section 2. Bacteriophage T7 15 Section 3. Concatemers 20 A. Molecular Models 2 0 B. Evidence Supporting Models 21 C. Evidence that Phage V i a b i l i t y Depends on Concatemer Formation 22 D. Phage T7 and the Watson Model 24 E. Concatemer Formation Using T7 Mutants 2 6 F. Summary 2 6 Chapter I I - — Methods and Materials 28 Strains 28 Chemicals and Isotopes 2 8 Media 2 9 Preparation of Labeled Bacteriophage 29 Heavy labeled Experimental System 31 Preparation of Density labeled Reference T7 DNA 31 Sucrose Gradient V e l o c i t y Sedimentation 3 2 CsCl Density Sedimentation 32 Determination of Label Uptake into Acid-Insoluble Material 33 Removal of Unattached Bacteriophage 33 Procedure for the Shear Experiment 34 DNA Extraction and P u r i f i c a t i o n .34 [ 3H] Thymidine Uptake 34 Preparation of the Double Mutants Used i n the Crosses.... 35 Genetic Crosses 35 Reconstruction Experiment 36 Chapter III - Results 38 Section 1. Molecular Recombination under Conditions Permissive and Nonpermissive for the T7 Exonuclease (Gene 6) 38 Section 2. Replication of the T7ts6 Phage at 43C 52 Section 3. Covalent Repair of Recombinant Molecules under Conditions Permissive and Nonper-missive for the Exonuclease (Gene 6)..... 58 V Page Section 4. Genetic Recombination under Conditions Permissive and Nonpermissive for the -T7 Exonuclease (.Gene 6) ................ .. ......... 65 Section 5. Molecular Recombination .under Conditions Permissive and Nonpermissive for the T7 Endonuclease (Gene 3).......................... 68 Section 6. Concatemer Formation under Conditions Permissive and Nonpermissive for Molecular Recombination i n T7.................. . 82 Chapter IV - Discussion and Conclusions...... 103 Section 1. The Role of T7 Exonuclease (Gene 6) i n Recombination. . . 103 Section 2. Involvement of the T7 Exonuclease (Gene 6) i n Covalent Repair......... 110 Section 3. The Role of T7 Endonuclease (Gene 3) i n Recombination 114 Section 4. The Involvement of T7 Recombination Enzymes i n Concatemer Formation 119 Bibliography 123 v i LIST OF FIGURES Page Figure 1. A schematic diagram showing the fate of parental DNA during a parent to progeny recombination experiment 4 Figure 2. A schematic diagram showing the e f f e c t that gene conversion has on the f i n a l products of meiosis i n ascomycetes 7 Figure 3. A schematic diagram showing the sequence of events expected to occur i n a branch migration model of recombination 10 Figure 4. CsCl sedimentation of native DNA from a density transfer experiment: T7 + versus T7am6 39 Figure 5. Comparison of theiCsCl sedimentation pat-terns of parental 3 2 P - l a b e l e d DNA from T7+ versus T7am6 for a la t e i n f e c t i o n time 41 Figure 6. Alk a l i n e sucrose gradient analysis of T7 + versus T7am6 parental 3 2 P - l a b e l e d DNA from a density transfer experiment 4 3 Figure 7. CsCl sedimentation of a l k a l i denatured r e p l i c a t i v e parental 3 2 P - l a b e l e d DNA from a density transfer experiment: T7 + versus T7am6 45 Figure 8. CsCl sedimentation of native DNA from a density transfer experiment incubated at both permissive and nonpermissive temper-atures: T7 + versus T7ts6 47 Figure 9. Comparison of the CsCl sedimentation pat-terns of parental 3 2 P - l a b e l e d r e p l i c a t i v e DNA for infections incubated at permissive and nonpermissive temperatures 50 Figure 10. Incorporation of [ 3H]thymidine into acid insoluble material for both T7 + and T7ts6 infections incubated at permissive and nonpermissive temperatures 53 Figure 11. CsCl sedimentation of native DNA from a T7ts6 strand exchange experiment 55 Figure 12. CsCl sedimentation of heat denatured T7ts6 r e p l i c a t i v e DNA i s o l a t e d i n a density transfer experiment 57 v i i Page Figure 13. CsCl sedimentation of native DNA from a density transfer experiment incubated at permissive and nonpermissive temperatures: T7 + versus T7ts6 59 Figure 14. CsCl sedimentation of heat denatured r e p l i -cative DNA i s o l a t e d i n a density transfer experiment: \T7 + versus T7ts6 61 Figure 15. Alkaline sucrose sedimentation of T7 + versus T7am6 DNAs from a density transfer experiment w 6 4 Figure 16. CsCl sedimentation of native T7am3 r e p l i -cative DNA from a density transfer experi-ment 69 Figure 17. Neutral sucrose sedimentation of T7am3 r e p l i c a t i v e DNA from a density transfer experiment 71 Figure 18. A schematic diagram of the postulated differences between concatemers synthe-sized i n T7 + versus T7am3 infections 73 Figure 19. Neutral sucrose sedimentation of unsheared and sheared r e p l i c a t i v e DNA from both T7+ and T7am3 infect i o n s 75 Figure 20. Superimposed CsCl sedimentation patterns of unsheared and sheared r e p l i c a t i v e DNA from both T7 + and T7am3 infections 77 Figure 21. Neutral sucrose sedimentation of sheared r e p l i c a t i v e DNA from a density transfer experiment: T7 + versus T7am3 79 Figure 22. CsCl sedimentation of both unsheared and sheared r e p l i c a t i v e DNA from a density transfer experiment: T7 + versus T7am3 81 Figure 23. Neutral sucrose sedimentation of parental 3 2 P - l a b e l e d r e p l i c a t i v e DNA from a T7+ versus a T7am6 i n f e c t i o n : early sampling times 84 Figure 24. Neutral sucrose sedimentation of parental 3 2 P - l a b e l e d r e p l i c a t i v e DNA from a T7+ versus a T7am6 i n f e c t i o n : late sampling times 85 Figure 25. Neutral sucrose sedimentation of progeny DNA labeled continuously with [ 3HJthymi-dine: T7 + versus T7am6 87 v i i i Page F i g u r e 26. N e u t r a l sucrose sedimentation of progeny DNA l a b e l e d c o n t i n u o u s l y with [ 3H]thymi-d i n e : T 7 + versus T7ts6 - ....89 F i g u r e 27. N e u t r a l sucrose sedimentation of p a r e n t a l 3 2 P - l a b e l e d r e p l i c a t i v e DNA from a T 7 + versus a T7ts6 i n f e c t i o n , both i n c u b a t e d a t the nonpermissive temperature of 43C 92 F i g u r e 28. N e u t r a l sucrose sedimentation of T7ts6 progeny DNA " p u l s e - l a b e l e d " w i t h [ 3 H ] t h y -midine 94 F i g u r e 29. N e u t r a l sucrose sedimentation of p a r e n t a l 3 2 P - l a b e l e d r e p l i c a t i v e DNA from a T 7 + versus a T7am3am6 i n f e c t i o n 97 F i g u r e 30. N e u t r a l sucrose sedimentation a n a l y s i s of T7+, T7am3, T7am6, and T7am3am6 progeny DNAs c o n t i n u o u s l y l a b e l e d w i t h [ 3H]thymi-dine 99 F i g u r e 31. N e u t r a l sucrose sedimentation a n a l y s i s of T 7 + , T7am3, T7am6, and T7am3am6 progeny DNAs " p u l s e - l a b e l e d " w i t h [ 3H] thymidine 101 ix ACKNOWLEDGEMENT I would l i k e to thank my supervisor, Professor R. C. M i l l e r , for his continual i n s p i r a t i o n and assistance throughout t h i s project. D. Taylor, H. Smith, R. Elwood, and B. Molson provided excellent technical assistance, and R. A. J . Warren and V. Paetkau provided many help f u l discussions. D. G. Scraba and R. D. Bradley did the electron microscopy, and F. W. Studier generously donated the phage mutants. My husband, C l i n t , gave invaluable assistance i n the preparation of t h i s manuscript. F i n a l l y , I would l i k e to thank the National Research Council of Canada for t h e i r f i n a n c i a l assistance during the course of my research. 1 CHAPTER I - INTRODUCTION Section 1. Recombination Genetic recombination can be defined as the set of enzymatic processes whi-eh f a c i l i t a t e e the establishment of new linkages between genes within a chromosome. The three recognized sub-classes of genetic recombination are described by Radding (89) as follows: 1) general recombination, 2) s i t e s p e c i f i c recombination, and 3) nonhomologous recombination. 1) General recombination i s the exchange of genetic material which can occur anywhere along the length of homologous chromosomes. Some gene coded products which are thought to p a r t i c i p a t e i n general recombination are the V exonuclease and 6 proteins coded for by the lambda red genes (17, 18, 19, 86, 87, 88, 102, 103), the recBC nuclease of Escherichia y c o l i (4, 27, 81), and the gene 46 and 47 encoded proteins of T4 (7, 9, 12, 84). 2) Site s p e c i f i c recombination occurs at a gene t i c a l l y determined region and i s independent of general recombination. An example of s i t e s p e c i f i c recombination i s the integration of bacteriophage lambda DNA into the host chromosome. Integration i s at least p a r t l y promoted by the i n t genes of lambda (33, 104, 125). Site s p e c i f i c recombination can mediate some general recombination but only between markers which f a l l on both sides of the integration s i t e (104, 125). 3) Nonhomologous recombination i s defined as exchanges between genomes which themselves are nonhomologous. The random i n s e r t i o n of the bacterio-phage Mu into the host chromosomes i s an example of nonhomologous recombination (13, 28). The d i v i s i o n of recombinational events into, d i s t i n c t classes 2 suggests that a va r i e t y of mechanisms are involved i n promoting recombination. Considerable research has been devoted to ascer-taining the molecular mechanisms involved i n generating recom-binants and i n determining the s p e c i f i c enzymes which mediate these processes (26, 27, 46, 74, 78, 79, 89). Experiments designed to study general recombination mechanisms of T7 w i l l be described i n t h i s thesis. A. Methods of Analysis Both genetic and molecular techniques have been used to study recombination. Several genetic techniques have been successful i n i ?d^rit2ifyiin.gj mutants which are recombination defec-t i v e . The e f f e c t that a s p e c i f i c mutation has on the recombi-nation frequencies occuring between other genetic markers can be studied (79, 52, 75). S e n s i t i v i t y to u l t r a v i o l e t ora-x-irradiation i s c h a r a c t e r i s t i c of some recombination defective mutants (26, 27, 40, 53, 101). The i n a b i l i t y of a s p e c i f i c mutant to form conjugates with a Hfr s t r a i n i s also c h a r a c t e r i s t i c of recombi-nation d e f i c i e n t mutants (26, 27). On a molecular l e v e l , genetic recombination can be defined as the physical exchange of material between two d i f f e r e n t DNA molecules. This conception of recombination has been supported by experimental evidence from T4 (2, 3, 55, 99, 118, 119), lambda (69), <(.X174 (5, 54), and T7 (71). Molecular techniques used to study phage recombination include both biparental and parent to progeny recombination experiments. Parent to progeny recombination experiments can be performed by i n f e c t i n g a density labeled host with r a d i o a c t i v e l y labeled 3 parental phage. The f i r s t round of r e p l i c a t i o n r e s u l t s i n the synthesis of phage DNA of hybrid density, one strand being of parental o r i g i n and the other strand being composed of completely substituted density labeled DNA. Subsequent rounds of r e p l i c a t i o n produce progeny DNA molecules both strands of which are composed of completely substituted DNA. Formation of recombinant molecules i s detected by CsCl density gradient analysis of the re p l i c a t e d DNA. Parental l a b e l that sediments at a density intermediate to that of pure progeny and hybrid i s recombinant. The terms used to describe various stages of r e p l i c a t i o n and recombination are shown i n Figure l.„ In a j o i n t molecule, the parental l a b e l sediments at a recombinant density under neutral conditions. When denatured, the parental contribution to the j o i n t molecule sediments at the l i g h t density. When the j o i n t molecule has been covalently repaired, then under denaturing conditions, the parental l a b e l w i l l sediment at a density greater than that of the conservative nonreplicated molecule. Two types of j o i n t molecules may be formed by recombi-nation. The recombinant heterozygote i s recombinant for outside markers. The i n s e r t i o n heterozygote has a heterozygous area inserted into: a molecule which contains genes from only one parent as outside markers (74, 78, 79). Biparental recombination experiments can be performed by mixedly i n f e c t i n g bacteria with density labeled phage and " l i g h t " r a d i o a c t i v e l y labeled phage. Recombination between parental molecules r e s u l t s i n the formation of molecules that have a density intermediate to that of either parent. To preserve the i n t e g r i t y of the biparental recombinants these experiments are 4 Figure 1. A schematic diagram showing the fate of parental DNA during a parent to progeny recombination experiment. x > T M or pH>12 Conservative, Pa ren ta l I Hybr i d Hybr id X ~ V ^ V W W V ^ . ^ ^ Progeny _ _ J . "^V", J o i n t Molecules ^ Repair "^-iT^S ... ~ J*LWV^ Cova len t 1 Recombinant PH>12 * * * OC A. Recombinant Heteroduplex B. Insert ion Heteroduplex 5 usually conducted with mutants defective i n DNA synthesis. When using either biparental or parent to progeny approaches to study recombination, one frequently uses chloramphenicol (CM) to i n h i b i t protein synthesis. In t h i s way maturation i s i n h i b i t e d , so that intermediates of r e p l i c a t i o n and recombination can be iso l a t e d . By adding CM at various stages of the r e p l i c a t i o n and recombination cycle an early intermediate of r e p l i c a t i o n or recombination can be accumulated, and formation of l a t e r i n t e r -mediates i n h i b i t e d . Thus an order i n which molecular events may occur i s indicated. The use of CM can also a s s i s t i n determining whether s p e c i f i c molecular events of phage DNA metabolism are dependent on phage coded enzymes. Adding CM at c r i t i c a l times i n T4 i n f e c t i o n can completely i n h i b i t T4 DNA r e p l i c a t i o n (56), permit r e p l i c a t i o n while i n h i b i t i n g j o i n t molecule formation (56, 57), or permit j o i n t molecule formation while i n h i b i t i n g t h e i r covalent repair,(57, 59). When CM i s added l a t e enough af t e r i n f e c t i o n , covalent repair of recombinants w i l l occur but maturation of the DNA into phage size molecules i s i n h i b i t e d (57, 72). The a b i l i t y to i n h i b i t the d i f f e r e n t stages of T4 development by the addition of CM to infected c e l l s indicates that these d i f f e r e n t t r a n s i t i o n s are at least p a r t l y dependent on the synthesis of phage coded enzymes. B. Molecular Models Experimental observations have resulted i n the formulation of numerous models to. explain the mechanisms by which recombi-nation i s £a-ciiita:tdd^ (46, 53, 74, 78, 89). The general model which i s s t i l l favored and which has generated many variants i s 6 the breakage and reunion mechanism of recombination (11, 44, 70, 72, 107, 116, 123). In t h i s model DNA i n a chromosome i s frag-mented and reincorporated into, other DNA molecules. O r i g i n a l l y the breakage and reunion mechanism of recombination was unable to: explain gene conversion and was not thought precise enough to explain genetic data. However, i n 1964 Holliday (44) proposed a model for recombination by breakage and reunion which included a mechanism which could explain conversion. In t h i s model i t i s assumed that a mutation involves a single base pair substitution. At c e r t a i n areas i n paired homologous chromosomes the DNA molecules unravel to expose single strands. These anneal with complementary strands from the other chromosome. If the annealed region includes a pointsof heterozygosity, a mutant s i t e , then mis-p a i r i n g of.bases occurs. If the mismatched base pairs are unstable then a repair mechanism could occur which would replace one or the other of the mismatched bases i n an otherwise homo-zygous duplex. This process, l i k e that suggested for the copy choice model (44, 89), i s not necessarily r e c i p r o c a l . An example of t h i s process i s shown i n Figure 2. This mechanism of gene conversion i s analogous to. the repair of UV induced thymine dimers. The general features of t h i s model also have the advant-age of s a t i s f y i n g other known features of recombination. DNA r e p l i c a t i o n does not need to occur during or after p a i r i n g of chromosomes for recombination to occur. I t allows for both semi-conservative (68) r e p l i c a t i o n and for breakage and reunion as the mechanism by which reassortment of genetic information can occur. Several recent models of the breakage and reunion type are 7 Figure 2. A schematic diagram showing the e f f e c t that gene conversion has on the f i n a l products of meiosis i n ascomycetes. A. Crossing over i s not accompanied by gene conversion so that the r a t i o of the o r i g i n a l genetic markers remains 2:2. B. Crossing over i s accompanied by gene conversion so that the r a t i o of the o r i g i n a l Q/q markers i s now 3:1. Crossing Over 3™E G Q X 9 q g B. Crossing Over & Gene Conversion (Q-G q g Reduction Division 2-2 Ratio for Both Genes " F T " G Q X g q G 33: 9^ g -5> 2-2 Ratio for G 3=1 Ratio for Q 8 mainly concerned with describing the possible steps involved i n the exchange of DNA between homologous chromosomes (11, 17, 72, 116). These models have several features i n common. They require that recombination be i n i t i a t e d by the introduction of discontin-u i t i e s into: the DNA molecules. These d i s c o n t i n u i t i e s are generally i n the form of single strand nicks. However, i n the cut and s t r i p model of Thomas (116, 123), a double stranded s c i s s i o n i s postulated followed by exonuclease digestion at the ends of the fragments to produce single strand regions. Double strand d i s c o n t i n u i t i e s do not accumulate i n the wildtype T4 i n f e c t i o n even under conditions i n which maturation i s i n h i b i t e d . Single strand d i s c o n t i n u i t i e s w i l l accumulate when maturation i s i n h i b i t e d (57, 58, 59). Mutants, i n which double strand d i s c o n t i n u i t i e s accumulate are nonviable*, (59, 67) . The exposure of single strand regions by exonuclease following the introduction of single strand nicks i s a charac-t e r i s t i c step postulated i n several models of recombination (11, 17, 46). These include the model postulated by Carter and Radding (17) as well as the branch migration model of Broker and Lehman (11). Two models which do not require exonuclease exposure of single strand regions; to: f:acili!.ta.te..i p a i r i n g between homol-ogous chromosomes are the diagonal cut model described i n a paper by Miller, et a l (72) and the as$pme.tr±ct.tBan;s"feernno$€l recently postulated by Meselson and Radding (70). In the diagonal cut ; model trans nicks are introduced followed by l o c a l melting across the nicks. Thus single strand regions are exposed which can pair with complementary single strands on other DNA molecules. In 9 contrast to the Thomas model i n which the cut generates two o r i g i n a l neighbors which are unable to recombine, the diagonal cut model allows o r i g i n a l neighbors to reunite. The repair of nicks then r e s u l t s i n a perfect molecule (72). In the Meselson and Radding model a nick i n one DNA molecule r e s u l t s i n a strand displacement due to DNA synthesis. The displaced single strand then invades another DNA molecule at a region of complementarity and induces a single strand break i n the second DNA molecule. Exonuclease digestion may occur to increase the extent of strand transfer, but the exonuclease a c t i v i t y i s not required for the i n i t i a l formation of the heteroduplex. In a l l of these models, once heterozygotes are established, then a combination of nuclease, polymerase, and ligase a c t i v i t y mediates the covalent repair of recombinants. A recombination model of the breakage and reunion type i s the branch migration model of Broker and Lehman (11). In t h i s model the following steps are postulated (See Figure 3). 1) An endonuclease mediated single strand nicking of the DNA duplexes occurs at homologous regions. 2) This i s followed by exonuclease mediated enlargement of the nicks to gaps or by exposure of single strand ends. 3) Pairing of complementary single strands i s promoted by DNA binding proteins. 4) Branch migration occurs to e s t a b l i s h heterozygotes. 5) Branched molecules are reduced to l i n e a r duplexes by nucleases. 6) The repair of gaps and nicks by DNA polymerase and ligase r e s u l t s i n the formation of covalently repaired recombinants. There i s considerable experimental evidence to support t h i s model. 10 Figure 3. A schematic diagram showing the sequence of events expected to occur i n a branch migration model of recombination. This figure has been modified from that of Broker and Lehman (11). 11 C. Evidence Supporting the Branch M i g r a t i o n Model of Recombination E l e c t r o n micrograph s t u d i e s of T4 r e p l i c a t i v e i n t e r m e d i a t e s show t h a t branches, gaps, whiskers, and s i n g l e s t r a n d ends do e x i s t i n r e p l i c a t i n g molecules (10, 11, 12). Branched s t r u c t u r e s i n r e p l i c a t i n g DNA molecules have been observed i n <fX174 (5) and i n f r e q u e n t l y i n phage T7 (50, 96). When CM i s added e a r l y i n i n f e c t i o n the s i n g l e s t r a n d d i s c o n t i n u i t i e s which normally occur i n T4 r e p l i c a t i v e DNA f a i l t o form and recombination i s i n h i b i t e d (56, 57). The i m p l i c a t i o n i s t h a t the i n t r o d u c t i o n of n i c k s i s r e q u i r e d t o i n i t i a t e recombi-n a t i o n . T h i s suggests t h a t an endonuclease i s needed t o i n i t i a t e recombination. Endonuclease n e g a t i v e mutants known to a f f e c t the f o r m a t i o n of j o i n t molecules have not been d i s c o v e r e d i n T4. However, the enzyme encoded by the T4 V + gene i s an endonuclease. Mutants d e f e c t i v e i n t h i s enzyme are s e n s i t i v e t o UV l i g h t presumably because they are unable to r e p a i r damaged DNA. I f the w i l d t y p e T4 V + phage are i r r a d i a t e d w i t h UV then recombination f r e q u e n c i e s i n c r e a s e (53, 74, 101). Since the presence of the V + gene product i s thought to f u n c t i o n by i n t r o d u c i n g d i s c o n t i n u i t i e s when e x c i s i n g damaged DNA, the presence of d i s c o n t i n u i t i e s i s c o r r e l a t e d w i t h g e n e t i c recombi-n a t i o n and thus endonuclease i s c o r r e l a t e d w i t h recombination. Mutants d e f e c t i v e i n exonuclease a c t i v i t y - T4 (gene 46 and 47) (7, 9), E s c h e r i c h i a c o l i (recBC encoded) (4, 27, 81), and lambda (red encoded) (102, 103) - show decreased g e n e t i c recombi-n a t i o n f r e q u e n c i e s . In T4 p o l l i g phage the s i n g l e s t r a n d i n t e r r u p t i o n s which accumulate i n the i n f e c t i n g p a r e n t a l DNA 12 r e q u i r e both DNA polymerase and l i g a s e t o be r e p a i r e d i n v i t r o . I f the phage i s a l s o d e f e c t i v e i n gene 46 or 47 the DNA can be r e p a i r e d l h v i t r o w i t h l i g a s e alone (3, 84). Thus, i n the T4 p o l — l i g mutant gaps accumulate because of the a c t i o n of the gene 46 coded exonuclease a t n i c k s , whereas i n the T4 p o l l i g 46 DNA gaps cannot accumulate because the gene 46 product i s absent. Furthermore, the p o l l i g 46 DNA molecules do not accumulate any of the s i n g l e s t r a n d and branched s t r u c t u r a l f e a t u r e s expected as i n t e r m e d i a t e s i n recombination, and b i p a r e n t a l recombination experiments w i t h t h i s mutant do not r e s u l t i n the f o r m a t i o n of recombinant d e n s i t y i n t e r m e d i a t e s (12, 74, 84). The i m p l i c a t i o n i s t h a t gaps may be a p r e r e q u i s i t e t o recombination. T4 mutants d e f e c t i v e i n gene 32 encoded b i n d i n g p r o t e i n show decreased g e n e t i c recombination f r e q u e n c i e s (1, 7, 80). B i p a r e n t a l recombination experiments u s i n g gene 32 mutants do not show the formation, of j o i n t molecules (58, 119), thus i n d i c a t i n g t h a t b i n d i n g p r o t e i n s are r e q u i r e d f o r the f o r m a t i o n of j o i n t m olecules. Although t h i s mutant i s d e f e c t i v e i n DNA r e p l i c a t i o n , T4 p o l l i g mutants which are a l s o d e f e c t i v e i n DNA r e p l i c a t i o n are a b l e to form j o i n t molecules i n b i p a r e n t a l experiments (3). Both DNA polymerase d e f e c t i v e and l i g a s e d e f e c t i v e mutants of T4 are found to have i n c r e a s e d g e n e t i c recombination f r e q u e n c i e s (7, 8, 9, 32, 60, 66). In the absence of DNA polymerase gaps accumulate i n the DNA (3, 11). Thus, s i n c e gaps have been shown to be recombinogenic, one might expect t h a t a mutation which i n c r e a s e s the l i f e t i m e of these d i s c o n t i n u i t i e s 13 would a l l o w more r e c o m b i n a t i o n a l events to occur (3, 12, 84). By a temperature s h i f t experiment w i t h 43 mutants (DNA polymerase negative) d u r i n g parent to progeny recombination, T4 DNA polymerase has been shown to be r e q u i r e d f o r the c o v a l e n t r e p a i r of j o i n t molecules (73). In an i n f e c t i o n by a l i g a s e n e g a t i v e mutant o f T4 both s i n g l e and double s t r a n d d i s c o n t i n u i t i e s accumulate i n the DNA, and the mutant i s a b l e t o undergo e f f i c i e n t p arent to progeny recombination. Some c o v a l e n t r e p a i r of these recombinant molecules a l s o o c c u r s , but whether i t i s mediated by phage or h o s t enzymes has not been determined (59). An r l l m utation p h e n o t y p i c a l l y r e v e r s e s a l i g a s e mutation. I t i s thought t h a t the r l l product fa'c:dilii.t;a.tesvs n i c k i n g of the DNA (An r l l i n f e c t i o n shows decreased DNAase a c t i v i t y . ) . T h i s i n c r e a s e s recombination when the l i g a s e i s m i s s i n g p r o b a b l y because the l i g a s e would normally r e p a i r many of these n i c k s . L i g a s e i s t h e r e f o r e , t h o u g h t to modulate the amount of n i c k i n g which occurs d u r i n g T4 i n f e c t i o n (6, 16, 32, 74). T h i s suggests t h a t l i g a s e a l s o may f u n c t i o n t o r e p a i r n i c k s i n recombinant DNA. By m o l e c u l a r , g e n e t i c , and b i o c h e m i c a l c r i t e r i a ' s 1 many mutants which are d e f e c t i v e i n the s y n t h e s i s of a s p e c i f i c gene product have been found to be d e f e c t i v e i n recombination. A n a l y s i s of recombination d e f e c t i v e mutants has r e s u l t e d i n d e t e r m i n i n g t h a t the i n d i v i d u a l mutants are u s u a l l y d e f e c t i v e i n a single'enzyme. Since recombination i s an e p i s t a t i c event which r e q u i r e s the i n t e r a c t i o n of s e v e r a l d i f f e r e n t p r o t e i n s , the data7ria-ve jgener'a)lil\y i n d i c a t e d t h a t c e r t a i n enzymes are i n v o l v e d i n recombination but not n e c e s s a r i l y the mechanism of t h e i r a c t i o n . T h i s t h e s i s w i l l attempt to s p e c i f y a mechanism 14 of action for T7 enzymes involved i n recombination based on a l l three c r i t e r i a . 15 S e c t i o n 2. Bacteriophage T7 The recombination system of T7 has not been s t u d i e d i n d e t a i l . However, phage T7 o f f e r s s e v e r a l advantages. I t has been w e l l c h a r a c t e r i z e d by p h y s i c a l , chemical, and p h y s i o l o g i c a l i n v e s t i g a t i o n s and has a l s o undergone c a r e f u l g e n e t i c a n a l y s i s (112, 113, 114). A b r i e f review of the s a l i e n t f e a t u r e s of T7 f o l l o w . T7 i s a v i r u l e n t phage which produces a t a s i n g l e b u r s t approximately 250 phage p a r t i c l e s . I t i s one of the s m a l l e s t T-phage; the phage p a r t i c l e has a molecular weight of between (38 and 45) times 10 (112). T7 i n c o n t r a s t t o T2 shows no l y s i s i n h i b i t i o n , m u l t i p l i c i t y r e a c t i v a t i o n , nor s u p e r - i n f e c t i o n breakdown. T7 i s s t a b l e to s o n i c a t i o n and a l s o t o osmotic shock (112). The main advantage of T7 i s i t s s i z e , which probably l i m i t s i t s coding c a p a c i t y t o those f u n c t i o n s d i r e c t l y r e l a t e d t o DNA metabolism and phage assembly. The T7 DNA molecule i s a s m a l l l i n e a r duplex w i t h a m o lecular weight of 26 times 10 . I t has a 48% GC r a t i o and i s composed of the f o u r unmodified bases (112). Most of the T7 DNA i s s y n t h e s i z e d from host DNA breakdown products (62, 63, 85). T7 DNA i s t e r m i n a l l y redundant and i s not c i r c u l a r l y permuted (91). Nineteen complementation groups i n T7 have been i d e n t i f i e d by i s o l a t i o n and c h a r a c t e r i z a t i o n of amber mutants (112). R e l a t e d f u n c t i o n s are found to be c l o s e l y l i n k e d on the g e n e t i c map. S e v e r a l other genes a l s o have been mapped (114). The T7 genome has an expected coding c a p a c i t y of 25-30 genes. Most of the T7 gene coded p r o t e i n s have been i d e n t i f i e d on s l a b g e l s and these 16 proteins have been characterized (113, 114). T7 does not r e p l i c a t e through a c i r c u l a r intermediate. DNA r e p l i c a t i o n i s i n i t i a t e d 17% from the lefthand end of a l i n e a r molecule. Replication i s b i d i r e c t i o n a l , and i t infrequently r e i n i t i a t e s DNA synthesis p r i o r to the completion of one round of r e p l i c a t i o n (31). Replication i s mediated by a discontinuous mechanism involving the synthesis of DNA fragments which are then joined to form a covalently sealed daughter strand (48, 126). Replication of T7 DNA involves a concatemeric intermediate (47, 49, 96) . T7 has been found to undergo parent to progeny recombination to an extent comparable to that demonstrated by T4. The T7 parental DNA molecule becomes fragmented and i s inserted as a subunit covalently bonded to the adjacent progeny strand (71, 9-9) . Both T7 parental strands are transfered with equal p r o b a b i l i t y to the progeny molecule (15, 115). Six complementation groups of T7 are known to be involved in T7 DNA metabolism. Gene 1 codes for an RNA polymerase (25) which i s required for t r a n s c r i p t i o n of most of the T7 genome including the other genes required for DNA synthesis. It may a f f e c t DNA r e p l i c a t i o n only i n d i r e c t l y (113, 114). The function of gene 2 protein i s not known but i t may be involved i n the formation of long single strands (24). DNA synthesis i s greatly reduced i n i t s absence (110, 112). Gene 4 codes for a protein which i s apparently necessary for the synthesis of DNA with L-strand p o l a r i t y (109). Evidence from in. v i t r o experiments suggest that the gene 4 product may be necessary for a chain 17 i n i t i a t i o n s tep i n T7 DNA r e p l i c a t i o n (95). Gene 5 codes f o r T7 DNA polymerase which i s r e q u i r e d f o r the s e m i - c o n s e r v a t i v e r e p l i c a t i o n of T7 DNA (39, 42). The a c t i v i t y of the T7 DNA polymerase i s dependent on a t l e a s t one host product, t h i o r e d o x i n (76, 77). Gene 3 (endonuclease; 20, 21, 22, 82) and gene 6 (exonuclease; 50, 51) mutants show reduced l e v e l s of DNA s y n t h e s i s (112). Since these enzymes are r e q u i r e d to degrade host DNA to a c i d s o l u b l e products (92) , the reduced s y n t h e s i s has been a t t r i b u t e d to the absence of DNA p r e c u r s o r s (62, 63, 85). T7 concatemers accumulate i n the absence of the T7 endonuclease so i t i s thought to be necessary f o r maturation of T7 DNA (110). Mutants i n e i t h e r nuclease are a b l e to undergo s e m i - c o n s e r v a t i v e r e p l i c a t i o n . L i g a s e (gene 1.3 i n T7) and unwinding p r o t e i n are a l s o thought to be necessary f o r normal DNA s y n t h e s i s . Host l i g a s e can s u b s t i t u t e f o r T7 l i g a s e i n v i v o (67). A p r o t e i n , l i k e the T4 gene 32 product (1) has been i s o l a t e d from T7 i n f e c t e d c e l l s . i n v i t r o the DNA unwinding p r o t e i n of the host can s u b s t i t u t e f o r the T7 unwinding p r o t e i n (90, 94), t h e r e f o r e the absence of the T7 p r o t e i n may not be l e t h a l . A t the time when t h i s p r o j e c t was i n i t i a t e d , T7 mutants d e f e c t i v e i n recombination had not been s p e c i f i e d . The o b j e c t i v e s of t h i s p r o j e c t were to examine recombination i n T7 to determine i f i t was dependent on phage coded products and t o a s c e r t a i n which phage coded products were necessary. Since both exonuclease and endonuclease have been i m p l i c a t e d i n recombination i n other systems (4, 7, 9, 18/ 74, 81), t h e i r r o l e i n recombi-n a t i o n of T7 was examined. 18 Two observations which suggest that T7 recombination may be independent of host enzymes are: 1) T7 undergoes normal levels of recombination as determined by the frequency of wildtype recombinants a r i s i n g from an amberNl cross amberN2 i n f e c t i o n i n recA -, recB -, recB~C~, or recA~B~C~ hosts (52, 83), and 2) T7 s p e c i f i e s a protein which, l i k e the gam protein (38, 120, 121) of lambda, s p e c i f i c a l l y i n h i b i t s the recBC nuclease a c t i v i t y of the host (122). T7 recombination may s t i l l depend on host proteins which are not part of the recA,B,C, recombination pathway. Since the biochemical properties of an enzyme can help to determine i t s mechanism i n vivo, a b r i e f summary of the biochem-i c a l properties of the T7 endonuclease (gene 3) and exonuclease (gene 6) i s included. The T7 induced exonuclease (gene 6) has a s p e c i f i c i t y for double stranded DNA. It begins hydrolysis at the 5'-termini and hydrolyzes at 5'-phosphoryl and 5'-hydroxyl terminated DNA at equal rates giving a f i n a l product of 5'-deoxyribonucleotides. The T7 exonuclease can i n i t i a t e hydrolysis at nicks as well as at external termini (50, 51). The T7 induced endonuclease (gene 3) hydrolyzes both single and double stranded DNA. i n v i t r o , double stranded DNA i s 4 hydrolyzed to li m i t e d fragments of approximately 2 times 10 . It degrades single stranded DNA even more extensively to 4 fragments smaller than 10 . In duplex DNA hydrolysis proceeds by the introduction of ligase repairable single strand sc i s s i o n s . Double strand scissions are also made (21, 22). There i s evidence that a second endonuclease may be synthesized by T7 19 (23, 93). Mutants defective i n t h i s enzyme (endonuclease II) have not been i s o l a t e d . Endonuclease II has been analyzed extensively, i n v i t r o , but i t s possible i n vivo function i s not known. 20 S e c t i o n 3. Concatemers Concatemers are molecules which are l o n g e r than a mature s i z e DNA molecule. They are found as i n t e r m e d i a t e s i n the r e p l i c a t i o n o f many phage (53, 117) i n c l u d i n g lambda (53, 105, 106), T4 (35, 72), and T7 (47, 49). The concatemeric DNA i s f r e q u e n t l y many phage e q u i v a l e n t u n i t s (PEU) i n l e n g t h . Concatemers are found i n two forms. In T7 s i n g l e s t r a n d i n t e r -r u p t i o n s one PEU apart are u s u a l l y p r e s e n t (15, 96, 97). In T4 s i n g l e s t r a n d i n t e r r u p t i o n s are o f t e n absent i n concatemers so t h a t when the long duplexes are denatured, the s i n g l e s t r a n d s are a l s o l o n g e r than one PEU (35, 72). S p e c i f i c models which p o s t u l a t e how d i f f e r e n t t y p es o f mature molecules are generated from concatemers have been formulated (49, 111, 117). The "he a d f u l h y p o t h e s i s " o f S t r e i s i n g e r (111) or l e n g t h s p e c i f i c c u t t i n g i s p o s t u l a t e d to. generate t e r m i n a l l y r e p i t i o u s , c i r c u l a r l y permuted molecules such as those of T4, T2, and P i . Sequence s p e c i f i c n i c k i n g f o l l o w e d by r e s i d u a l DNA s y n t h e s i s i s a mechanism suggested f o r the ge n e r a t i o n o f t e r m i n a l l y redundant molecules w i t h unique sequences (117). In T7 the duplex i s made complete. In lambda s t i c k y ends are l e f t on the molecule. A. M o l e c u l a r Models A more p u z z l i n g q u e s t i o n i s how concatemers themselves are formed i n v i v o . S e v e r a l models to e x p l a i n the formation o f concatemers have been proposed. Concatemers have been p o s t u l a t e d to be formed as a r e s u l t o f r o l l i n g c i r c l e r e p l i c a t i o n by the continuous e x t r u s i o n o f newly r e p l i c a t e d DNA from a r o l l i n g c i r c l e 21 (37). Recombination also has been postulated as a mechanism by which concatemers are generated. Two types of recombination mechanisms which have been suggested are: 1) by general recombi-nation across the terminal redundancies and 2) by a discontinuous synthesis recombination mechanism postulated by Watson (124) to explain the o r i g i n of T7 concatemers. The p o s s i b i l i t y exists that d i f f e r e n t phage use d i f f e r e n t mechanisms to generate concatemers. In t h i s thesis the role of a general recombination system i n the formation of concatemers i n T7 i s examined. B. Evidence Supporting Models Evidence exists which suggests that i t i s recombination that i s responsible for concatemer formation dm T4 and not the r o l l i n g c i r c l e mechanism (30, 72). When concatemer formation i s examined i n T4 i t i s found that the addition of CM at 5 minutes af t e r i n f e c t i o n prevents both molecular recombination and long single strand formation, although DNA r e p l i c a t i o n i s not impaired. Between 50-100 PEU of DNA are synthesized under these conditions. When CM i s added l a t e r i n the i n f e c t i o n during a period when recombination i s permitted long single strands are generated. Furthermore, under conditions when maturation i s not i n h i b i t e d , long single strands do not appear u n t i l about 2 0 PEU of DNA has been synthesized. Their appearance coincides with the expected onset of recombination and repair of recombinants (72). Therefore, the r o l l i n g c i r c l e model, i n a l l likelyhood, does not apply to T4. In contrast experimental evidence from lambda favors r o l l i n g c i r c l e r e p l i c a t i o n as the mechanism for generating concatemers (34, 105, 106). This conclusion i s based p a r t l y on the observation 22 that concatemer formation i s independent of the known recombi-nation mechanisms i n lambda and i s also independent of the func-tions of genes i n the A-J segment of the lambda genetic map (105) . Also, lambda DNA molecules of one PEU containing c i r c u l a r permutations of the mature sequence have been observed i n electron micrographs of l a t e r e p l i c a t i n g DNA. It i s proposed that these molecular forms are generated from a r o l l i n g c i r c l e which has f a l l e n apart at i t s growing point (106). C. Evidence That Phage V i a b i l i t y Depends on Concatemer Formation Phage v i a b i l i t y may be dependent on the formation of concatemers i n lambda (34, 108). In lambda defects i n i n t r a c e l -l u l a r phage DNA maturation have been correlated with the i n a b i l i t y of/the phage to form DNA concatemers. Under conditions which diminish DNA synthesis i n lambda, the production of viable phage, that contain f u l l y conserved parental DNA depends on the action of the genetic recombination systems. If r e p l i c a t i o n i s permitted so that more DNA molecules are synthesized, dependence on recombination for v i a b i l i t y i s diminished (108). Thus, perhaps the reduction of lambda DNA to mature size molecules from larger molecules i s e s s e n t i a l for phage production. The c o r r e l a t i o n between v i a b i l i t y and concatemer formation also i s indicated i n experiments which investigated'the i n t e r -action between gam, rec, and red genes (34). When either the red or gam lambda mutants i n f e c t a recA + or a recA host, DNA synthesis i s reduced. However, the red mutant produces DNA which i s s t r u c t u r a l l y normal and i s e f f i c i e n t l y packaged. In contrast the DNA from a gam i n f e c t i o n i s s t r u c t u r r a l l y abnormal, containing mainly c i r c u l a r forms and some concatemers at late times of i n f e c t i o n . There i s viable phage produced from t h i s i n f e c t i o n . When a gam mutant i n f e c t s a recA B host DNA synthesis occurs at normal wildtype l e v e l s , the DNA i s struc-t u r a l l y normal and i s e f f i c i e n t l y matured and packaged. The in f e c t i o n of a red mutant i n a recA B host i s q u a l i t a t i v e l y s i m i l a r to the i n f e c t i o n observed in the recA host. The gam product appears to function i n a l t e r i n g synthesis from "early" c i r c u l a r r e p l i c a t i o n to the " l a t e " concatemer forming r e p l i c a t i o n . However, i t i s not required to mediate t h i s t r a n s i t i o n when the recB nuclease i s missing. The implication i s that the gam gene product serves a negative role by antagonizing the recB nuclease which would i n h i b i t the normal i n i t i a t i o n of " l a t e " r e p l i c a t i o n . Other i n vivo and also i n v i t r o evidence indicates that the gam protein does i n t e r f e r e with the recB nuclease (38, 121). Since red DNA development i s r e l a t i v e l y normal whether the red phage i s i n f e c t i n g a recA +, recA , or a recA B host, the red product does not appear to function i n generating " l a t e " r e p l i c a t i v e structures. I f a gam red phage in f e c t s a recA host then concatemeric structures do not appear at a l l and phage production i s severely reduced. The i n f e c t i o n of a. recA + host by either a gam or a gam red phage i s q u a l i t a t i v e l y s i m i l a r . The impl i -cation i s that when the normal t r a n s i t i o n from "early" to " l a t e " r e p l i c a t i o n i s impaired, some formation of concatemers can be promoted by recombination or repair of intermediates. Either the recA or red gene products can mediate th i s process. Thus, the occurence of abnormal r e p l i c a t i o n i n which there i s poor conca-24 terner formation coincides with severe impairment of i n t r a c e l l u l a r phage DNA maturation. The following observation also indicates that there may be a c o r r e l a t i o n between concatemer formation and v i a b i l i t y . The T4w mutants show reduced recombination as well as reduced v i a b i l i t y (41). There i s very i n e f f i c i e n t packaging of DNA i n these mutants and the amount of concatemers synthesized i s decreased. Therefore, reduced v i a b i l i t y i s correlated with a reduction i n the formation of concatemers. Since recombination also is' reduced i n t h i s mutant the question arises whether the gene w product i s involved i n both recombination and concatemer formation. This would suggest a possible c o r r e l a t i o n between recombination and v i a b i l i t y . D. Phage T7 and the Watson Model It has been established that mutants of both T7 endonuclease (gene 3) and T7 exonuclease (gene 6) are defective i n molecular recombination (64, 65). Mutants i n both also show reduced genetic recombination frequencies (52, 75, 83). Therefore, both of these enzymes are required for general recombination i n T7. Since concatemer formation may be mediated by recombination i n T7, the question arises whether either of these enzymes i s necessary for concatemer formation i n T7. However, in the recombination d e f i c i e n t mutant of T4 which is.. defective, i n exonuclease (gene 46), concatemer formation occurs but the concatemers are not stable (10, 45, 98, 100). This suggests that exonuclease a c t i v i t y may not be involved i n the formation of concatemers i n T4. 25 Watson (124) suggests that r e p l i c a t i o n of T7 through a concatemeric intermediate may be es s e n t i a l for phage v i a b i l i t y . The argument rests on two fa c t s . 1) A l l known DNA polymerases are unable to i n i t i a t e de novo DNA synthesis and are unable to catalyze the synthesis of DNA i n a 3 5 1 d i r e c t i o n (40, 53). 2) T7 i s a terminally redundant l i n e a r molecule which does not re p l i c a t e through a c i r c u l a r intermediate (127). He postulates that under these constraints the absence of concatemeric interme-diates would be l e t h a l . The r e p l i c a t i o n of a l i n e a r molecule of T7 with the r e s t r i c t i o n s imposed on the DNA polymerase would r e s u l t i n the synthesis of molecules with unreplicated, small, single strand regions at the 3' termini. The subsequent rounds of r e p l i c a t i o n i n the absence of concatemerization would produce smaller and smaller DNA molecules. Eventually e s s e n t i a l genetic information would be l o s t which would be l e t h a l . Also, i f packaging of the DNA i s dependent on the size of the molecule, then these shorter molecules would not be packaged. However, these conditions can lead to the formation of concatemeric intermediates. The presence of terminal redundancies i n the molecule means that two molecules having 3' single strand t a i l s can base pa i r to form a dimer. The process of dimerization may require a preliminary exonuclease step to enlarge the single strand t a i l . If the single strand ends extend beyond the terminal redundancies, then a gap would be produced when the dimer i s formed. The gaps produced could be f i l l e d by a 5'-^3' DNA polymerase. The exonuclease could function also i n removing redundant single strands probably by strand displacement since / I I / 26 i t cannot h y d r o l y z e s i n g l e s t r a n d DNA (50, 51). Ma t u r a t i o n of T7 DNA from concatemers can be mediated by the i n t r o d u c t i o n of staggered n i c k s across the t e r m i n a l redundancies. These are formed so t h a t o n l y growth i n a 5'—?3' d i r e c t i o n needs to occur f o r the completion o f the duplex molecule. T h i s model suggests t h a t concatemers are an e s s e n t i a l i n t e r -mediate due to the necessary mode of r e p l i c a t i o n . The enzymes i n v o l v e d i n recombination may a l s o p a r t i c i p a t e i n concatemer formation s i n c e they may be needed t o make a proper " j o i n t " between the ends of adjacent molecules. T h i s model i n d i c a t e s t h a t an exonuclease may perform an important r o l e i n s t a b i l i z i n g concatemers and a l s o may c o n t r i b u t e t o t h e i r f o rmation. E. Concatemer Formation Using T7 Mutants Experimental r e s u l t s of S t r a t l i n g e t al_. (110) and others (43) i n d i c a t e t h a t i n an i n f e c t i o n by a gene 3 (endonuclease) mutant s t a b l e concatemers accumulate a t a time when i n the w i l d t y p e i n f e c t i o n they are b e i n g broken down to mature s i z e DNA molecules. I n f e c t i o n by a gene 6 (exonuclease) mutant l e a d s t o the accumulation of double s t r a n d d i s c o n t i n u i t i e s i n the newly s y n t h e s i z e d DNA (36). I n t e r p r e t a t i o n of the r e s u l t s i s compli-cated by the f a c t t h a t o n l y f a i r l y l a t e samples were looked a t . A r e - e v a l u a t i o n o f the p o s s i b l e r o l e of exonuclease and endonu-c l e a s e i n concatemer formation i s undertaken i n t h i s t h e s i s . Concatemer formation a l s o w i l l be examined under c o n d i t i o n s nonpermissive f o r both the endonuclease and exonuclease. F. Summary In summary, t h i s t h e s i s e v a l u a t e s the r o l e s of_T7. '•_ endonuclease (gene 3) and exonuclease (gene 6) i n recombination of bacteriophage T7 DNA, and t h e i r role i n concatemer formation. It i s found that both the endonuclease and exonuclease are required for molecular recombination. The exonuclease i s require for genetic recombination, and also may play a minor role i n the covalent repair of recombinants. Results suggest that i n the absence of the exonuclease, recombination i s defective at the breakage and reunion step, since j o i n t molecules are not formed. However, nicks as well as double strand d i s c o n t i n u i t i e s accumu-late i n the DNA of the exonuclease defective mutant. Endonu-clease mutants may be impaired at the breakage step i n recombi-nation, since j o i n t molecule formation i s r e s t r i c t e d i n the absence of endonuclease a c t i v i t y . Neither enzyme i s required for the formation of concatemers. However, the exonuclease i s apparently needed to s t a b i l i z e concatemeric DNA when the endonuclease i s present but not i n the absence of the endonu-clease, since stable concatemers accumulate i n the absence of both enzymes. Therefore, the general recombination system of T7 i s apparently not involved i n concatemer formation i n T7. CHAPTER II - METHOD'S' AND' MATERIALS 28 Strains 1. Bacteria: A l l experiments were conducted i n Escherichia  c o l i B23 (nonpermissive host for the,amber mutants). O i l ' i s a derivative of Escherichia c o l i B-containing an amber suppressor and requiring thymine for growth (112);jit was used for the preparation of the amber phage. A l l b a c t e r i a l cultures were g f r e s h l y grown for two generations to 3 X 10 c e l l s per ml p r i o r to resuspension and growth i n defined media. 2. Bacteriophage: Strains of T7 were a g i f t from F. W. Studier. The wildtype s t r a i n i s designated T7 +. T7am6-233, an amber i n gene 6, which s p e c i f i e s an exonuclease (50, 51), i s designated T7am6. T7ts6-13 6, a temperature sensitive mutant of gene 6, i s designated T7ts6. T7am3-29, an amber i n gene 3, which s p e c i f i e s an endonuclease (20, 21, 22), i s designated T7am3. T 7 t s 5 - l l , a temperature sensitive mutant of gene 5, which s p e c i f i e s a DNA polymerase (39), i s designated T7ts5. T7aml-193, an amber mutant of gene 1, which s p e c i f i e s an RNA polymerase (25), i s designated T7aml. T7am2-64 designated T7am2, T7aml2-3 designated T7aml2, T7aml6-325 designated T7aml6, and T7aml6-260 were also used. Chemicals and Isotopes L-Chloramphenicol (CM) and 5-fluQrodeoxyuridine (FdU) were purchased from the Sigma Chemical Company. 5-Bromodeoxyuridine (5-BrdU) was purchased from the Calbiochemical Company. 3 3 32 J Methyl- Hj] thymidine, 5-[ 6- HJ bromodeoxyuridine, and P as 32 H T vP0 4 were purchased from New England Nuclear Corporation. 29 Media 1. A l l radio-actively labeled phage were prepared, and experiments were performed i n TCG medium (0.1 M T r i s ' C l , .0.16 mM Na 2S0 4, 1.0 mM MgS04, 0.05% NaCl, 0.1% glucose, 0.1 mM CaCl 3 , 3 uM F e C l 3 , 0.05% vitamin-free Casamino Acids, 40 ug of trypto-phane per ml, f i n a l pH 7.4; 54). TCG was supplemented with phosphate by the addition of 0.1 M KH 2P0 4 as 0.65 ml per 100 ml for normal P0 4 TCG, 0.128 ml per 100 ml for 1/5 P0 4 TCG, and 6.4 ml per 100 ml for 10 X PC>4 TCG; 10 X P0 4 TCG contains 250 yg of P per ml. 5-BrdU TCG contains 200 yg of 5-BrdU per ml, 5 yg of FdU per ml, and 25 yg of u r a c i l per ml. 2. H-broth contains 5 g of peptone (Difco), 1 g of glucose, 5 g of NaCl, and 8 g of nutrient broth (Difco) a l l i n 1000 ml of d i s t i l l e d water, f i n a l pH 7.0. 3. TNE contains 0.01 M T r i s ' C l , 0.015 M EDTA, and 0.15 M NaCl at pH 7.4. 4. T7 T r i s s a l t i s 0.01 M Tris«Cl plus 1.0 M NaCl at pH 7.4. Preparation of Labeled Bacteriophage 32 8 1. P: Bacteria were grown for two generations to 3 X 10 32 c e l l s per ml i n [ P]orthophosphate TCG (at 5-10 yCi/yg). O i l ' growth medium was supplemented with 10 yg/ml of thymidine. C e l l s were infected with bacteriophage at a m u l t i p l i c i t y of i n f e c t i o n (M. 0. I.) of 5. Infection by T7 + and T7 amber phage was carr i e d out at 37C. Infection by T7 temperature sensitive phage was car r i e d out at 30C. After 90 minutes NaCl was added to 1 M, the c e l l s were lysed with chloroform, the lysate was treated with DNAase, and the phage p u r i f i e d by d i f f e r e n t i a l centrifugation. The phage then were p u r i f i e d further by gel f i l t r a t i o n through a 30 column (18 X 0.8 cm) of Bio-Gel A-50M, 100-200 mesh (Bio-Rad Laboratories). . The column was eluted with T7 T r i s s a l t . Samples (0.5 ml) were co l l e c t e d , and portions were transferred to glass f i b e r f i l t e r s , dried, overlayed with toluene-based l , 4 - b i s - [ 2 ] -(5-phenyloxazolyl)benzene and 2,5-diphenyloxazolyl s c i n t i l l a n t , and counted for r a d i o a c t i v i t y i n an Isocap 300 s c i n t i l l a t i o n spectrometer. 3 2. H: Bacteria were grown i n normal PO^ TCG containing thymidine (5 yg/ml), 5-fluorodeoxyuridine (5 yg/ml), and u r a c i l (25 ug/ml). This medium was supplemented with 1-2 yCi of 3 [Methyl- H]thymidine per yg. The bacteria were infected as des-cribed above. The bacteriophage were p u r i f i e d by d i f f e r e n t i a l centrifugation. 3. 5-BrdU: Bacteria were grown for two hours i n the dark g from an i n i t i a l density of 0.75 X 10 bacteria per ml at 30C i n 5-BrdU 10 X P0 4 TCG. The c e l l s were infected as described above. The bacteriophage were concentrated by d i f f e r e n t i a l centrifugation and then were p u r i f i e d through CsCl. The phage were shown to be density labeled by either of two methods. 1) A sample of the 32 phage was sedimented through CsCl together with P-labeled l i g h t reference phage. The gradient was c o l l e c t e d into s h e l l v i a l s . Portions of each sample then were t i t r e d to determine the d i s t r i b u t i o n of plaque forming units and also the d i s t r i b u t i o n of l a b e l i n the samples was determined. 2) A small portion of 3 the infected c e l l s was., labeled with [ H]5-BrdU at 6 minutes af t e r i n f e c t i o n and the density d i s t r i b u t i o n of the r e s u l t i n g 32 progeny phage was compared to the density of P-light T7 r e f e r -3 ence phage. A l t e r n a t i v e l y , the H-labeled phage was extracted and the density of the DNA compared with P-labeled l i g h t reference DNA. Heavy Labeled Experimenta1 System Bacteria were incubated i n the dark for two hours at 30C 32 in 5-BrdU 10 X P0 4 TCG. C e l l s then were infected with P-labeled phage at a m u l t i p l i c i t y of 7. At various times aft e r i n f e c t i o n samples were ice c h i l l e d or treated with CM at 100 pg/ml to i n h i b i t maturation and incubated further and then ice c h i l l e d . Samples to be analyzed were d i l u t e d into 2 volumes of cold TNE. The i n f e c t i v e centers were pellet e d and resuspended i n a small volume of TNE. The DNA was p u r i f i e d from these samples and analyzed as described. Preparation of Density-Labeled Reference TT DNA Bacteria were grown i n normal PO^ 5-BrdU TCG for 2 hours i n the dark at 30C. They were infected at a m u l t i p l i c i t y of 5 with 3 T7 phage, and 0.1 yCi of [ H]5-BrdU per yg was added at 6 minutes after i n f e c t i o n . CM at 100 yg/ml was added at 15 minutes a f t e r i n f e c t i o n . T h i r t y minutes a f t e r i n f e c t i o n the sample was ice c h i l l e d i n cold TNE. The i n f e c t i v e centers were sedimented and resuspended i n a small volume of TNE. The DNA was extracted and then dialyzed with a continuous flow negative pressure d i a l y s i s apparatus against 0.01 M potassium phosphate pH 7.4, plus 0.001 M EDTA. The density of the progeny DNA was analyzed against 32 P-labeled l i g h t T7 DNA by CsCl centrifugation to show that the DNA was density labeled. The DNA was determined to be T7 s p e c i f i c by standard hybridization technique (29). Since the average density of the 5-BrdU labeled T7 DNA varied somewhat from experiment to experiment, whenever possible, a density-labeled DNA reference was made at the same time that the experiment for which i t was to be used was performed. Sucrose Gradient V e l o c i t y Sedimentation 1. A l k a l i n e sucrose gradients: Infective centers or labeled 3 DNA p u r i f i e d from infected bacteria was mixed with either H- or 32 P- labeled reference T7 phage i n TNE; NaOH was added to a f i n a l concentration of 0.2 M. This mixture was incubated at 37C for 20 to 30 minutes and then layered on a 5 ml 5 to 20% a l k a l i n e sucrose gradient (The sucrose was dissolved i n a solvent composed of 0.2 M NaOH, 1 ,'M NaCl, and 0.001 M EDTA). Gradients were centrifuged at 35,000 rpm for three hours i n a Beckman SW 50.1 rotor. Between 35-40 fractions were co l l e c t e d d i r e c t l y onto glass f i b e r f i l t e r s i n parental labeled experiments and onto 2.5 cm1 "Whatman 3-MM f i l t e r disks i n progeny labeled experiments i n which the acid i n -soluble counts were analyzed. Radioactivity was determined as described above. 2. Neutral sucrose gradients: P u r i f i e d i n f e c t i v e center DNA 3 32 was mixed with either H- or P- labeled reference T7 phage DNA in TNE. This mixture was layered on a 4.6 ml 5 to 20% neutral sucrose gradient underlayed by a 0.4 ml saturated sucrose pad, (The sucrose was dissolved in a solvent composed of 1 M NaCl, 0.001 M EDTA, and 0.01 M T r i s * C l , f i n a l pH 7.4). Gradients were centrifuged at 30,000 rpm for three hours on a Beckman SW 50.1 rotor. Gradients were co l l e c t e d and r a d i o a c t i v i t y determined as described above. CsCl Density Sedimentation 32 1. Native P-labeled DNA was analyzed by CsCl density 33 gradient centrifugation: 1.4 ml of- sample was mixed with 5 ml of saturated CsCl (in d i s t i l l e d water at room temperature) , or. 1 ml of the DNA preparation was mixed with 4 ml of saturated CsCl i n polyallomer tubes. The 6.4 ml gradients were overlayed with p a r a f f i n o i l and centrifuged i n an SB283 rotor on a B60 Inter-national centrifuge at IOC for 48-72 hours at 30,000 rpm. The 5 ml gradients were centrifuged i n an SW 50.1 rotor under the same conditions. Preparative CsCl gradients were co l l e c t e d into s h e l l v i a l s i n 0.1 ml portions. Aliquots (5 y l i t e r s ) were transferred to glass f i b e r f i l t e r s , and the r a d i o a c t i v i t y was determined. Replicative bands of labeled DNA were is o l a t e d from the gradients, dialyzed as described above, and resedimented with a l i g h t and/or 3 heavy H-labeled T7 DNA reference i n CsCl at a neutral pH. 2. Denatured DNA CsCl gradients were prepared by heat dena-3 turmg the DNA from the r e p l i c a t i v e peaks together with H-labeled T7 reference DNA i n the presence of 2% formaldehyde for 5 minutes at 95-98C; the samples then were added to the CsCl i n denatured DNA treated polyallomer tubes. Some denatured CsCl gradients were made by denaturing the DNA i n a l k a l i . These were made up by adding Na^PO^ to the gradients to give a 0.2 M solution. Gradients then were centrifuged as described. A n a l y t i c a l gradients were col l e c t e d d i r e c t l y onto glass f i b e r f i l t e r s and the r a d i o a c t i v i t y determined. Determination of Label Uptake into Acid Insoluble Material Samples were pipetted or gradients were co l l e c t e d d i r e c t l y onto 2.5 cm Whatman 3-MM f i l t e r disks and washed batchwise i n 5% t r i c h l o r o a c e t i c acid (TCA) at 4C. After three 15 minute washes 34 in TCA the f i l t e r s were washed twice i n anhydrous ethanol. The f i l t e r s were dried and counted as described. Removal of Unattached Bacteriophage 32 Bacteria were infected with P-labeled phage. At 6.5 . *" minutes after i n f e c t i o n unattached phage were removed by f i l t r a -t i o n through a 0.45 ym M i l l i p o r e f i l t e r . F i l t e r e d c e l l s then were washed and resuspended i n an equivalent volume of media which was equilibrated to the experimental temperature. Procedure for the Shear Experiment The shear experiments were performed using a V i r t i s homo-geniser. The homogeniser was standardized for the amount of shear produced by testing i t at d i f f e r e n t speeds against both T7 and T4 DNA. The DNA was di l u t e d to 5-10 yg per ml i n TNE. A l l shear experiments were performed at 4C i n TNE. A l l experimental samples were sheared for 10 minutes as t h i s length of time was found to be s u f f i c i e n t for shear to reach completion. DNA samples were analyzed before and after shearing by neutral sucrose v e l o c i t y sedimentation. DNA Extraction and P u r i f i c a t i o n Infective centers i n TNE were treated with 0.5% sodium dodecyl sulfate (SDS) for 5 to 10 minutes at 37C and then incu-bated with predigested pronase (2 mg/ml) for 7-14 hours. Samples were extracted with water saturated phenol followed by three washes with water saturated ether. When i t was necessary to remove a l l of the phenol because the DNA samples were to be analyzed by o p t i c a l density at 2 60 nm, further ether washes were performed, followed by d i a l y s i s against 0.01 M potassium phosphate, 35 0.001 M EDTA, and 0.1 M NaCl at pH 7.4. Extraction of the DNA : from phage was done d i r e c t l y with phenol followed by several ether washes. 3 [ H]Thymidine Uptake 1. Continuous lab e l i n g of progeny DNA: Bacteria were grown i n normal P0 4 TCG containing 5 yg/ml FdU, 5 yg/ml thymidine, and 25 yg/ml u r a c i l for two generations to 3 X 10' bacteria per ml at 30C. They were infected at an M. 0. I. of 7 with unlabeled 3 parental phage. At 6.5 minutes a f t e r i n f e c t i o n [ H]thymidine (1 yCi/yg) was added. Uptake was monitered by determining the acid insoluble counts present at d i f f e r e n t times a f t e r i n f e c t i o n . 2. Pulse-chase of progeny DNA: Bacteria were grown and infected as described above. At various times aft e r i n f e c t i o n 3 [ H]thymidine (1 yCi/yg) was added for 1 minute. Labeling medium was removed by f i l t r a t i o n of c e l l s through a 0.45 ym M i l l i p o r e f i l t e r . Washed c e l l s were resuspended i n the o r i g i n a l incubation mixture supplemented with 200 yg/ml of cold thymidine. No further uptake of t r i t i u m occurred after the pulse as determined by moni-toring the amount of acid insoluble l a b e l just p r i o r to and aft e r the chase. The labeled DNA was determined to be T7 s p e c i f i c by hybridization (29). Preparation of Double Mutants Used i n the Crosses Double mutants were prepared by multiply i n f e c t i n g E. c o l i O i l 1 at an M. 0. I. of 10 with each single mutant at 30C i n H-broth. The r e s u l t i n g progeny were plated on O i l 1 and plaques were picked and analyzed for t h e i r a b i l i t y to grow on B23 at room temperature. When one of the parental types was temperature 36 sensitive the progeny were plated also on B23 at the nonpermissive temperature of 43C. Those phage plaques which were thought to represent the double mutants were analyzed for reversion frequency and by complementation to confirm that they were the double mutant required. Genetic Crosses Double mutants of the type amNtsM were used to i n f e c t O i l 1 at 30C i n the following way: amNltsM X amN2tsM at an M. 0. I. equal to 10 for each double mutant. At 5 minutes a f t e r i n f e c t i o n the c e l l s were d i l u t e d into two portions of T7 antisera, one portion of which had been pre-incubated at 30C and the other portion of which was pre-incubated at the "semi-permissive" temperature of 37C. At 11 minutes aft e r i n f e c t i o n the c e l l s were d i l u t e d out of antisera and a sample was taken to test for the number of i n f e c -t i v e centers. A sample also was treated with chloroform and the number of viable background phage (unattached) remaining was determined. The infected c e l l s were incubated u n t i l 60 minutes afte r i n f e c t i o n . Then the progeny were analyzed for the amount of recombination which had occurred between the two amber muta-tions at the d i f f e r e n t temperatures. The control cross, amNl X amN2, was performed concurrently under the same conditions and the data were compared. Reconstruction Experiment E. c o l i B2 3 was grown i n 5-BrdU normal PO^ TCG for 2 hours i n the dark at. 30C. At one minute p r i o r to i n f e c t i o n a sample of the c e l l s was c h i l l e d and the DNA p u r i f i e d . This DNA then was analyzed by preparative CsCl sedimentation, c o l l e c t e d , and the 37 samples then were analyzed on a Zeiss spectrophotometer at 2 60 nm to determine the d i s t r i b u t i o n of material which absorbed at t h i s wavelength. The r e s t of the culture was infected with 5-BrdU labeled T7 at an M. 0. I. of 10. At 6 minutes afte r i n f e c t i o n the c e l l s were washed free of 5-BrdU and were resuspended i n media supplemented with thymidine. At 8 minutes a f t e r i n f e c t i o n 3 [ H]thymidine (1 uCi/ug) was added to the infected c e l l s and they were incubated further. Samples were removed at various times and the DNA p u r i f i e d . This DNA was analyzed i n preparative CsCl gradients. The samples co l l e c t e d from the gradient were analyzed both for the d i s t r i b u t i o n of 260 nm absorbing material and the d i s t r i b u t i o n of acid insoluble counts. The d i s t r i b u t i o n of 260 nm absorbing material from the uninfected c e l l s was compared with the d i s t r i b u t i o n of t r i t i u m and 260 nm absorbing material i n the infected samples. The labeled T7 DNA which was i s o l a t e d from the neutral CsCl gradients as a r e p l i c a t i v e progeny DNA peak was considered to be free of E. c o l i DNA i f there was l i t t l e or no 260 nm absorbing material from the uninfected sample which over-lapped with both the 260 nm absorbing and t r i t i u m containing peak i n the infected samples. 38 CHAPTER III - RESULTS Section 1. Molecular Recombination under Conditions Permissive and Nonpermissive for the T7 Exonuclease (Gene 6) If the T7 exonuclease (gene 6) i s required for molecular recombination, then under nonpermissive conditions an amber mutant of gene 6 should r e p l i c a t e only to hybrid. To tes t t h i s hypothesis the k i n e t i c s of r e p l i c a t i o n and recombination by both the T7 + and T7am6 phage i n a density s h i f t experiment was examined. E. c o l i B2 3 was grown i n 5-BrdU 10 X P0 4 TCG for 2 hours at 30C. The culture was divided; one portion was infected 32 + 32 with P-labeled T7 , and the other was infected with P-labeled T7am6. At various times after i n f e c t i o n a portion of each culture was treated with CM and incubated further. The DNA from the various samples was p u r i f i e d and analyzed by neutral CsCl density sedimentation. The res u l t s are shown i n Figure 4. Panels A, B, C, and D represent the T7 + i n f e c t i o n i n which CM was added at 8, 10, 12, and 16 minutes after i n f e c t i o n , respectively. Panels E, F, G, and H represent the T7am6 i n f e c t i o n under the same conditions. When CM was added at 8 minutes a f t e r i n f e c t i o n neither infected culture showed detectible r e p l i c a t i o n (A and E). However, when CM was added at 10 minutes afte r i n f e c t i o n detectible r e p l i c a t i o n occured i n both infections„(B and F). In the T7 + i n f e c t i o n , when CM was added at 10 minutes after i n f e c t i o n (B), some of the DNA sedimented at the density of recombined DNA. The addition of CM at 12 minutes (C) or 16 minutes (D) after i n f e c t i o n permited more of the wildtype parental l a b e l to sediment at the density of recombined DNA. In contrast, i n the mutant Figure 4. CsCl sedimentation of native DNA-from a density transfer experiment: T7 versus . T7am6. Panels A-D represent the T7 i n f e c t i o n to which CM was added at 8> 10, 12, and 16 minutes after i n f e c t i o n respectively. Panels E-nH represent the T7am6 in f e c t i o n for the same sampling times. Tritium labeled T7 DNA was used as a reference. Percent of Recovered Radioactivity i n f e c t i o n delaying the addition of CM from 10 minutes (F) to 12 minutes (G) or 16 minutes (H) after i n f e c t i o n did not r e s u l t i n a s h i f t of r e p l i c a t i v e parental l a b e l from the density of hybrid DNA to the density of recombined DNA. The T7am6 mutant was unable to r e p l i c a t e beyond hybrid, and therefore i t was appar-ently defective i n parent to progeny molecular recombination. To c l a r i f y the difference in the r e p l i c a t i o n patterns observed i n the T7am6 i n f e c t i o n compared to the T7 + i n f e c t i o n , the graphs obtained from the 16 minute CM treated samples were superimposed (Figure 5). The arrows indicate the positions of " l i g h t " reference DNA and completely substituted heavy reference DNA. As can be seen, at a time when a s i g n i f i c a n t amount of the r e p l i c a t i v e DNA from the wildtype i n f e c t i o n was sedimenting at the density of recombined DNA, the r e p l i c a t i v e DNA from the T7am6 in f e c t i o n remained at a hybrid location of the gradient. The conclusion drawn from t h i s experiment i s that i n the absence-of T7 gene 6 encoded exonuclease, T7 i s unable to undergo molecular recombination. In a density s h i f t experiment the sedimentation of parental l a b e l at a recombined density does not guarantee that the DNA has undergone parent to progeny recombination. The presence of recombinant density DNA can be a r e f l e c t i o n of the presence of concatemeric DNA i n which the progeny DNA contribution i s greater than the parental DNA contribution. Therefore i t was necessary to show that the DNA having a recombinant density i n the wildtype i n f e c t i o n represented DNA which had recombined. Concatemeric DNA from a T7 + i n f e c t i o n i s found to contain single 41 Figure 5. A comparison of the CsCl sedimentation patterns of parental , 3 2P-labeled DNA from T7 versus T7am6 for a l a t e i n f e c t i o n time. HH LL Percent of Length of Gradient 42 strand d i s c o n t i n u i t i e s one phage equivalent unit apart (15, 96, 97), although there i s some evidence that long single strands may be formed (24, 47). However, al k a l i n e sucrose gradient analysis of T7 + and T7am6 parental labeled DNA from a density s h i f t experiment indicated that long single strands are not present under these experimental conditions. Figure 6 shows alk a l i n e sucrose analysis of DNA from both a T7 + (A) and a T7am6 (B) i n f e c t i o n i n which CM was added at 16 minutes aft e r i n f e c t i o n . The parental labeled DNA from both infections sedimented as one phage equivalent or smaller under these conditions. Thus, i f the r e p l i c a t i v e DNA from a T7 + density s h i f t experiment i s analyzed, and the DNA has undergone j o i n t molecule formation as well as covalent repair, then some of the parental labeled DNA should sediment i n a denatured CsCl gradient at a density greater than that of the " l i g h t " reference DNA. This single strand recombinant density DNA then would be t r u l y representative of parent to progeny molecular recombination. To show that molecular recombination did occur under conditions permissive for the exonuclease, the parental labeled DNA from a density transfer experiment was examined for the presence of covalently repaired j o i n t molecules using the following procedure. E. c o l i was grown and infected as described for Figure 4. Samples from both a T7 + and a T7am6 i n f e c t i o n were treated with CM at 16 minutes a f t e r i n f e c t i o n and incubated further. The DNA p u r i f i e d from these samples was analyzed i n preparative neutral CsCl density gradients. The d i s t r i b u t i o n of lab e l was determined, and the r e p l i c a t i v e DNA (any DNA which i s cn Figure 6. Alkaline sucrose gradient analysis of T7 versus T7am6 parental 3 2 P - l a b e l e d DNA from a density transfer experiment. Panel A i s the T7 sample. Panel B i s the T7am6 sample. Tritium labeled T7.phage DNA was used as a reference. 44 displaced from the conservative unreplicated peak) from each gradient was i s o l a t e d and reanalyzed i n alkaline CsCl density gradients. The d i s t r i b u t i o n of l a b e l from the denatured CsCl gradients i s shown i n Figure 7. The r e p l i c a t i v e DNA from the wildtype i n f e c t i o n contained a large amount of parental l a b e l which sedimented at a density greater than the l i g h t reference DNA (A). In contrast, a l l of the denatured r e p l i c a t i v e DNA from the T7am6 i n f e c t i o n sedimented at the conservative (light) density (B). The p o s i t i o n of the l i g h t reference DNA i s indicated by the arrow. A l l of the parental l a b e l i n the T7am6 sample must return to the l i g h t location of the gradient under denaturing conditions since the DNA only r e p l i c a t e s to hybrid, as shown by neutral CsCl sedimentation. That i s , the T7am6 r e p l i c a t i v e DNA would not contain any recombined DNA. In contrast, i n the T7 + i n f e c t i o n , a large amount of the denatured r e p l i c a t i v e DNA did sediment at the density of recombined DNA. As can be observed in Figure 5, even at a late sampling time, some of the T7 + r e p l i c a t i v e DNA s t i l l sedimented as hybrid and therefore would not be expected to sediment at the density of recombined DNA i n a denatured CsCl density gradient. This r e s u l t confirms the conclusion that under conditions permissive for the exonuclease parent to progeny molecular recombination has occur reel. From the experiment described i n Figures 4 and 5 i t was concluded that the T7 exonuclease i s necessary for molecular recombination since i n i t s absence parental DNA only r e p l i c a t e d to hybrid, whereas i n the wildtype i n f e c t i o n recombination was observed. However, i t has also been shown that DNA synthesis i s Figure 7. CsCl sedimentation of a l k a l i denatured r e p l i c a t i v e parental 3 2 P - l a b e l e d DNA from a density transfer experiment: T7+ versus T7am6. Panel A represents a T7+ sample which was treated with CM at 16 minutes after i n f e c t i o n . .Panel B . represents a T7am6 sample for the same time. The arrow indicates the po s i t i o n of a l i g h t reference DNA. -p > -P o Percent of Length of Gradient r e s t r i c t e d i n the T7am6 mutant (112) . Although a s i g n i f i c a n t amount of DNA s y n t h e s i s i s known to occur i n t h i s mutant, i t i s p o s s i b l e t h a t i n the absence o f the exonuclease not enough r e p l i -c a t i o n takes p l a c e t o a l l o w recombination, i . e . , i f the mating p o o l of i n t r a c e l l u l a r DNA does not i n c l u d e a s u b s t a n t i a l amount of completely s u b s t i t u t e d progeny DNA, recombination might not be de t e c t e d . T h e r e f o r e , the experiment p r e v i o u s l y d e s c r i b e d was repeated u s i n g the T7ts6 mutant. R e p l i c a t i o n was permited to proceed u n t i l the p a r e n t a l DNA had r e p l i c a t e d t o h y b r i d p r i o r to a s h i f t t o a nonpermissive temperature. Under these c o n d i t i o n s some breakdown of host DNA was expected (92). The experiment was conducted i n the f o l l o w i n g way. De n s i t y 32 + 3 2 l a b e l e d E. c o l i were i n f e c t e d w i t h P - l a b e l e d T7 or P - l a b e l e d T7ts6 phage a t the p e r m i s s i v e temperature o f 30C. Samples a t v a r i o u s times a f t e r i n f e c t i o n were i c e c h i l l e d . At the same time samples from both i n f e c t i o n s were t r e a t e d w i t h CM; h a l f of each CM t r e a t e d sample was l e f t a t 30C and the r e s t o f the sample was s h i f t e d t o the nonpermissive temperature of 43C. At 30 minutes a f t e r i n f e c t i o n the CM t r e a t e d samples were i c e c h i l l e d . The DNA from a l l samples was e x t r a c t e d and analyzed by C s C l d e n s i t y g r a d i e n t sedimentation at a n e u t r a l pH. The g r a d i e n t s were c o l l e c t e d , and the d i s t r i b u t i o n o f l a b e l determined. T h i s experiment gave e s s e n t i a l l y the same r e s u l t s as o b t a i n e d i n the T7am6 experiment. These r e s u l t s are presented i n F i g u r e 8. Panels A, B, and C r e p r e s e n t the d i s t r i b u t i o n of p a r e n t a l l a b e l from the T 7 + i n f e c t i o n as f o l l o w s : A) a t 12 minutes a f t e r i n f e c t i o n ; B) CM 47 Figure 8. CsCl sedimentation of native DNA from a density trans-. fe r experiment incubated at both permissive and nonper- : missive temperatures: T7 + versus T7fcs6. Panels A-C represent the T7+ i n f e c t i o n at 12 minutes, at 12 min-utes + CM incubated at 30C, and 12 minutes + CM incu-bated at 43C, respectively. Panels D-F represent the T7ts6"infection under the same conditions. Panels G-L represent the r e p l i c a t i v e peaks from A-F resedi-mented with l i g h t and completely substituted (5-BrdU) 3H reference DNAs. P E R C E N T OK R E C O V E R E D R A D I O A C T I V I T Y 48 added at 12 minutes, then incubated to 30 minutes at 30C; and C) CM added at 12 minutes, then incubated to 30 minutes at 43C. Panels D, E, and F represent the T7ts6 i n f e c t i o n under the same conditions as for the T7 + i n f e c t i o n . Since about 50% of the parental labeled DNA i n a density s h i f t experiment always f a i l s to r e p l i c a t e , t h i s unreplicated DNA acts as an in t e r n a l reference i n preparative gradients. The r e p l i c a t i v e DNA from each of the gradients A-F was pooled, dialyzed, and reanalyzed i n a second set of CsCl density gradients supplemented with both completely substituted (5-BrdU) and l i g h t reference H-labeled T7 DNAs (Figure 8G-L). This experiment compared the extent of r e p l i c a t i o n and recombination of both the T7 + and T7ts6 DNA before and after incubation under conditions permissive and nonpermissive for the exonuclease. In both the T7 (A and G) and the T7ts6 (D and J) infectio n s the DNA was repl i c a t e d to the density of hybrid DNA at 12 minutes a f t e r i n f e c t i o n before the addition of CM and the s h i f t i n temperature. The addition of CM at 12 minutes and continued incubation at 3 0C of both the T7 + (B and H) and the T7ts6 (E and K) infecti o n s leads to recombination, i . e . 32 P-labeled DNA i n a l l these gradients banded at a density heavier than hybrid. Therefore between 12 and 30 minutes a f t e r i n f e c t i o n both the T7 and T7ts6 rep l i c a t e d and recombined extensively at the permissive temperature. However, when the T7 + and the T7ts6 infec t i o n s were sh i f t e d to the nonpermissive temperature of 43C a quite d i f f e r e n t r e s u l t was obtained. The T7 + i n f e c t i o n recombined extensively at 43C (Figure 8C and I ) . Denatured CsCl density analysis of T7 + r e p l i c a t i v e DNA for both 49 12 minute samples at 3 0C and at 43C indicated that the wildtype i n f e c t i o n underwent covalent repair to about the same extent at both temperatures. Similar r e s u l t s were obtained for the T7ts6 sample incubated at 30C. The T7ts6 DNA did not recombine to a s i g n i f i c a n t extent at the nonpermissive temperature (F and L). A comparison of panel L with panel K or of panel L with panel I indicates that the r e p l i c a t i v e T7ts6 DNA at the nonpermissive conditions i s s i g n i f i c a n t l y less dense than i t s counterpart under permissive conditions. The density of the T7ts6 DNA after 30 minutes incubation at 43C had not changed s i g n i f i c a n t l y from the density at 12 minutes (compare Figure 8D and J with F and L) . I t remained hybrid. Note that the density of the T7 + r e p l i c a t e d DNA when the i n f e c t i o n i s sh i f t e d to 43C (I) was greater than that shown by the re p l i c a t e d DNA which was l e f t at 30C (H) after the addition of CM. The rep l i c a t e d DNA from Figure 81 was also 3 denser than the [ H]5-BrdU reference which was made at 30C. The DNA polymerase at 43C may have l o s t a preference which i t had at 30C for thymidine over 5-BrdU. This r e s u l t does not a f f e c t the conclusion about the necessity of the exonuclease for recombis.c nation, because the r e p l i c a t i v e DNA of the T7ts6 at 43C (L) did not s h i f t to a recombined density with respect to either heavy reference. To c l a r i f y the extent to which recombination i s in h i b i t e d i n the T7ts6 i n f e c t i o n when i t was sh i f t e d to a nonpermissive temperature appropriate graphs from Figure 8 were superimposed. Figure 9 panel A represents the combined graphs of both the T7 + and T7ts6 infec t i o n s which had been s h i f t e d to 43C at 12 minutes 50 Figure 9.. Comparison of the CsCl sedimentation patterns of parental 3 2 P - l a b e l e d r e p l i c a t i v e DNA for in f e c t i o n s incubated at permissive and nonpermissive tempera-tures. A. T7+ versus T7ts6 both at 43C. B. T7ts6 at 30C versus T7ts6 at 43C. Bottom 50 Percent of Lengthnof Gradient 51 a f t e r i n f e c t i o n . Panel B r e p r e s e n t s the combined graphs of the T7ts6 i n f e c t i o n s which were incubated a t both 30C and 43C a f t e r the a d d i t i o n of CM. The d i s t r i b u t i o n of p a r e n t a l l a b e l i n F i g u r e 9B i s v e r y s i m i l a r t o t h a t i l l u s t r a t e d by F i g u r e 5, which a l s o compared the e x t e n t of recombination under p e r m i s s i v e and nonpermissive c o n d i t i o n s f o r the exonuclease. Thus i n the absence of exonuclease T7 i s unable to r e p l i c a t e beyond h y b r i d ; parent t o progeny molecular recombination i s d e f e c t i v e . 52 S e c t i o n 2. R e p l i c a t i o n o f the T7ts6 Phage DNA a t 43C Since t h e r e was s t i l l concern t h a t the p o o l of completely s u b s t i t u t e d progeny DNA molecules may have been too s m a l l i n the T7ts6 experiment to a l l o w the g e n e r a t i o n of de.tectib.le parent to progeny recombinant molecules, the f o l l o w i n g two experiments were performed. 3 ij)) The uptake of [ HJ-thymidine i n t o a c i d i n s o l u b l e m a t e r i a l was t e s t e d a t both p e r m i s s i v e and nonpermissive temperatures i n both the T 7 + and T7ts6 i n f e c t i o n . C e l l s were grown at 30C f o r one g g e n e r a t i o n t o 3 X 10 c e l l s per ml i n normal PO^ TCG supplemented w i t h 5 jig/ml Fdu, 5 V<3,/ml dThd, and 25 y g/ml u r a c i l . The c e l l s were d i v i d e d and one p o r t i o n was i n f e c t e d w i t h T 7 + and the other w i t h T7ts.6 phage, both a t an M.O.I, of 10. At 6 minutes a f t e r 3 i n f e c t i o n [ HJ-thymidine (1 u Ci/p g) was added to. each i n f e c t e d c u l t u r e . At 7 minutes a f t e r i n f e c t i o n h a l f of each c u l t u r e was s h i f t e d to. 43C. The uptake was monitered by t a k i n g samples a t 3 f r e q u e n t i n t e r v a l s and dete r m i n i n g the amount o f H-l a b e l e d a c i d i n s o l u b l e m a t e r i a l p r e s e n t . These r e s u l t s are shown i n F i g u r e 10 (64). Both T 7 + and T7ts6 i n f e c t i o n s were ab l e to i n c o r p o r a t e 3 [ HJ-thymidine i n t o a c i d i n s o l u b l e m a t e r i a l a t s i m i l a r r a t e s f o r e i t h e r temperature of i n c u b a t i o n . T h i s does not n e c e s s a r i l y mean t h a t t h e same amount o f DNA i s b e i n g s y n t h e s i z e d i n both cases. However, i t does i n d i c a t e t h a t DNA i n the- T7ts6 i n f e c t i o n i ncubated a t 43C i s b e i n g s y n t h e s i z e d at a r a t e comparable to t h a t shown by the w i l d t y p e . The i n f e c t i v e c e n t e r (I.C.) DNA s y n t h e s i z e d at 43C from both i n f e c t i o n s was p u r i f i e d by the SDS-pronase-phenol method and then the DNA was h y b r i d i z e d u s i n g a standard h y b r i d i z a t i o n 53 Incorporation of [ 3H]thymidine into acid insoluble material for both T7+ and T7ts6 infec t i o n s incubated at permissive and nonpermissive temperatures. (The hybridization data of the DNA synthesized at the nonpermissive temperature (43C) is-presented i n the inset.) TIME (min) 54 technique (29) to confirm that the DNA was T7 s p e c i f i c . The hybridization data are-preserited i n the inset;.of Figure 10. This r e s u l t supports the conclusion that the exonuclease i s d i r e c t l y responsible for recombination and that i t s absence does not i n d i r e c t l y i n h i b i t recombination by eliminating the progeny DNA pool. i i ) Since the pools of nucleoside triphosphates may be d i f f e r e n t i n the wildtype i n f e c t i o n than i n the i n f e c t i o n non-permissive for the exonuclease, i t was desireable to: show d i r e c t l y that at a s h i f t to a nonpermissive temperature the exonuclease negative i n f e c t i o n could s t i l l undergo another round of DNA r e p l i c a t i o n . Therefore the following experiment was performed simultaneously with the experiment described i n Figure 8. E. c o l i B23 was grown i n a 5-BrdU labeling media at 30C and 32 infected with P-labeled T7ts6 phage. At 12 minutes after i n f e c t i o n , the infected bacteria were washed free of 5-BrdU and shi f t e d to 4 3C. A sample of the infected c e l l s was taken both before washing the culture and d i r e c t l y after the s h i f t to. 4 3C. These samples were lysed, extracted, and analyzed by CsCl density gradient sedimentation to: show that the bacteriophage DNA had r e p l i c a t e d successfully. Since the time required for washing and resuspending the infected c e l l s did not faffect the observed lab e l d i s t r i b u t i o n pattern i n the 12 minute samples, only the sample taken a f t e r resuspension at 4 3C i s shown (Figure 11A). The r e s t of the culture was supplemented with thymidine (200 jig/ml), CM was added, and the culture was incubated to 30 minutes after i n f e c t i o n at 43C. Another sample was lysed, extracted, and / 55 / Figure 11. CsCl sedimentation of native DNA from a,_TJ.ts<'6 strand exchange experiment. A. Sedimentation pattern p r i o r to the addition of thymidine to the medium. B. Sedimentation pattern a f t e r incubation i n the presence of CM and thymidine. Tritium labeled DNA was used as a reference. 50 Bottom Percent of Length of Gradient analyzed by CsCl density centrifugation (Figure 11B). Nearly a l l of the l a b e l at the hybrid location i n Figure 11A had returned 32 to the l i g h t l o c ation i n Figure 11B. The P-labeled DNA had exchanged a previously 5-BrdU-labeled strand for one containing thymidine. Therefore e s s e n t i a l l y a l l of the r e p l i c a t i v e DNA i n the T7ts.6 i n f e c t i o n was shown to be able to undergo a further round of r e p l i c a t i o n a f t e r a s h i f t to.tthe nonpermissive temperature of 43C. Under these same conditions the T7ts6 i n f e c t i o n was unable to undergo parent to progeny molecular recombination. 3 2 Note that the small amount of P-label which remained displaced toward the heavy location was i n excellent agreement with the amount of recombinants found at 12 minutes afte r i n f e c t i o n (before the temperature sh i f t ) (Figure 8D). To v e r i f y that recombinant DNA was indeed present i n the 12 minute r e p l i c a t i v e DNA that was analyzed i n Figure 8D, a sample of the r e p l i c a t i v e peak was heat denatured and then was analyzed by CsCl density, sedimentation (Figure 12). As one can see, a small amount of the DNA i s covalently repaired recombinant DNA. The amount of recombinant DNA present i n Figure 12 appears to. be i n excess of that observed i n Figure 11B. However, t h i s i s not the case singe:in Figure 11 the t o t a l lysate i s being examined including both r e p l i c a t e d and unreplicated DNA and i n Figure 12 only re p l i c a t e d DNA was analyzed. 57 Figure 12. CsCl sedimentation of heat denatured T7ts6 r e p l i -cative DNA i s o l a t e d i n a density transfer experiment. A 12 minute T7ts6 sample i s analyzed. Tritium labeled DNA was used as a reference. -P •H > •H •p o Percent of Length of Gradient 58 Section 3. Covalent Repair of Recombinant Molecules under Conditions Permissive and Nonpermissive for the Exonuclease (Gene 6) Exonuclease digestion to: remove redundant single strands or to reduce branched molecules to. l i n e a r duplexes has been suggested as a necessary step p r i o r to. the covalent re p a i r of some j o i n t : molecules (11, 17, 74). The following experiment was conducted to see i f recombinants formed i n the presence of the exo.nuclease (gene 6) could be repaired i n i t s absence. The procedure used was sim i l a r to that described for Figure 8, i n fact the experiments were performed at the same time. However, the temperature s h i f t was delayed to allow j o i n t molecule formation before a s h i f t to the nonpermissive temperature. Separate portions of density labeled E. c o l i were infected with 3 2 P - l a b e l e d T7 + or T7ts6 phage at 30C. At 14 minutes after i n f e c t i o n a sample of. each culture was c h i l l e d , lysed, extracted, and analyzed by CsCl density gradient sedimentation. The rest of each infected culture was treated with CM and half was l e f t at 30C and the rest was s h i f t e d to 43C and incubated further. Portions of these CM treated samples then were lysed, extracted, and also analyzed by neutral CsCl density sedimentation. The gradients were c o l l e c t e d and the d i s t r i b u t i o n of l a b e l was determined. The re s u l t s are shown i n Figure 13. Panels A, B, and C represent the T7 + i n f e c t i o n as follows: (A) at 14 minutes; (B) at 14 minutes with CM added fehen incubated at 30C; and (C) at 14 minutes with CM added and s h i f t e d to 43C. Panels D, E, and F represent the T7ts6 i n f e c t i o n under the same conditions. / 5 9 Figure 13. CsCl sedimentation of native DNA from a—density transfer experiment incubated at permissive and nonpermissive temperatures: T7 + versus T7ts6. Panels A-C represent the T7 + i n f e c t i o n at 14 minutes, at 14 minutes + CM incubated at 30C, and at 14 min-utes + CM incubated at 43C, respectively. Panels D-F represent the T7ts6 i n f e c t i o n for the same sampling times. 60 The arrows i n the f i g u r e i n d i c a t e the expected p o s i t i o n o f DNA of h y b r i d d e n s i t y d u r i n g a d e n s i t y s h i f t experiment -at 30C as determined by the companion experiment. Most of the p e p l i c a t i v e DNA i n a l l the samples sedimented to the d e n s i t y of recombined DNA. There i s l i t t l e d e t e c t i b l e d i f f e r e n c e between the amount of r e p l i c a t i v e DNA which i s sedimented at t h e d e n s i t y of recombined DNA iri;dibhe^l4aminute samplestandatfioseOsampies ^  which were -incubated f u r t h e r w i t h CM at 3 0C-—compare A w i t h B and D w i t h E. The r e p l i c a t i v e DNA from these g r a d i e n t s was p o o l e d , d i a l y z e d , and resedimented i n C s C l w i t h a p p r o p r i a t e r e f e r e n c e s . As expected e s s e n t i a l l y a l l of the DNA was recombined by 14 minutes and no s i g n i f i c a n t d i f f e r e n c e s were noted i n the g r a d i e n t s . Again the w i l d t y p e i n f e c t i o n i n c u b a t e d a t 43C (C) d i s p l a y e d a l a r g e s h i f t : to a d e n s i t y g r e a t e r than t h a t of the heavy r e f e r e n c e which was s y n t h e s i z e d a t 30C. To a s c e r t a i n the extent o f c o v a l e n t r e p a i r of j o i n t molecules the r e p l i c a t i v e DNA from these g r a d i e n t s then was heat denatured and rebanded i n C s C l . S e v e r a l d i f f e r e n c e s , were noted when comparing the T 7 + (Figure 14A-C, corres p o n d i n g to F i g u r e 13A-C) w i t h the T7ts6 DNA (Figure 14D-F, corres p o n d i n g to F i g u r e 13D-F). One can observe t h a t a t 14 minutes a f t e r i n f e c t i o n most of the recombinant DNA was u n r e p a i r e d (Aaand D). F u r t h e r i n c u b a t i o n at 30C l e a d s t o e x t e n s i v e r e p a i r o f both the T 7 + and T7ts6 r e p l i c a t i v e DNA (B and E ) . However, i n c u b a t i o n of the recom-b i n a n t s at 43C leads to a n o t i c e a b l e d i f f e r e n c e between r e p a i r of T 7 + DNA and T7ts6 DNA <('C and F) . There i s more u n r e p a i r e d DNA (DNA o v e r l a p p i n g w i t h H - l a b e l e d T7 r e f e r e n c e DNA) from the 61 Figure 14. CsCl sedimentation of heat denatured r e p l i c a t i v e DNA is o l a t e d i n a density transfer experiment: T7 + versus T7ts6. Panels A-C represent the T7+ i n f e c t i o n at 14 minutes, at 14 minutes + CM incubated at 30C, and 14 minutes + CM incubated at 43C, respectively. Tritium labeled T7 DNA was included as a reference.-Panels D-F represent the T7ts6 i n f e c t i o n for the same sampling times. P e r c e n t of R e c o v e r e d R a d i o a c t i v i t y 62 T7ts6 infected c e l l s than from the T7 + infected c e l l s . There i s also less repair with the T7ts6 at 43C than with the T7ts6 at 3 0C (E and F). In addition, determining the percent of l a b e l found i n the covalently repaired recombinant DNA peak of the T7 + i n f e c t i o n indicates that there i s an increase at 43C compared to the same i n f e c t i o n incubated at 30C of about 18%. Comparison of the percentage of covalent r e p a i r of the T7ts6 i n f e c t i o n incu-bated at 43C with incubation at 30C indicates that there i s a 23% decrease i n the amount of covalent repair at the higher temperature. This means that i n the absence of exonuclease a c t i v i t y , there i s a 41% decrease i n the amount of covalent repair occuring at 43C when compared with the wildtype i n f e c t i o n under the same conditions. These re s u l t s suggest that the exonuclease i s necessary for a limited amount of covalent repair. Since some covalent repair i s permitted aft e r the s h i f t to a temperature nonpermissive for the exonuclease (compare D with E), some j o i n t molecules do not require the presence of the exonuclease to mediate t h e i r repair. Other enzymes are also involved i n t h i s process. For example, the T4 DNA polymerase i s required to mediate covalent r e p a i r of j o i n t molecules by f i l l i n g i n gaps (73). Therefore, one would not expect a great deal of repair to be necessariliy^ mediated by an exonuclease. However, the d i s p a r i t y between the amount of covalent repair permitted when conditions are permissive for the exonuclease compared to when conditions are nonpermissive suggests that at l e a s t some j o i n t molecules require an active exonuclease for covalent repair. In addition, another observation which may r e f l e c t the extent to which repair i s mediated by exonuclease i s that an infection, of a nonpermissive host by T7am6 phage leads to: the accumulation of d i s c o n t i n u i t i e s i n the parental DNA. This was shown i n an experiment i n which density labeled E. c o l i was 32 + 32 infected with P-labeled T7 or P-labeled T7am6 phage at an M.O.I, of 7. CM was added tossamples of the infected c e l l s at various times after i n f e c t i o n and samples were incubated u n t i l 30 minutes post i n f e c t i o n . The i n f e c t i v e centers then were incubated i n a l k a l i and the labeled DNA was analyzed by alk a l i n e sucrose gradient sedimentation. The r e s u l t s are presented i n Figure 15. Panels A, B, and C represent the T7 + i n f e c t i o n to which CM was added at .8, 12, and 16 .minutes after i n f e c t i o n , respectively. Panels D, E, and F represent the T7am6 i n f e c t i o n under the same incubation conditions. In the T7am6 i n f e c t i o n under conditions nonpermissive for the exonuclease an increasing amount of the parental T7am6 DNA accumulated as fragments smaller than one phage equivalent uni t as incubation continued, i . e . , 32 more and more P sedimented toward the top of the gradient the longer the infected c e l l s were allowed to incubate p r i o r to the addition of CM. This pattern of sedimentation can also be demonstrated i n the T7ts6 i n f e c t i o n under the appropriate conditions. The amount of single strand d i s c o n t i n u i t i e s apparent i n the T7 + DNA remained f a i r l y constant throughout the i n f e c t i o n . Therefore, under conditions nonpermissive for the exonuclease T7 apparently i s p a r t i a l l y defective i n repair of i t s DNA. 64 Figure 15. Alkaline sucrose sedimentation of T7 + versus T7am6 DNAs from a density transfer experiment. Panels A-C represent a T7+ in f e c t i o n to which CM was added at 8, 12, and 16 minutes a f t e r i n f e c t i o n . Panels D-F represent the T7am6 i n f e c t i o n for the same conditions. 'Tritium labeled T7 DNA was used as a reference. . P e r c e n t o f R e c o v e r e d R a d i o a c t i v i t y Section 4. Genetic Recombination under Conditions Permissive and Nonpermissive for the T7 Exonuclease (Gene 6) In the absence of exonuclease, fragments of T7 parental DNA are not r e d i s t r i b u t e d into progeny DNA molecules as i s expected when molecular recombination i s permitted. Therefore, i n the absence of exonuclease genetic recombination also would be expected to be defective i n T7. To te s t t h i s hypothesis the following experiment was performed. Double mutants of the type amNts6 were constructed. These mutants were crossed i n the following way: amNlts6 X amN2ts6 were used to i n f e c t O i l ' at 30C. At 5 minutes after i n f e c t i o n the infected c e l l s were d i l u t e d into T7 antisera and half of the antisera treated sample was shifted to the "semi-permissive" temperature of 37C and the rest was l e f t at 30C. (At a "semi-permissive" temperature incubation r e s u l t s i n a decreased phage burst compared to that observed at a permissive temperature (30C) but a higher phage burst than that r e s u l t i n g when incubation i s at a nonpermissive temperature '•(43C).) At 11 minutes the infected c e l l s were d i l u t e d out of antisera and incubated u n t i l 60 minutes a f t e r i n f e c t i o n . Control crosses of the type amNl X amN2 were conducted simultaneously under i d e n t i c a l conditions. The number of viable unattached phage remaining aft e r treatment of the infected c e l l s with antisera was determined for each cross. The number of phage -produced by a single i n f e c t i v e center was also calculated. The r e s u l t s showed that the number of viable unattached phage remaining i n the incubation mixture aft e r antisera treatment was less than J.% of the t o t a l number of progeny produced. The crosses involved mutations close to the l e f t and r i g h t ends of v the T7 genetic :map, i n the middle of the map, between adjacent genes, between distant genes, and between two mutations within the same gene. A l l of these r e s u l t s (75) showed that recombi-nation frequencies between any two mutants tested were s i g n i f i -cantly decreased when the cross was homozygous for a defective T7 exonuclease (gene 6) at a "semi-permissive" condition (Table 1). There was also a concomitant decrease i n the average y i e l d of progeny phage from a cross "semi-permissive" for,the exonuclease. Each of these crosses was performed at least twice with similar r e s u l t s i n each case. To show that the decreased recombination frequency was not caused by the decreased progeny y i e l d per se, analogous crosses of the type amNlts5 X amN2ts5 were conducted (75) . These crosses were performed under conditions permissive (30C) and "semi-permissive" (37C) for the T7 DNA polymerase (gene 5). In t h i s experiment the average phage y i e l d was brought down to the l e v e l comparable to that seen when the T7 exonuclease was defective. However, the recombination frequency observed between amNl and amN2 remained unaffected, even though DNA r e p l i c a t i o n was severely l i m i t e d causing a corresponding low average y i e l d . Therefore a decrease i n the number of progeny phage produced does not d i r e c t l y a f f e c t the extent to which recombination occurs. 67 Table I. E f f e c t of T7 exonuclease '(gene 61 on genetic r ec omb ina t io n. Cross Temp. (°C) Recombination % Phage/ I.C. am2 X am3 30 2.9 116 37 2.2 86.8 am2ts6 X am3ts6 30 3.63 132 37 0.63 0.5 ami X ami2 30 5.4 199 39 4.8 74 amlts6 X aml2ts6 30 8.8 175 39 2.6 1.3 aml2ts6 X aml6ts6 30 23.6 65 37 1.3 0.4 aml6-260 X aml6-325 30 5.1 185 37 5.2 132 aml6-260ts6 X aml6-325ts6 30 6.0 169 37 0.7 3.2 ami X am3 30 13.5 342 37 14.8 271 amlts6 X am3ts6 30 12.1 228 37 1.8 3.4 amlts5 X am3ts5 30 12.2 64 37 14.4 0.34 , . 2 X p.f.u. on B23 X 100% Percentage Recombinatxon = — s ... ., , ^ p.f.u. on O i l ' p.f.u., plaque-forming unit I . e . , i n f e c t i v e center 68 S e c t i o n 5. M o l e c u l a r Recombination under C o n d i t i o n s P e r m i s s i v e and Nonpermissive f o r the T7 Endonuclease (Gene 3) I f the T7 endonuclease (gene 3) i s r e q u i r e d f o r m o l e c u l a r recombination, then i n a d e n s i t y s h i f t experiment under c o n d i t i o n s nonpermissive f o r the endonuclease an amber mutant of gene 3 should o n l y be ab l e t o r e p l i c a t e t o h y b r i d . To t e s t t h i s hypo-t h e s i s the k i n e t i c s o f r e p l i c a t i o n and recombination by a T7am3 mutant i n a d e n s i t y t r a n s f e r experiment was examined. E. c o l i B23 was grown i n 5-BrdU 10 X P0 4 TCG f o r 2 hours a t 30C. The c u l t u r e 32 was i n f e c t e d w i t h P - l a b e l e d T7am3 a t an M.O.I, of 7. At v a r i o u s times a f t e r i n f e c t i o n samples were c h i l l e d , the c e l l s l y s e d , and the DNA e x t r a c t e d . A t 16 minutes a f t e r i n f e c t i o n a sample was t r e a t e d w i t h CM and incubated t o 30 minutes a f t e r i n f e c t i o n . The DNA from t h i s sample was e x t r a c t e d a l s o . The DNA from a l l samples was analyzed i n p r e p a r a t i v e C s C l d e n s i t y g r a d i e n t s a t a n e u t r a l pH. The g r a d i e n t s were c o l l e c t e d and the d i s t r i b u t i o n of p a r e n t a l l a b e l determined. The r e p l i c a t i v e DNA from these g r a d i e n t s was i s o l a t e d and r e a n a l y z e d i n n e u t r a l C s C l t o g e t h e r w i t h " l i g h t " H-T7 r e f e r e n c e DNA. The r e s u l t s of t h i s experiment are shown i n F i g u r e 16. Panels A, B, and C r e p r e s e n t the sedimentation of T7ara3 r e p l i c a t i v e DNA a t 12, 14, and 16 minutes a f t e r i n f e c t i o n , r e s p e c t i v e l y . Panel D r e p r e s e n t s the r e p l i c a t i v e DNA from the sample t r e a t e d w i t h CM a t 16 minutes a f t e r i n f e c t i o n and incubated t o 30 minutes a f t e r i n f e c t i o n . The p o s i t i o n expected f o r the h y b r i d DNA i s i n d i c a t e d by the arrow. At 12 minutes a f t e r i n f e c t i o n (A) e s s e n t i a l l y a l l of the r e p l i c a t i v e DNA sediments a t the d e n s i t y of h y b r i d DNA. T h i s i s 69 Figure 16. CsCl sedimentation of native T7am3 r e p l i c a t i v e DNA from a density transfer, experiment. Panels A^c / represent 12, 14, and 16 minutes aft e r i n f e c t i o n , , respectively. Panel D represents the 16 minute sample of infected c e l l s to which CM was added and then incubation continued. Tritium labeled T7 DNA was used as a reference. 70 comparable to the sedimentation of T7 + r e p l i c a t i v e DNA under the same conditions (Figure 8A and G). As the i n f e c t i o n was allowed to proceed the r e p l i c a t i v e DNA shi f t e d from the density of hybrid DNA to the density expected of DNA which had recombined. There-fore i n the absence of the endonuclease the parental DNA was able to r e p l i c a t e to the density of recombined DNA. It has been shown that under conditions nonpermissive for the endonuclease that concatemers accumulate (110). The gener-ation of r e p l i c a t i v e DNA which has a density c h a r a c t e r i s t i c of "recombined" DNA can be a function of the presence of concatemers which contain more progeny than parental DNA. Therefore i n the T7am3 experiment the presence of DNA of a recombinant density may not r e s u l t from molecular recombination but from concatemer formation. To determine i f the T7am3 in f e c t i o n did produce concatemeric DNA the r e p l i c a t i v e DNA which was analyzed i n Figure 16 also was analyzed by neutral sucrose gradient sedimen-ta t i o n . These r e s u l t s are shown i n Figure 17. Panels A, B, and C represent the T7am3 r e p l i c a t i v e DNA at 12, 14, and 16 minutes after i n f e c t i o n . Panel D represents DNA is o l a t e d from the sample which was treated with CM at 16 minutes and incubated to 3 0 minutes a f t e r i n f e c t i o n . At a l l sampling times a majority of the 3 r e p l i c a t i v e DNA sedimented faster than the H-T7 mature phage DNA reference. Therefore the presence of recombinant density DNA i n an experiment nonpermissive for the endonuclease did not guarantee that molecular recombination had i n fact taken place. In a T7 + i n f e c t i o n i n a parent to,progeny recombination 32 experiment, P-parental DNA i s fragmented and red i s t r i b u t e d among 71 Figure 17. Neutral sucrose sedimentation of. T7am3 r e p l i c a t i v e DNA from a density transfer experiment. Panels A-C represent the i n f e c t i o n at 12, 14, and 16 minutes after i n f e c t i o n , respectively. Panel D represents the 16 minute sample of infected c e l l s to which CM was added and then incubation was continued. Tritium labeled T7 DNA was used as a reference. Percent of Length of Gradient the 5-BrdU-labeled progeny DNA .molecules (55, 71, 73). The recombined molecule thus formed has a density, intermediate to that of DNA of hybrid density and.DNA which i s completely s u b s t i -tuted with 5-BrdU. Replicative DNA which has the density expected of DNA which has recombined also can be generated i n the following 32 manner; concatemers can be formed which contain the P la b e l as a unit size molecule joined end to end as a hybrid molecule attached to a f u l l y heavy progeny phage DNA molecule. Since the endonuclease i s postulated to be necessary to i n i t i a t e recombi-nation, j o i n t molecule formation would not be expected to occur under conditions nonpermissive for the endonuclease (11, 17, 58, 116). Therefore, under conditions nonpermissive for the endonu-clease, i t i s postulated that the r e p l i c a t i v e DNA from a parent to progeny recombination experiment which sediments at a density c h a r a c t e r i s t i c of recombined DNA occurs because of concatemer :' formation and not because the parental DNA i s fragmented and dispersed among the progeny DNA pool. If t h i s hypothesis i s y.-. correct then under conditions i n which the concatemeric DNA i s sheared to one phage equivalent unit i n length, the parental l a b e l would be li b e r a t e d as a molecule having the density of hybrid DNA (Figure 18). In contrast, i f the parental l a b e l has been fragmented and dispersed into progeny DNA as occurs i n the T7 + i n f e c t i o n , then the r e p l i c a t i v e DNA which i s sheared w i l l r e t a i n the density c h a r a c t e r i s t i c of DNA which has recombined. To show that the r e p l i c a t i v e DNA from a T7am3 i n f e c t i o n sedi-mented at the density of recombined DNA because of concatemer formation rather than recombination the following density transfer Figure 18. A schematic diagram of the postulated differences between concatemers synthesized i n T7+ versus T7am3 infectio n s . T7 amber 3 c o n c a t e m e r s I si ze 1 I i T 7 + c o n c a t e m e r s s i z e 1 experiment was performed. E..; c o l i B23 was grown i n 5-BrdU 10 X PO^ TCG a t 30C. The c u l t u r e was s p l i t and.one p o r t i o n was 3 2 ' *4* ' 32 i n f e c t e d w i t h P-T7 and the. other w i t h P-T7am3 phage. The :'. i n f e c t e d c e l l s from both i n f e c t i o n s were incubated u n t i l 14 minutes a f t e r i n f e c t i o n and then CM was added; the c e l l s were incubated u n t i l 3 0 minutes a f t e r i n f e c t i o n . The DNA was e x t r a c t e d from both samples and analyzed by p r e p a r a t i v e C s C l sedimentation. The r e p l i c a t i v e DNA was i s o l a t e d and r e a n a l y z e d by both n e u t r a l sucrose and C s C l sedimentation. The s i z e and d e n s i t y d i s t r i b u t i o n of the r e p l i c a t i v e DNA was determined. The r e p l i c a t i v e DNA then was sheared a t a speed which had p r e v i o u s l y been shown t o shear the DNA to approximately one phage e q u i v a l e n t u n i t i n l e n g t h . T h i s sheared DNA a l s o was analyzed by both n e u t r a l sucrose and C s C l sedimentation. The r e s u l t s of n e u t r a l sucrose v e l o c i t y s edimentation f o r both the sheared and unsheared samples are presented i n F i g u r e 19. Panels A and B r e p r e s e n t the T 7 + r e p l i c a t i v e DNA b e f o r e and a f t e r shear, r e s p e c t i v e l y . Panels C and D r e p r e s e n t the T7am3 r e p l i c a t i v e DNA bef o r e and a f t e r shear. R e p l i c a t i v e DNA from both the T 7 + (A) and T7am3 (C) i n f e c t i o n s c o n t a i n e d l a r g e amounts of f a s t sedimenting DNA p r i o r to shear. A f t e r s s h e a r i n g (B and D) r e p l i c a t i v e DNA from both i n f e c t i o n s sedimented a t approximately s i z e one. To show t h a t under c o n d i t i o n s nonpermissive f o r the endonu-c l e a s e t h a t the form a t i o n o f recombinants was i n h i b i t e d , both the sheared and unsheared r e p l i c a t i v e DNA was analyzed by C s C l d e n s i t y sedimentation a t a n e u t r a l pH. The g r a d i e n t s were c o l l e c t e d and the d i s t r i b u t i o n o f l a b e l determined. To compare 75 Figure 19. Neutral sucrose sedimentation of unsheared and sheared r e p l i c a t i v e DNA from both T7+ versus T7am3 inf e c t i o n s . A. Unsheared T7 +, B. Sheared T7 +, C. Unsheared T7am3, D. Sheared T7am3. Tritium labeled T7 DNA was used as a reference. the r e s u l t s -more e a s i l y , the graphs of sheared and unsheared DNA from each i n f e c t i o n were superimposed and are presented i n Figure 20. Panel B represents the r e p l i c a t i v e DNA from the T7 + i n f e c t i o n before and a f t e r shear t o one phage equivalent u n i t . Panel D represents the T7am3 r e p l i c a t i v e DNA under the same conditions. Panel A indicates the d i s t r i b u t i o n of the " l i g h t " and completely substituted (5-BrdU) T7 reference DNAs. Panel C indicates the d i s t r i b u t i o n of the " l i g h t " and completely substituted T7am3 reference DNAs. The v e r t i c a l l i n e s were included to indicate the positions of the references i n the r.eplicative DNA gradients (B and D). Pr i o r to shear a l l of the T7 + (B) and most of the T7am3 (D) r e p l i c a t i v e DNA sedimented at a density c h a r a c t e r i s t i c of recombined DNA. As expected, under conditions permissive for the endonuclease, sheared r e p l i c a t i v e DNA retains the density of recombined DNA. Under conditions nonpermissive for the endonu-clease, a majority of the r e p l i c a t i v e DNA does not r e t a i n the density of recombined DNA when sheared to size one (D). On the contrary there i s a dramatic s h i f t of the DNA to the density of hybrid DNA. This r e s u l t i s consistent with the hypothesis that under conditions nonpermissive for the endonuclease the parental DNA i s not fragmentedfobut remains i n t a c t ; parent to progeny molecular recombination i s i n h i b i t e d . In addition, a substantial amount of the T7am3 parental labeled DNA only r e p l i c a t e s to hybrid under conditions m which the T7 DNA i s e s s e n t i a l l y a l l recom-bined (prior to shear). This observation supports the conclusion that recombination i s i n h i b i t e d under conditions nonpermissive for the endonuclease. Figure 20. Superimposed CsCl sedimentation patterns of unsheared and sheared . r e p l i c a t i v e DNA from a T7 + versus a T7am3 in f e c t i o n . A. Light and - -heavy 3H -T7+ DNAS, Bi; Unsheared and sheared T7 + r e p l i c a t i v e DNA, C.. Light and heavy 3H-T7am3 DNAs, D. Unsheared and sheared T7am3 r e p l i c a t i v e DNA. 78 A l l of the T7am3 r e p l i c a t i v e DNA d i d not s h i f t from the d e n s i t y of recombined t o the d e n s i t y of h y b r i d DNA when sheared to one phage e q u i v a l e n t u n i t . T h e r e f o r e the f o l l o w i n g q u e s t i o n s were asked: 1) what shear reduces a l l of the T7am3 r e p l i c a t i v e DNA t o h y b r i d , and 2) does the T 7 + r e p l i c a t i v e DNA s t i l l r e t a i n i t s * recombinant d e n s i t y a t t h a t shear? The experiment performed was s i m i l a r to t h a t d e s c r i b e d f o r F i g u r e s 19 and 20. D e n s i t y 32 + 32 l a b e l e d E. c o l i B23 i n f e c t e d w i t h P-T7 or P-T7am3 phage were incubated u n t i l 16 minutes a f t e r i n f e c t i o n ; CM then was added and the c e l l s i n c u b a t e d u n t i l 3 0 minutes a f t e r i n f e c t i o n . The p u r i f i e d DNA from both i n f e c t i o n s was analyzed i n p r e p a r a t i v e C s C l d e n s i t y g r a d i e n t s , the d i s t r i b u t i o n o f p a r e n t a l l a b e l d e t e r -mined, and the r e p l i c a t i v e DNA then was analyzed by n e u t r a l sucrose v e l o c i t y sedimentation t o determine i f f a s t sedimenting DNA was pr e s e n t p r i o r t o shear. Both the T 7 + and T7am3 r e p l i -c a t i v e DNA samples c o n t a i n e d s u b s t a n t i a l amounts of f a s t sedimenting DNA. Samples of the r e p l i c a t i v e DNA from both i n f e c t i o n s were sheared a t s e v e r a l speeds. The n e u t r a l sucrose sedimentation p a t t e r n s of the sheared samples are presented i n F i g u r e 21. Panel A r e p r e s e n t s the T 7 + DNA sheared t o s i z e one, panel B r e p r e s e n t s the T 7 + DNA sheared t o a h s i z e molecule, p a n e l C r e p r e s e n t s the T7am3 DNA sheared t o s i z e one, and panel D r e p r e s e n t s the T7am3 DNA sheared t o a h s i z e molecule (14). To show t h a t the T7am3 r e p l i c a t i v e DNA c o u l d be reduced completely to h y b r i d a t a shear s i z e which was i n s u f f i c i e n t t o reduce the T 7 + DNA to h y b r i d , the unsheared and sheared r e p l i c a -t i v e DNA was resedimented i n C s C l a t a n e u t r a l pH. These r e s u l t s 79 Figure 21. Neutral sucrose sedimentation of sheared r e p l i c a t i v e DNA"from a density transfer experiment: T7+ versus' T7am3. A and B. Sheared r e p l i c a t i v e T7+.DNA. C and D. Sheared r e p l i c a t i v e T7am3 DNA. DNA was used as a reference. Tritium labeled T7 PERCENT OF RECOVERED RADIOACTIVITY to o 80 are p r e s e n t e d i n F i g u r e 22. Panels A, B, and C r e p r e s e n t the d i s t r i b u t i o n o f l a b e l from the T 7 + i n f e c t i o n as f o l l o w s : (A) unsheared r e p l i c a t i v e DNA; CB) r e p l i c a t i v e DNA sheared to s i z e one; and (C) r e p l i c a t i v e DNA sheared t o h. Panels D, E, and F re p r e s e n t the T7am3 r e p l i c a t i v e DNA under the same c o n d i t i o n s . The v e r t i c a l l i n e s i n d i c a t e the p o s i t i o n of completely s u b s t i -t u t e d (5-BrdU) r e f e r e n c e DNA, a sample of which i s i n c l u d e d i n panel A. S e v e r a l o b s e r v a t i o n s from these g r a d i e n t s i n d i c a t e t h a t T7am3 i s unable t o Eecombine t o the extent observed i n the T 7 + i n f e c t i o n . 1) Only h a l f o f the r e p l i c a t i v e DNA i s converted from h y b r i d t o recombined d u r i n g the i n f e c t i o n (D) whereas a l l of the T 7 + DNA i s recombined (A). 2) When the r e p l i c a t i v e DNA from the T7am3 experiment i s reduced t o s i z e one a t l e a s t h a l f of the "recombined" T7am3 DNA i s s h i f t e d t o h y b r i d (E). In c o n t r a s t , t h e r e i s no s i g n i f i c a n t change i n the d e n s i t y of the T7 + r e p l i c a t i v e DNA which i s reduced t o s i z e one (B). 3) Shear of the T7am3 r e p l i c a t i v e DNA to h s h i f t s a l l o f the p a r e n t a l l a b e l t o the d e n s i t y of h y b r i d DNA (F). However, not even s h e a r i n g the + 32 + T7 DNA to s i z e \ y i e l d s a s i g n i f i c a n t s h i f t i n the P-T7 DNA from recombined t o a h y b r i d l o c a t i o n (C). T h e r e f o r e , under c o n d i t i o n s nonpermissive f o r the endonuclease p a r e n t a l l a b e l i s not fragmented t o a s i g n i f i c a n t extent and r e i n c o r p o r a t e d i n t o progeny mole c u l e s . The p a r e n t a l DNA i s u s u a l l y found i n t a c t under these c o n d i t i o n s . 81 Figure 22. CsCl sedimentation of both unsheared and sheared r e p l i c a t i v e DNA from a density transfer experiment: T7 versus T7am3. Panels'A-C represent T7+ DNA unsheared, sheared to siz e one, and sheared to 1 h, respectively. Panels Dr-F represent T7am3 DNA under the same conditions. Tritium labeled T7 DNA was used as the references. P E R C E N T OF L E N G T H O F G R A D I E N T 82 Section 6. Concatemer Formation under Conditions Permissive and Nonpermissive for Molecular Recombination i n T7 Double stranded molecules longer than one phage equivalent unit i n length occur as intermediates of r e p l i c a t i o n i n several phage systems (117). These r e p l i c a t i v e intermediates c a l l e d concatemers were o r i g i n a l l y postulated to be necessary for the generation of c i r c u l a r l y permuted phage DNA such as found i n T4 (111). Various investigators also have shown that concatemers may be e s s e n t i a l for phage v i a b i l i t y either as a s t r u c t u r a l requirement for phage maturation as indicated for lambda (108), or to. r e p l i c a t e the ends of terminally 'redundant molecules as postulated for T7 (124). The mechanism by which concatemers are generated has been investigated but l i t t l e evidence has been found to implicate particular, enzymes as e s s e n t i a l to mediate concatemer formation. However, evidence favors recombination as the mechanism by which concatemers are generated i n T4 (30, 72). Since r o l l i n g c i r c l e r e p l i c a t i o n does not occur i n T7 (127) a possible mechanism for T7 concatemer formation i s also v i a a general recombination pathway. I t has been shown that the T7 exonuclease (gene 6) i s required for molecular recombination (64) as well as genetic recombination (52, 75, 83). Furthermore, newly r e p l i c a t e d T7 DNA i s found to accumulate fragments, smaller than one phage equivalent under conditions nonpermissive for the exonuclease at a time when concatemers are found i n the wildtype i n f e c t i o n (36). Therefore i t was possible that an exonuclease was required for concatemer formation as well as recombination. Experiments were performed to determine whether T7 concatemers 83 are formed at any time during the T7 i n f e c t i o n cycle under conditions nonpermissive for the exonuclease. To determine i f concatemers containing parental DNA were generated under conditions nonpermissive for the exonuclease a 32 + density transfer experiment was performed i n which P-T7 or 32 P-T7am6 phage were used to i n f e c t density, labeled E. c o l i B23. The DNA p u r i f i e d from samples taken at various times during these infect i o n s was analyzed by preparative neutral CsCl density sedimentation. The d i s t r i b u t i o n of parental l a b e l was determined, and the r e p l i c a t i v e peaks of DNA were i s o l a t e d from the gradients. The r e p l i c a t i v e DNA was reanalyzed by neutral sucrose .sedimentation. The res u l t s are shown i n Figure 23. Panels A, B, and C represent the sucrose gradient analysis of the T7 + r e p l i c a t i v e DNA at 9, 11, and 13 minutes after i n f e c t i o n , respectively. Panels D, E, and F represent the T7am6 r e p l i c a t i v e DNA for the same sampling times. Although there i s fast-sedimenting material, evident i n the T7am6 i n f e c t i o n at 9 minutes (D), by 13 minutes (F) a l l of the parental la b e l sediments as one phage equivalent unit or smaller. The T7 + i n f e c t i o n contains f a s t sedimenting material at a l l sampling times (A-C). Late r e p l i c a t i v e DNA from both infections was also analyzed and the res u l t s are shown i n Figure 24. Panels A, B, and C represent the DNA from T 7 + samples to: which CM was added at 12, 14, and 16 minutes after i n f e c t i o n and the sample incubated further. Panels D, E, and F represent the T7am6 i n f e c t i o n for the same sampling times. None of the T7am6 r e p l i c a t i v e DNA was fast-sedimenting at late times of i n f e c t i o n . Furthermore the 84 F i g u r e 23. N e u t r a l sucrose sedimentation of p a r e n t a l 3 2 P - l a b e l e d r e p l i c a t i v e DNA from a T 7 + versus a T7am6 i n f e c t i o n : e a r l y sampling times. Panels A-C r e p r e s e n t 9, 11, and 13 minute samples o f the T 7 + i n f e c t i o n , r e s p e c -t i v e l y . Panels D-F r e p r e s e n t s i m i l a r samples from the T7am6 i n f e c t i o n T r i t i u m l a b e l e d T7 DNA was used as a r e f e r e n c e . PERCENT OF LENGTH OF GRA DIE NT 85 F i g u r e 24, o < o Q < CC Ui > N e u t r a l sucrose sedimentation o f p a r e n t a l 3 2 P - l a b e l e d ( r e p l i c a t i v e DNA from a T 7 + versus a T7am6 i n f e c t i o n : j l a t e sampling times. Panels A-C r e p r e s e n t a T 7 + j irife"ct i 6 h -to which CM was added a t 12, 14, and 16 minutes a f t e r i n f e c t i o n , r e s p e c t i v e l y . Panels D-F j r e p r e s e n t a T7am6 f o r the same sampling times. T r i t i u m l a b e l e d T7 DNA was used as a r e f e r e n c e . 30 1<H 20' A • \\ 3 2P ; : ^ ^ ^ ^ B • j \ Bottom PERCENT OF LENGTH OF GRADIENT 86 DNA i s highly fragmented. In the T7 + i n f e c t i o n a majority of the r e p l i c a t i v e DNA i s fast-sedimenting. At the l a t e s t T7 + sampling time (C) a peak of DNA of one phage equivalent unit i n length i s forming. This probably reflects, the s t a r t of DNA maturation/ The results, of these experiments indicated that concatemers form early i n i n f e c t i o n under conditions nonpermissive for the exonuclease, but that they are unstable and are broken down to: fragments smaller than that of mature phage. The formation and s t a b i l i t y of concatemers under conditions permissive and nonpermissive for the exonuclease also was examined i n a progeny label, experiment. E. c o l i B23 was grown i n normal P0 4 TCG supplemented with 5 yg/ml FdU, 5 yg/ml dThd, and 2 5 yg/ml + u r a c i l at 30C. One portion of the culture was infected with T7 and the other with T7am6 phage at an M.O.I, of 7. At 7 minutes 3 after i n f e c t i o n [ H]thymidine (1 yCi/yg) was added to each i n f e c t i o n . Samples were taken at various times after i n f e c t i o n and the p u r i f i e d DNA was analyzed by neutral sucrose sedimentation. The re s u l t s are shown i n Figure 25. Panels A, B, and C represent the T7 + i n f e c t i o n at 9, 11, and 13 minutes after i n f e c t i o n , respectively. Panels D, E, and F represent the T7am6 i n f e c t i o n for the same sampling times. In the T7 + i n f e c t i o n at the e a r l i e s t sampling time (A) e s s e n t i a l l y a l l of the DNA i s f a s t -sedimenting; by 13 minutes (C) same of the T7 + DNA has broken down to one phage equivalent unit. In contrast the f a s t -sedimenting DNA present at .9 minutes i n the T7am6 i n f e c t i o n i s unstable and becomes highly fragmented by 13 minutes afte r i n f e c t i o n . This i s consistent with the res u l t s obtained i n the 87 Figure.25. N e u t r a l sucrose sedimentation o f progeny DNA l a b e l e d c o n t i n u o u s l y w i t h [ 3H]thymidine: T 7 + versus T7am6. Panels A-C r e p r e s e n t the T 7 + a t 9, 11, and 13 minutes a f t e r i n f e c t i o n , r e s p e c t i v e l y . Panels D-F r e p r e s e n t the T7am6 under the same c o n d i t i o n s . T7 DNA was used as a r e f e r e n c e . 3 2 P l a b e l e d Percent o f Recovered R a d i o a c t i v i t y w o rt r+ 0 3 1. fl) i-< O (D 3 rt O Hi tr1 (D 3 rt O Hi O H PJ OJ H-(!) 3 rt r 15 \ * 88 parental label, experiment and indicates that i t i s the s t a b i l i t y of the concatemers that i s affected by the absence of the exo-nuclease. rather, than t h e i r formation. Because neutral sucrose gradient sedimentation can be an unreliable i n d i c a t i o n of the molecular weight of r e p l i c a t i v e DNA ?(57) i t was desirable to examine progeny r e p l i c a t i v e DNA d i r e c t l y by electron microscopy. Progeny T7 r e p l i c a t i v e DNA was i s o l a t e d i n the following way: E. c o l i B23 was grown for two .generations i n 5-BrdU 10 X PO^ TCG and portions were infected with either 5-BrdU-labeled T7 + or 5-BrdU-labeled T7ts6 phage at an M.O.I, of 10. At 6 minutes afte r i n f e c t i o n the c e l l s were washed free of 5-BrdU and were resuspended at 43C i n media 3 supplemented with dThd. At 8 minutes a f t e r i n f e c t i o n H-dThd (1 uCi/ug) was added to each •ieaiktu£e>;ri. Then samples from each-infections were taken at various times. The p u r i f i e d DNA was analyzed i n preparative neutral CsCl density gradients. The p u r i f i e d r e p l i c a t i v e DNA at the unsubstituted (LL) region of a neutral CsCl density gradient was i s o l a t e d . A reconstruction experiment showed that no E. c o l i DNA (HH) or parental phage DNA (HH) contaminated the progeny phage DNA (LL) loca t i o n . The r e p l i c a t i v e DNA i s o l a t e d from CsCl was analyzed by neutral sucrose sedimentation for the presence of fast-sedimenting material. Figure 26A represents the T7 + i n f e c t i o n at 9 minutes at which time CM was added and the sample incubated further. Figure 26B represents the T7ts6 i n f e c t i o n for the same conditions. Both DNA samples contained fast-sedimenting material. Therefore, concatemers were expected to be present i n both i n f e c t i o n s . As 89 F i g u r e 26. N e u t r a l sucrose sedimentation of progeny DNA l a b e l e d c o n t i n u o u s l y w i t h [ 3H]thymidine: T 7 + v e r s u s T7ts6". A. T 7 + sample t r e a t e d with CM a t 9 minutes. B. T7ts6 under the same c o n d i t i o n s . . 3 2P l a b e l e d T7 DNA was used as a r e f e r e n c e . Bottom 50 Percent of Length o f Gradient 90 expected under, these conditions a large amount of the T7ts6 DNA i s fragmented. Both the T7 + and T7ts6 r e p l i c a t i v e DNA were examined by electron microscopy, to determine i f DNA longer than one phage equivalent unit was present i n the samples. The electron microscopic examination of t h i s DNA was performed by R. D. Bradley and D. G. Scraba (Department of Biochemistry, University of Alberta). The DNA sample from the T7ts6 i n f e c t i o n was found to contain molecules longer than one phage equivalent unit (75). Therefore, some T7 concatemers are formed at early times after i n f e c t i o n i n the absence of T7. exonuclease; the fast-sedimentation i s not simply an a r t i f a c t of some complex r e p l i c a t i v e form. A histogram of the concatemers from the T7ts6 sample indicated that the concatemeric DNA had an average length of 2±.62 phage equiv-alents, with 44% of the molecules measured being greater than two phage equivalents i n length (75) . Two other T7ts:6 samples taken at d i f f e r e n t times than thersample described above and which contained fast-sedimenting material were also found to contain DNA molecules longer than one phage equivalent unit. (The wildtype samples examined by electron microscopy also contained many molecules longer than one phage equivalent i n length as expected.) It was necessary to show that the patterns of neutral sucrose sedimentation of the r e p l i c a t i v e DNA under conditions nonpermissive for the exonuclease were sim i l a r i n both the T7am6 and T7ts6 i n f e c t i o n s , since the T7ts6 i n f e c t i o n was used to determine the presence of concatemers by electron micros-91 copy. The sedimentation c h a r a c t e r i s t i c s of the T7ts6 r e p l i c a t i v e DNA under conditions nonpermissive for the exonuclease was examined from both parental and progeny label experiments. Parental DNA was examined by i n f e c t i n g portions of density labeled E. c o l i B23 with 3 2 P - T 7 + or 3 2P-T7ts6 phage. At 5% minutes aft e r i n f e c t i o n the infected c e l l s were f i l t e r e d , washed, and resuspended i n 5-BrdU media preincubated to 4 3C. Samples were c h i l l e d at various times aft e r i n f e c t i o n , the DNA extracted, and then analyzed i n preparative neutral CsCl gradients. The r e p l i c a t i v e DNA was i s o l a t e d from these gradients, dialyzed, and then reanalyzed by neutral sucrose sedimentation. The r e s u l t s are shown i n Figure 27. Panels A, B, and C represent the T7 + i n f e c t i o n at 9 minutes, 13 minutes, and 13 minutes with CM added and incubation continued u n t i l 30 minutes, respectively. Panels D, E, and F represent the T7ts6 i n f e c t i o n for the same sampling times. At 9 minutes after i n f e c t i o n both the T7 + (A) and the T7ts6 (D) samples contained f a s t sedimenting r e p l i c a t i v e DNA. This pattern of sedimentation was very similar to that observed i n the T7am6 i n f e c t i o n (Figure 2 3D). By 13 minutes a f t e r i n f e c t i o n although the T7 + r e p l i c a t i v e DNA contains a large amount of fast sedimenting material (B), the r e p l i c a t i v e DNA from the T7ts6 i n f e c t i o n was a l l one phage equivalent unit i n length and smaller (E). In the T7am6 i n f e c t i o n (Figure 23F) the r e p l i c a t i v e DNA was fragmented also and free of f a s t sedimenting material by 13 minutes. At the late sampling time most of the T7 + r e p l i c a t i v e DNA had matured into unit length molecules (C), however, the T7ts6 DNA had become even more fragmented (F). j i. 92 r F i g u r e 27. N e u t r a l sucrose sedimentation of p a r e n t a l 3 2 P - l a b e l e d \ r e p l i c a t i v e DNA from a T7 + ; versus a T7ts6 i n f e c t i o n , j both incubated a t the nonpermissive temperature of 43C. Panels A-C r e p r e s e n t the T 7 + a t 9 minutes, i v 13 minutes, and 13 minutes + CM, r e s p e c t i v e l y . Panels: D-F r e p r e s e n t the T7ts6 under the same c o n d i t i o n s . j T r i t i u m l a b e l e d T7 DNA was used as a r e f e r e n c e . j f i 40-. ! 30 A PERCENT OF LENGTH OF GRADIENT The sedimentation properties of progeny labeled T7ts6 r e p l i c a t i v e DNA was examined i n a pulse chase experiment. E. c o l was grown i n normal PO^ TCG supplemented with dThd (5. yg/ml) , FdU 8 (5 -yg/ml) r and u r a c i l (25 jpg/ml) to a c e l l density of 3 X 10 bacteria per ml at 30C. The c e l l s were infected at an M.O.I, of 10 with T7ts6 phage. At 6 minutes afte r i n f e c t i o n the c e l l s were s h i f t e d to 43C. Portions of the c e l l s were pulse labeled 3 with I Hjthymidine (1 pCi/ng) for the following one minute i n t e r v a l s ; 7-8 minutes, 9-10 minutes, and 11-12 minutes a f t e r i n f e c t i o n . Following the pulse the infected c e l l s were chased with cold thymidine at a concentration of 200 yg/ml. No further l a b e l was incorporated into acid insoluble material following the chase. Samples were taken from each portion of pulse labeled c e l l s at several i n t e r v a l s a f t e r the chase and the DNA was p u r i f i e d . The r e p l i c a t i v e DNA was then analyzed by neutral r; sucrose gradient sedimentation and the d i s t r i b u t i o n of label determined as shown i n Figure 28. Panels A, B, and C represent the 7-8 minute pulsed c e l l s at 9, 13, and 15 minutes a f t e r i n f e c -t i o n , respectively. Panels D and E represent the 9-10 minute pulsed c e l l s at 11 and 13 minutes a f t e r i n f e c t i o n . Panel F represents the 11-12 minute pulse at 13 minutes afte r i n f e c t i o n . Samples taken early a f t e r the chase for portions of c e l l s pulsed at early times a f t e r i n f e c t i o n contained some fast sedimenting material (A and D). However, samples taken l a t e r are found to contain r e p l i c a t i v e DNA which i s highly fragmented. Even the sample taken at 11 minutes from a 9-10 minute pulse (D) which contained some fast sedimenting "material also contained a large Figure 28. 94 i Neutral sucrose sedimentation of T7ts6 progeny DNA j "pulse-labeled" with' [ 3H]thymidine. Panels A-C are j samples taken at 9, 13, and 15 minutes for "cells pulsed from 7-8 minutes. Panels D-E are samples taken at 11 j and 13 minutes from a 9-10 minute pulse." Panel F i s |\ a 13 minute sample from a 11-12 minute pulse. j Hybridization data: 204 10 A > i -> o < < CC a UJ > o o u. o I-z Hi o ce UJ a E. T7ts6 I. C. DNA . c o l i . 12 T7 6850 tea**!! Bottom 5 0 PERCENT OF LENGTH OF GRADIENT amount of r e p l i c a t i v e DNA s m a l l e r than one phage e q u i v a l e n t i n l e n g t h . T h e r e f o r e , j u s t as was observed i n the T7am6 i n f e c t i o n (Figure 25D, E, and F ) , under c o n d i t i o n s nonpermissive f o r the exonuclease i n a T7ts6 i n f e c t i o n the concatemeric DNA which i s s y n t h e s i z e d e a r l y i s u n s t a b l e and i s q u i c k l y converted t o mole-c u l e s c o n s i d e r a b l y s m a l l e r than one phage e q u i v a l e n t u n i t i n l e n g t h . To show t h a t the f a s t sedimenting DNA p r e s e n t i n the T7ts6 i n f e c t i o n r e p r e s e n t e d T7 DNA , h y b r i d i z a t i o n s were performed. The h y b r i d i z a t i o n data f o r the 9 minute sample taken from the 7-8 minute p u l s e experiment a l s o i s presented i n F i g u r e 28. E s s e n t i a l l y a l l of the l a b e l was i n c o r p o r a t e d i n t o T7 DNA. Concatemers formed e a r l y i n a T7 i n f e c t i o n nonpermissive f o r i the exonuclease were found to be u n s t a b l e . T h e r e f o r e i t was not the s y n t h e s i s of concatemers but t h e i r s t a b i l i t y which was a f f e c t e d by c o n d i t i o n s nonpermissive f o r the exonuclease. In c o n t r a s t i t has been shown t h a t concatemers formed i n the absence of the T7 endonuclease (gene 3) are s t a b l e and accumulate a t a time when concatemers made i n the w i l d t y p e i n f e c t i o n are being broken down to one phage e q u i v a l e n t u n i t i n l e n g t h (110). The r e p l i c a t i v e concatemeric DNA from a T7am3 i n f e c t i o n has been shown t o be lon g e r than one phage e q u i v a l e n t u n i t by e l e c t r o n microscopy (65). I t was thought t h a t the i n s t a b i l i t y of the T7 concatemers under c o n d i t i o n s nonpermissive f o r the exonuclease may occur because d i s c o n t i n u i t i e s i n t r o d u c e d i n t o the DNA by the endonuclease were l e f t u n r e p a i r e d i n the absence of the exonu-c l e a s e . I f t h i s were t r u e then one would expect s t a b l e conca-temers t o form when-both enzymes were absent. To t e s t t h i s hypothesis experiments designed to study both the synthesis and s t a b i l i t y of concatemers "under conditions nonpermissive for both these enzymes were performed. An examination of the sucrose sedimentation pattern of r e p l i c a t i v e DNA. from infec t i o n s by the following phage was made: T7 +, T7am3, T7am6, T7am3am6 (defective' i n both T7 exonuclease (gene 6) and endonuclease (gene 3)). Experiments to study parental, continuous labeled progeny, and pulse chase progeny were performed. To examine parental labeled r e p l i c a t i v e DNA density labeled E. c o l i B23 was grown at 30C and portions were infected with 32 + 32 P-T7 or P- T7ara3am6 phage at an M.O.I, of 7. At 14 minutes a f t e r i n f e c t i o n samples of each culture were chilled,., the DNA was extracted, and then analyzed by neutral CsCl density sedimenta-t i o n . The r e p l i c a t i v e DNA was is o l a t e d , dialyzed, and then reanalyzed by neutral sucrose sedimentation. The re s u l t s are shown i n Figure 29. Panel A represents the sucrose sedimentation pattern of the T7 + r e p l i c a t i v e DNA, and panel B represents the T7am3am6 r e p l i c a t i v e DNA at the same sampling time. Under condi-tions nonpermissive for both the exonuclease and endonuclease fast sedimenting material was present. In a similar T7am6 in f e c -t i o n (Figure 23F, 13 minutes a f t e r infection) a l l of the r e p l i -cative DNA was fragmented.prior to 14 minutes a f t e r i n f e c t i o n . Under sim i l a r conditions nonpermissive for the endonuclease only (Figure 17B) 14 minutes a f t e r infection) a large amount of fast sedimenting DNA was present. Therefore when both enzymes were absent the DNA was more stable than when only the exonuclease was missing. These r e s u l t s suggested that the i n s t a b i l i t y of conca-97 Figure 29. Neutral sucrose sedimentation of parental ,3 2P—labeled r e p l i c a t i v e DNA from a T7+ versus a T7am3am6 i n f e c -t i o n . A. T7 +, Be T7am3am6. Tritium labeled T7 DNA was used as a reference. 9 8 terriers under conditions nonpermissive for the exonuclease was due to the presence of the endonuclease. To study further the af f e c t s of the absence of these enzymes on the synthesis and s t a b i l i t y of concatemers the k i n e t i c s of concatemer formation' was examined i n progeny l a b e l experiments. An experiment i n which progeny DNA was continuously labeled was performed i n the following way. E. c o l i B23 was grown at 3 0C to g a c e l l density of 3 X 10 c e l l s per ml. Portions of the c e l l s were infected with T7 +, T7am3, T7am6, and T7am3am6 phage at an 3 M.O.I, of 10. At lh minutes aft e r i n f e c t i o n [ H]thymidine (1 iiCi/jig) was added to each i n f e c t i o n . At various times samples were ice c h i l l e d . Some samples were incubated further with CM pr i o r to being i c e c h i l l e d . The i n f e c t i v e center DNA was extracted and analyzed by neutral sucrose sedimentation. These r e s u l t s are shown i n Figure 30. Panels A, C, E, and G represent 10 minute samples of the T7 + , T7am3, T7am6, and T7am3am6 infections,::.respec-t i v e l y . Panels B, D, F, and H represent 14 minute samples to which CM was added and incubation continued for the same i n f e c -tions as described above. At 10 minutes i n a l l infec t i o n s (A, C, E, and'"G) most of the r e p l i c a t i v e DNA i s found to be f a s t ' s e d i -menting and therefore representative of concatemerie DNA. At the lat e sampling time the T7 + r e p l i c a t i v e DNA mostly has been broken down to -molecules one phage equivalent unit i n length (B) . The T7am6 r e p l i c a t i v e DNA accumulates double strand d i s c o n t i n u i t i e s at l a t e i n f e c t i o n times, as shown previously, and i s thus unstable. Late r e p l i c a t i v e DNA from both the T7am3 CD) and T7am3am6 (H) infecti o n s continues to be fast sedimenting i n d i -. Neutral sucrose sedimentation analysis of T7 +, T7am3, T7am6, and T7am3am6 DNAs continuously labeled^with [3H] thymidine. Panels A, C, E, and G repre sent 10 minute samples of the T7+, T7am3, T7am6, and T7am3am6 i n f e c t i o n s , respectively. Panels B, D, F, and H.represent samples of these same infe c t i o n s treated with CM at. 14 minutes after i n f e c t i o n . 3 2 P - l a b e l e d T7 DNA was used as a reference. PERCENT OF LENGTH OF GRADIENT 100 eating the accumulation of stable concatemers. In order to confirm the above r e s u l t s concatemeric DNA was also examined i n a pulse chase progeny labeled DNA experiment. • + E. c o l i B23 was infected with T7 , T7am3, T7am6, or T7am3am6 3 at an M.O.T. of 10. At 8 minutes aft e r i n f e c t i o n I Hjthymidine Cl yCi/jig) was added to each i n f e c t i o n and the c e l l s were incu-bated u n t i l 9 minutes. The c e l l s then were f i l t e r e d , washed free of the l a b e l , and resuspended i n normal PO^ TCG media supplemented with 200 ug/ml of unlabeled thymidine. No further l a b e l was incorporated into acid insoluble material following the chase. Samples were c h i l l e d at various times aft e r the chase, the c e l l s lysed, the DNA extracted, and then analyzed by neutral sucrose sedimentation. The re s u l t s are shown i n Figure 31. Panels A, C, E, and G represent the 10 minute samples of T7 +, T7am3, T7am6, and T7am3am6 r e p l i c a t i v e DNA, respectively. Panels B, D, F, and H represent the 15 minute samples for the same i n f e c t i o n s . The res u l t s are very similar to those obtained for the continuous l a b e l experiment (Figure 30). In the T7 + i n f e c t i o n concatemers are made (A) but are e f f i c i e n t l y matured (B) into mature size phage DNA molecules. When conditions are nonpermissive for the exonuclease concatemers are unstable and are quickly converted to fragments smaller than mature phage DNA (E and F ) . When condi-tions are nonpermissive for the endonuclease (C and D) or both -the exonuclease and endonuclease (G and H) stable concatemers accumulate and are not -matured. B r i e f l y , the r e s u l t s indicate that mutations which have been found to severely i n h i b i t .molecular and genetic recombination i n Figure 31. Neutral sucrose sedimentation analysis of T7 , T7am3, T7am6, and T7am3am6 o progeny DNAs "pulse-labeled" with [ 3H]thymidine. Panels A, C, E, and G H represent-the 10 minute.samples of the T7 +, T7am3 T7am6, and T7am3am6 infec t i o n s , respectively. Panels B, D, F, and H represent the 15 minute samples for the same inf e c t i o n s . 3 2 P - l a b e l e d T7 DNA was used as a reference. j Percent of Length of Gradient i 102 T7--mutations i n e i t h e r the T7 gene 3 (endonuclease; 52, 65, 83) or T7 gene 6 (exonuclease; 52, 6 4 , 8 3 ) — d o not i n t e r f e r e w i t h the f o r m a t i o n of concatemers, a l t h o u g h s e l e c t e d T7 recombination d e f e c t i v e .mutations w i l l a f f e c t the s t a b i l i t y o f these concatemers. T h e r e f o r e g e n e r a l recombination i n T7 does not appear to be i n v o l v e d i n the formation of concatemers s i n c e concatemers are ;.. s y n t h e s i z e d under c o n d i t i o n s nonpermissive f o r recombination. 103 CHAPTER IV - DISCUSSION AND CONCLUSIONS The r e s e a r c h d e s c r i b e d i n t h i s t h e s i s was undertaken to: pr o v i d e some i n s i g h t i n t o the mechanism of recombination as i t : occurs i n phage T7. The p o s s i b l e r o l e of recombination i n the formation o f concatemers was examined a l s o . Two w e l l charac-t e r i z e d gene products of T7, the exonuclease (gene 6) and the endonuclease (gene 3), were i n v e s t i g a t e d w i t h regard to: t h e i r e s s e n t i a l r o l e s i n recombination and t h e i r p o s s i b l e . i n v o l v e m e n t i n concatemer formation and s t a b i l i z a t i o n . S e c t i o n 1. The Role o f T7 Exonuclease (Gene 6) i n Recombination The exposure o f s i n g l e s t r a n d r e g i o n s i n double s t r a n d DNA has been p o s t u l a t e d as a requirement f o r the es t a b l i s h m e n t o f j o i n t molecules i n recombination (11, 17, 58, 116). I f homologous s i n g l e s t r a n d r e g i o n s were exposed on two d i f f e r e n t chromosomes these molecules would be ab l e t o anneal and thus e s t a b l i s h a het e r d u p l e x molecule. Furthermore, i t has been p o s t u l a t e d t h a t an exonuclease mediates the exposure o f s i n g l e s t r a n d r e g i o n s (11, 17, 116). T h e r e f o r e , under c o n d i t i o n s nonpermissive f o r the exonuclease recombination of T7 DNA would be i n h i b i t e d i n v i v o because j o i n t molecules c o u l d not be formed. S e v e r a l p i e c e s of evidence from T7 experiments do i n d i c a t e t h a t the exonuclease i s needed f o r recombination and t h a t j o i n t molecule formation i s d e f e c t i v e under c o n d i t i o n s nonpermissive f o r the. exonuclease. The T7 exonuclease i s found t o be necessary f o r both g e n e t i c (52, 75, 83) and mol e c u l a r (64) recombination. Under 104 conditions nonpermissive for the exonuclease the DNA i s only able to r e p l i c a t e to. hybrid even though a substantial amount of DNA r e p l i c a t i o n i s permitted (64, 110); and thus j o i n t molecules are not formed. Although d i s c o n t i n u i t i e s accumulate i n T7 parental DNA when exonuclease i s defective, these single strand i n t e r -ruptions are not s u f f i c i e n t to: permit the parental DNA to become fragmented and reincorporated into progeny DNA molecules. I t i s concluded that the T7 exonuclease i s required for the breakage and reassociation of recombinant DNA. T7 exonuclease i s known to digest double stranded DNA i n a stepwise reaction from a 5'-terminus or from an i n t e r n a l single strand interruption (nick; 51). Thus, since nicks accumulate i n the absence of the exonu-clease, i t i s reasonable to assume that, i n vivo, i t s a c t i v i t y i s to produce a gap which leads to recombination, or that a gap i s converted to. a sticky end which can anneal with a comple-mentary molecule; a l t e r n a t i v e l y i t may produce a sticky end d i r e c t l y . To assess the experimental data which led to the above conclusions several aspects of the r e s u l t s need to be considered. In a density transfer experiment, the T7 parental DNA from an i n f e c t i o n of a nonpermissive host by an amber mutant of gene 6 (exonuclease) only r e p l i c a t e s to hybrid. This can occur for either of two reasons: 1) because the exonuclease i s d i r e c t l y involved i n recombination so that j o i n t molecule "formation i s not permitted i n i t s absence, or 2) because r e p l i c a t i o n i s severely r e s t r i c t e d so that most of the r e p l i c a t i v e DNA within the infected c e l l s i s hybrid and very l i t t l e progeny DNA has been 105 synthesized. Of course, i f both of these factors operate under v conditions nonpermissive for the exonuclease, only the r e s t r i c t i o n on DNA r e p l i c a t i o n could be observed and thus no conclusion concerning the involvement of the exonuclease i n recombination would be possible. However, i n a T7ts6 i n f e c t i o n i n which DNA r e p l i c a t i o n i s not r e s t r i c t e d to the extent that i t Is i n a T7am6 in f e c t i o n , the same r e s u l t i s obtained; the parental DNA only r e p l i c a t e s to. hybrid and j o i n t molecule formation i s i n h i b i t e d under conditions nonpermissive for the exonuclease. To conclude that the exonuclease i s d i r e c t l y involved i n recombination, i t s t i l l had to be shown that s u f f i c i e n t progeny DNA was synthesized. Two experiments to evaluate the extent of DNA synthesis under conditions nonpermissive for the exonuclease 3 were performed. In the f i r s t experiment the uptake of [ H]-thymidine into acid insoluble material was monitered under conditions permissive and nonpermissive for the exonuclease. The incorporation of label into T7 s p e c i f i c DNA was e s s e n t i a l l y the same for a l l conditions. In the second experiment the a b i l i t y of the T7ts6 mutant to synthesize a new DNA complement at the nonpermissive temperature was investigated. A new complement was synthesized under these conditions i n d i c a t i n g that the DNA was able to undergo at least one more round of r e p l i c a t i o n at the nonpermissive temperature. Although one more round of r e p l i c a t i o n was permitted under these conditions, molecular recombination did not take place. Both experiments indicated that s u f f i c i e n t r e p l i c a t i o n for de t e c t i b l e recombination was permitted. Therefore, these r e s u l t s support the conclusion v 106 that the T7 exonuclease i s d i r e c t l y involved i n recombination. The absence of the exonuclease does•not. .indirectly i n h i b i t recombination9beeause«DNA r e p l i c a t i o n i s r e s t r i c t e d . To show that molecular recombination was i n h i b i t e d under conditions nonpermissive for the exonuclease, i t was necessary to show that molecular recombination was permitted under permissive conditions. This was determined by analyzing the r e p l i c a t i v e 32 + P-DNA from a T7 density transfer experiment and showing that covalently repaired j o i n t molecules were present. If long single strands were formed by the covalent linkage of a unit length parental DNA strand with one or more adjacent 5-BrdU labeled DNA strands within a concatemer, then these long molecules also would sediment at a density c h a r a c t e r i s t i c of covalently repaired j o i n t molecules under denaturing conditions. However, i t was shown that the presence of covalently repaired j o i n t molecules was not an a r t i f a c t of long single strand formation. This analysis i s important for the following reasons: 1) some long single strands are generated during a T7 + i n f e c t i o n as deter-mined by an examination of r e p l i c a t i v e progeny T7 DNA labeled 3 with [ H]-thymidine (24, 47, 49); 2) although the appearance of long single strands i n a T4 i n f e c t i o n coincides with the expected onset of recombination and repair of recombinants (72), t h i s c o r r e l a t i o n between long single strand formation and the onset of recombination has not been made yet i n T7; and 3) although evidence indicates that recombination i s responsible for concatemer formation i n T4, recombination does not appear to be responsible for concatemer formation i n T7 (75). Thus, i n 107 T7, concatemer formation or long single strand formation cannot be used as an i n d i c a t i o n that recombination has commenced. The presence of long single strands could i n t e r f e r e with any assessment of the extent to which T7 has recombined. As shown by the results, presented i n t h i s thesis and previously by Carlson (15), long single strands are not formed when density l a b e l i n g conditions (5-BrdU) are used. This r e s u l t i s consistent with observations from T4 density labeled experiments. T4 i n f e c t i o n normally generates large amounts of long single stranded DNA at lat e i n f e c t i o n times, but, i n a density transfer experiment, long single strands are d r a s t i c a l l y reduced (72). Therefore, i t i s not surprising that i n a T7 i n f e c t i o n , i n which long single strands are formed less frequently, they would not appear under density, l a b e l i n g conditions. An important objective i n studying molecular recombination i s to: r e l a t e the amount of molecular recombination to observed genetic recombination frequencies i n recombination defective mutants. Since molecular recombination i s thought to be the manifestation of genetic recombination at a molecular l e v e l , any mutation which i n h i b i t s molecular recombination would be expected to decrease genetic recombination. Therefore, since molecular recombination i s i n h i b i t e d under conditions nonpermissive for the exonuclease i n T7, then genetic recombination frequencies should also decline. The experimental r e s u l t s obtained j u s t i f y t h i s assumption since under conditions "semi-permissive" for the exonuclease genetic recombination frequencies are reduced 3 to 18-fold. Using d i f f e r e n t techniques other investigators also 108 have shown that the exonuclease i s required for genetic recom-bination (52, 83). The major d i f f i c u l t y i n evaluating these experiments i s that the lowest recombination frequencies observed also involved the lowest phage y i e l d s . Thus, i t was necessary to determine the extent to which decreased recombination may a r i s e from low phage y i e l d s . Under conditions "semi-permissive" for the T7 DNA polymerase (gene 5), when DNA synthesis i s r e s t r i c t e d and t h i s r e s t r i c t i o n i s accompanied by a severe decrease i n the phage y i e l d , recombination frequencies are equivalent to: those observed i n the "wildtype" control. Therefore, a d r a s t i c decrease i n phage y i e l d does not d i r e c t l y i n h i b i t genetic recombination. The'j'T? DNA polymerase has been implicated i n genetic recombination using a similar technique to. that described i n t h i s thesis. However, the r e s u l t s are comparable over the temperature range used (52). In addition, Kerr and Sadowski (52) performed an experiment which confirms the conclusion that low genetic recombination frequencies do notooccur because of low phage y i e l d . They measured genetic recombination frequencies for crosses performed i n the presence of n a l i d i x i c acid, a drug that i n h i b i t s both phage production and DNA synthesis. When the l e v e l of n a l i d i x i c acid used reduced the phage y i e l d or DNA synthesis by greater than 9 9% there i s very l i t t l e change i n the recombination frequencies. Therefore, neither low phage y i e l d nor reduced DNA synthesis d i r e c t l y cause low recombination frequencies. This observation also supports the conclusion that the exonuclease i s involved d i r e c t l y i n molecular recombination since conditions 109 which severely r e s t r i c t DNA synthesis do not a f f e c t genetic recombination and, therefore, would not be expected to. i n h i b i t molecular recombination. Also the r e s t r i c t i o n on DNA synthesis which occurs when the exonuclease i s defective i s u n l i k e l y to a f f e c t genetic recombination frequencies since a substantial amount of DNA r e p l i c a t i o n i s permitted under conditions non-permissive for the exonuclease-, (64, 110). Studies with T4 are consistent with those of T7, in d i c a t i n g that the T7 exonuclease may be required for the formation of single strand regions p r i o r to j o i n t molecule formation. There are many s i m i l a r i t i e s between the exonuclease negative mutants of T7 (gene 6) and T4 (gene 46, 47). Neither degrade host DNA to acid soluble products (61, 92). Both show reduced genetic recombination frequencies (7, 9, 52, 75, 83), and both are defective i n j o i n t molecule formation (64, 84). An examination of the r e p l i c a t i v e DNA from either mutant by electron microscopy show that few single strand regions or branched structures are present (11, 12, 75). One reservation with t h i s comparison i s that although electron microscopic analysis of T4 + r e p l i c a t i v e DNA show abundant s i n g l e strand regions and branches (11), studies on thesstructural features of T7 + recombinant DNA are incomplete. However, the current data does favor the hypothesis that the T7. exonuclease functions i n recombination by exposing single strand regions as a prerequisite to j o i n t molecule formation. 110 Section 2. Involvement of the T7 Exonuclease (Gene 6) i n Covalent Repair The multifunctional properties of enzymes can make an evaluation of t h e i r i n d i v i d u a l roles i n metabolism d i f f i c u l t to es t a b l i s h . The T7 gene 6 exonuclease i s an example of an enzyme with several functions. I t i s required for the degradation of host DNA (92) as well as for the mediation of j o i n t molecule formation as previously described (64). Following the formation of hydrogen bonded j o i n t molecules, normally the j o i n t s are sealed to form covalent recombinants (11, 74). To evaluate the possible r o l e of the exonuclease i n the covalent repair of j o i n t molecules, a density, transfer experiment using the T7ts6 mutant was performed. I t was found that i t was possible to s h i f t the infected c e l l s to: the nonpermissive temperature aft e r the formation of j o i n t molecules but before s i g n i f i c a n t covalent repair takes place. Thus i t was possible to demonstrate that covalent r e p a i r of j o i n t molecules i s reduced somewhat when conditions are nonpermissive for the exonuclease. Two comments about t h i s experiment should be noted. 1) It i s possible that the i n f e c t i o n incubated under permissive c o n d i t i o n s — T 7 + at 30C or 43C and T7ts6 at 3 0 C — a c t u a l l y formed more j o i n t molecules upon further incubation whereas the T7ts6 culture s h i f t e d to: 43C could not. This would mean that under permissive conditions there are more j o i n t molecules available to be repaired than under nonpermissive conditions. However, there i s not a s i g n i f i c a n t change i n the density of the T7ts6 r e p l i c a t i v e DNA when incubated at either 30C or 43C compared to: the density I l l observed at the time of the temperature s h i f t (Figure 13D, E, and F). In a l l three samples a majority of the DNA sediments at the density c h a r a c t e r i s t i c of recombined molecules. 2) The exonu-clease may not be involved d i r e c t l y i n processing of j o i n t . molecules for repair but may act i n d i r e c t l y by providing more nucleotides by degrading host DNA.-. These nucleotides may be required i f a substantial amount of repair involves the f i l l i n g of gaps. However, the s h i f t to a nonpermissive temperature occurs l a t e enough i n the i n f e c t i o n cycle so that a majority of the host DNA should be degraded. One problem with t h i s assumption i s that the degradation of host DNA by phage T7 was studied under d i f f e r e n t experimental conditions. Host DNA was found to be degraded to acid soluble products: by the T7 exonuclease between 7.5 and 15 minutes aft e r i n f e c t i o n when the infected c e l l s were incubated at 37C and when density l a b e l was not used (92). The degradation of host DNA by the T7 exonuclease may be slower at 30C and i n 5-BrdU media. In 3?4 'a substantial amount of covalent re p a i r does depend on the f i l l i n g of gaps by DNA polymerase (73). Therefore, the exonuclease i s not necessarily expected to be the only enzyme needed for covalent repair of j o i n t molecules. Indeed i n T7 some j o i n t molecules must not depend on the exonuclease for repair since about 30% more of the r e p l i c a t i v e DNA i s covalently repaired following the s h i f t to a nonpermissive temperature (compare Figure 14D and F) . . The results: presented i n t h i s thesis do indicate that some j o i n t molecules may require the exonuclease to mediate t h e i r repair. 112 The accumulation of single and double strand d i s c o n t i -n u i t i e s i n T7 parental DNA under conditions nonpermissive for the exonuclease indicates that r e p a i r i s defective i n gene 6 mutants. However, i t i s possible that the accumulation of d i s c o n t i -n u i t i e s may r e f l e c t a block i n recombination, i . e . , an incomplete f i r s t step i n recombination which renders re p a i r impossible, and thus a gene 6 mutation may a f f e c t r e p a i r only i n d i r e c t l y . In any event, these results, suggest that some kind of s t r u c t u r a l feature oft'the r e p l i c a t i v e DNA renders the DNA unstable unless acted upon by the exonuclease. The p o s s i b i l i t y , exists that d i s c o n t i n u i t i e s accumulate i n the DNA i n the absence of the exonuclease because normally the exonuclease operates on a pre-recombination nick to form a gap. The i n t e r a c t i o n between the exonuclease and the endonuclease (gene 3) w i l l be discussed more f u l l y i n the section dealing with concatemers. Several models of recombination suggest that the removal of branches and redundant single strands i s a necessary step p r i o r to the covalent repair of j o i n t molecules. Furthermore, i t i s thought that an exonuclease may be required to reduce these redundant molecules to a l i n e a r duplex (11, 17, 18, 19). If the exonuclease i s needed to remove redundant s t r u c t u r a l features of j o i n t molecules, then i t i s postulated that the T7 exonuclease would function by a strand displacement mechanism simi l a r to: that postulated for the lambda exonuclease (18) since the T7 exonuclease also has a s p e c i f i c i t y for double stranded DNA (50, 51). However, i n contrast to. the lambda exonuclease (18) ,. the. exonuclease of T7 may leave a gap following the assimi-113 l a t i d n of the s i n g l e s t r a n d . The reason f o r t h i s d i f f e r e n c e i s that,, i n v i t r o , the hydro l y t i c a c t i v i t y of the lambda exonuclease i s a r r e s t e d once a redundant s i n g l e s t r a n d i s completely a s s i m i -l a t e d (19) . T h i s p r o p e r t y of the enzyme may be r e l a t e d to. another c h a r a c t e r i s t i c df the lambda exonuclease, i t s i n a b i l i t y , td: i n i t i a t e h y d r d l y s i s a t a n i c k : (17) . The T7. exonuclease can i n i t i a t e h y d r o l y s i s from a n i c k , in_ v i t r d , and, t h e r e f o r e , h y d r o l y s i s would not n e c e s s a r i l y be a r r e s t e d once a s i n g l e s t r a n d i s a s s i m i l a t e d . 114 S e c t i o n 3. The Role of T7 Endonuclease (Gene 3) i n Recombination Recent experimental evidence has shown t h a t T7 g e n e t i c recombination i s s e v e r e l y r e s t r i c t e d under c o n d i t i o n s nonpermissive f o r the endonuclease (gene 3) (52, 83). Thus, molecular recombi-n a t i o n i s also, expected t o be i n h i b i t e d under c o n d i t i o n s nonpermissive f o r the endonuclease. An endonuclease has been p o s t u l a t e d to. be the enzyme which i n i t i a t e s r ecombination by i n t r o d u c i n g s i n g l e s t r a n d d i s c o n t i n u i t i e s i n t o duplex DNA (11, 17, 70, 72, 116). I'f the endonuclease were d e f e c t i v e , s i n g l e s t r a n d i n t e r r u p t i o n s would not be expected t o form i n the DNA. The 32 molecules would remain i n t a c t . : Under these c o n d i t i o n s a P-l a b e l e d p a r e n t a l DNA molecule of u n i t l e n g t h r e p l i c a t i n g semi-c o n s e r v a t i v e l y i n a density, t r a n s f e r , experiment might o n l y be found t o sediment t o a d e n s i t y c h a r a c t e r i s t i c of h y b r i d DNA. There would be no fragmentation of the p a r e n t a l s t r a n d and no r e i n c o r p o r a t i o n i n t o the completely s u b s t i t u t e d (5-BrdU) progeny DNA. T h e r e f o r e , i f the T7 endonuclease (gene 3) i s necessary to. i n i t i a t e r e combination, then under c o n d i t i o n s nonpermissive f o r the endonuclease, the T7 p a r e n t a l DNA strand s would remain i n t a c t ; they would not be fragmented and r e d i s t r i b u t e d among the progeny DNA molecules. To show t h a t molecular recombination was r e s t r i c t e d under c o n d i t i o n s nonpermissive f o r the endonuclease a shear technique was used to analyze p a r e n t a l l a b e l e d r e p l i c a t i v e T7 DNA. Although s h e a r i n g the T7am3 r e p l i c a t i v e DNA to s i z e one d i d not reduce a l l of the p a r e n t a l l a b e l to h y b r i d , a l a r g e percentage of i t was s h i f t e d from the d e n s i t y o f recombined DNA to the 115 density of hybrid. Therefore, a substantial amount df the T7am3 r e p l i c a t i v e DNA which sedimented at the density c h a r a c t e r i s t i c df recdmbined DNA was found to: be cdmpdsed df concatemers which contained more progeny than parental DNA; i t was ndt necessarily 32 P-DNA which had been fragmented and reincorporated into progeny 32 molecules. Furthermore, since a substantial amount of the P was reduced to. hybrid by shear to size one, a large percentage df the parental labeled DNA must have been l e f t i n t a c t as part df a hybrid unit length md.lecu.le. Since only abdut half df the parental l a b e l was sh i f t e d to a hybrid density by shearing the r e p l i c a t i v e DNA to size one i n the T7am3 experiment, the s t r u c t u r a l features df the r e p l i c a t i v e DNA were examined further by increasing the amount of shear i n the DNA samples. This was done to determine whether a s i g n i f i c a n t amount df the r e p l i c a t i v e DNA from a T7 + experiment was reduced to: hybrid by the same amount df shear required to reduce a l l df 32 + the P-T7am3 r e p l i c a t i v e DNA to hybrid. In a T7 experiment the amount Of parental DNA which i s incorporated into progeny DNA molecules i n a density transfer experiment has been calculated to: represent 17% Of the t o t a l molecule (71). Therefore, i n a T7 + i n f e c t i o n the recombinant molecules would need to. be sheared to: h df one phage equivalent unit dr smaller before a s i g n i f i c a n t amount df the parental l a b e l would sediment as hybrid. For thi s reason i n the second shear experiment samples df the T7 + and T7am3 r e p l i c a t i v e DNA were reduced td: \ sized molecules' and analyzed for the presence df parental l a b e l which retained the density df recdmbined DNA. I t was found that shearing the T7am3 116 32 r e p l i c a t i v e DNA to s i z e h s h i f t e d a l l o f the P - l a b e l e d DNA to h y b r i d (Figure 22F). There are two p d s s i b l e reasons why t h i s amount o f s h e a r i n g i s r e q u i r e d ; 1) not a l l o f the concatemers are even numbered m u l t i p l e s of u n i t l e n g t h , which would prevent the " c l e a n " l i b e r a t i o n df a h y b r i d fragment, and 2) some fragmen-t a t i d n o f the p a r e n t a l T7am3 DNA may take p l a c e i n the absence df the T7 enddnuclease (gene 3) , I.e.', some other enzyme may operate a t a low l e v e l i n a d d i t i o n to; the enddnuclease (gene 3). T h i s may e i t h e r be a ho s t encoded enzyme or sdme other T7 encoded enddnu-c l e a s e (23, 93). In any case i t i s c e r t a i n t h a t t h e l e v e l s df recombination i n the T7am3 DNA under nonpermissive c o n d i t i o n s are f a r below those o f T 7 + DNA. T h i s i s shown i n s e v e r a l ways. F i r s t , o n l y h a l f as much T7am3 DNA i s converted frdm h y b r i d molecules to recombined DNA i n the c e l l (compare F i g u r e 22A wit h D). Second, at l e a s t h a l f df the "recombined" T7am3 DNA i s s h i f t e d t d h y b r i d a f t e r s h e a r i n g to s i z e one. T h i s r e s u l t i n d i c a t e s t h a t the p a r e n t a l s t r a n d i s l e f t i n t a c t i n the T7am3 i n f e c t i o n . T h i s i s not t r u e df the T 7 + i n f e c t i o n s i n c e the r e p l i c a t i v e DNA i s not s h i f t e d td: h y b r i d when the DNA i s sheared to s i z e one. T h e r e f o r e , a majdrity. df the T 7 + p a r e n t a l DNA has undergone an undetermined amount df fragmentation. F i n a l l y , not even s h e a r i n g the T 7 + t d 32 + s i z e k y i e l d s a s i g n i f i c a n t s h i f t i n the P-T7 DNA from a recombined l o c a t i o n (Figure 22C) to: a h y b r i d l o c a t i o n . Under these c o n d i t i o n s a l l df the T7am3 DNA i s s h i f t e d t d h y b r i d ^ F i g u r e 22F). T h e r e f o r e , m d lecular recombination i s s e v e r e l y .reduced under c o n d i t i o n s nonpermissive f d r the. enddnuclease 117' (gene 3) i n T7;. the parental DNA i s not fragmented but i s l e f t i n t a c t as a strand one phage equivalent unit i n .length. These re s u l t s support the cdnclusion that the T7. enddnuclease (gene 3) i s required for genetic recombination (52, 83). In addition, using a d i f f e r e n t technique, F r d h l i c h , et- al'... (36) also have implicated the enddnuclease i n molecular recdmbinatidn. They 32 have shown that the parental P-labeled molecules i n a density transfer experiment ndnpermissive fdr the enddnuclease maintain t h e i r integrity, and are not fragmented. The kndwn i n v i t r d function df the T7 enddnuclease i s td introduce l i g a s e repairable single strand nicks into double strand DNA. I t a l s d w i l l produce double strand scissions (22). Furthermore, under conditions nonpermissive for the enddnuclease j d i n t mdlecule formation i s reduced dr eliminated. In contrast to the exonuclease negative i n f e c t i o n , d i s c o n t i n u i t i e s do not accumulate i n the r e p l i c a t i v e DNA under conditions ndnpermissive fdr the enddnuclease. Results described i n t h i s thesis indicate that the parental DNA i s l e f t i n t a c t i n the T7am3 i n f e c t i d n . In addition, analysis df T7am3 r e p l i c a t i v e DNA by electron micro-scopy shows none df the single strand or branched s t r u c t u r a l features expected df recdmbining molecules as i s seen i n T4 recombinant DNA f(65, 11). However, the true significance df t h i s observation can only be evaluated upon completion df the studies on the structure df T7 + recombinant DNA. These observations suggest that the T7 enddnuclease (gene 3) i s necessary at the breakage step df recdmbinatidn. Therefore i t i s proposed that, i n v i t r d , the enddnuclease functions by intrdducing nicks into 118 the DNA which i s a prerequisite for recombination. F i n a l l y , i n a density transfer experiment the density of the progeny DNA synthesized under conditions nonpermissive for the enddnuclease i s greater than that synthesized under permissive conditions (compare Figure 20A and C). The following explanation i s proposed. In a density t r a n s f e r experiment the host c e l l s are grown i n density la b e l i n g media fdr about 2 generations. Therefore, 25% df the hdst DNA i s not substituted with 5-BrdU. In the wildtype i n f e c t i o n the host DNA i s e f f i c i e n t l y degraded td acid sdluble products. This means that the pool df 5-BrdU i s dil u t e d by the presence df thymidine which i s released when the hdst DNA i s degraded. Thus, any T7 DNA synthesized from t h i s mixed pool wduld be only p a r t l y density labeled. In contrast the pool df 5-BrdU present i n the i n f e c t i d n nonpermissive for the enddnuclease i s not d i l u t e d by an i n f l u x df hdst DNA breakdown products because the hdst DNA i s not degraded under these condi-^' tidns (92). Therefdre, the progeny DNA frdm a T7am3. experiment wduld be expected td be more highly substituted with 5-BrdU than that synthesized i n a T7 + i n f e c t i d n . The difference i n the densities df the progeny DNA i n the wildtype cdmpared td the T7am3 i n f e c t i d n has been noted by other invetigatdrs (36). They offered a similar, explanation. 119 S e c t i o n 4. The Involvement df T7 Recdmbinatidn Enzymes i n Concatemer Formation Experimental evidence i n d i c a t e s t h a t recdmbinatidn i s r e s p o n s i b l e f o r concatemer fo r m a t i o n i n T4 (30, 72). However, T4 enzymes shown td be e s s e n t i a l f d r recdmbinatidn are not n e c e s s a r i l y i n v o l v e d i n concatemer formation. A T4 gene 46 dr 47 (exonuclease) mutant which i s d e f e c t i v e i n recdmbinatidn (7, 9, 84) fdrms some 'concatemers but t h e s e concatemers are u n s t a b l e (45, 98, 100). N e i t h e r T4 nor T7 undergo r o l l i n g c i r c l e r e p l i -c a t i o n (30, 72, 126, 127) and, thus, concatemers are not formed by t h i s mechanism. T h e r e f o r e , cdncatemer fdrmatidn i n T7 wduld most l i k e l y be promoted by recdmbinatidn ac r o s s the t e r m i n a l redundancies (111) dr by the d i s c o n t i n u o u s s y n t h e s i s mechanism proposed by Watson (124). It i s w e l l e s t a b l i s h e d t h a t both the T7 exonuclease (gene 6) (52, 64, 75, 83) and enddnuclease (gene 3) (52, 65, 83) are necessary f d r both molecular and g e n e t i c recdmbinatidn. To determine i f recdmbinatidn i s the mechanism r e s p o n s i b l e f d r cdncatemer formation i n T7, s e l e c t e d T7 gene 3 and gene 6 mutants were examined f d r t h e i r a b i l i t y to produce concatemers. The r e s u l t s presented here show t h a t nd concatemers are prese n t i n phage i n f e c t e d c e l l s a f t e r 11 minutes pdst i n f e c t i o n w i t h a T7 exonuclease (gene 6) d e f e c t i v e phage. These r e s u l t s have been o b t a i n e d u s i n g both amber and temperature s e n s i t i v e mutations i n gene 6. The r e s u l t s ; are the same whether p a r e n t a l , continuous progeny, dr p u l s e chase progeny DNA l a b e l i s . examined. Some cdncatemers are found e a r l y a f t e r i n f e c t i o n by a l l three 120 l a b e l i n g procedures w i t h both phage mutants. T h i s was determined by both n e u t r a l sucrose sedimentation and by. e l e c t r o n microscopy. However, under c o n d i t i o n s ndnpermissive f o r the exonuclease the concatemers are 'unstable and the DNA becomes fragmented at a time when l a r g e amounts, df concatemers are s t i l l present,under p e r m i s s i v e c o n d i t i o n s . Not only are the concatemers prematurely broken down, but t h e DNA becomes fragmented and accumulates as molecules' c o n s i d e r a b l y s m a l l e r than mature s i z e T7 DNA. These r e s u l t s are s i m i l a r t d those o b t a i n e d d u r i n g an i n f e c t i d n w i t h gene 46 or 47 mutants df T4 (45, 98, 100). The c o n c l u s i o n reached based on the above data i s t h a t the exonuclease, although not d i r e c t l y i n v o l v e d i n cdncatemer f o r m a t i o n , i s needed t d s t a b i l i z e the concatemers. S i n c e d i s c o n t i n u i t i e s accumulated i n r e p l i c a t i v e DNA under c o n d i t i o n s ndnpermissive f o r the exonuclease, a T7am3am6 double mutant was examined t d determine i f the enddnuclease (gene 3) was r e s p o n s i b l e f d r the i n s t a b i l i t y d f the DNA i n the exonu-c l e a s e n e g a t i v e i n f e c t i d n . Under c o n d i t i o n s ndnpermissive f o r both, the exonuclease and enddnuclease, s t a b l e concatemers are generated as determined by both p a r e n t a l and progeny l a b e l experiments:. These r e s u l t s support t h e c d n c l u s i d n t h a t t he exonuclease i s not i n v o l v e d d i r e c t l y i n cdncatemer fdrmatidn but t h a t i t p r o b a b l y i s needed t d s t a b i l i z e concatemers. The premature breakdown df the T7am6 concatemers i s mediated by the T7 enddnuclease (gene 3) because s t a b l e cdncatemers are formed d u r i n g i n f e c t i d n by a T7am3am6 double mutant. The f a i l u r e df these concatemers to mature t o the s i z e of progeny phage i s 121 presumably because the enddnuclease i s important i n the maturation process (110). Since the products df both gene 3 and gene 6 are necessary f d r g e n e t i c recdmbinatidn and m o l e c u l a r recdmbinatidn, the f o l l o w i n g mechanism df a c t i o n i s proposed f o r these enzymes. The enddnuclease (gene 3) and exonuclease (gene 6) a c t s e q u e n t i a l l y d u r i n g recdmbinatidn. The enddnuclease n i c k s the DNA and the exonuclease uses the n i c k e d DNA as a s u b s t r a t e to form gapped molecules which, i n c o n j u n c t i o n w i t h other enzymes, leads to the formation df j o i n t molecules (11, 17, 74). In the absence df the enddnuclease the DNA s t r a n d s remain i n t a c t and are not : fragmented. In the absence df the exdnuclease the DNA accumu-l a t e s n i c k s and becomes fragmented. E l i m i n a t i o n df the endonu-c l e a s e (gene 3) e l i m i n a t e s t h i s fragmentation. R e s u l t s d e s c r i b e d here show t h a t T7am6 DNA does accumulate., n i c k s , . and; the endonu-c l e a s e i s shown to be r e s p o n s i b l e f o r the fragmentation of the T7am6 cdncatemers. T h e r e f o r e , the T7. exonuclease (gene 6) i s necessary f d r c o n t i n u a t i o n df the recombination process and td. m a i n t a i n s t a b l e cdncatemers f d r the proper maturatidn df progeny phage DNA. Since s t a b l e cdncatemers are formed i n the combined absence df the exdnuclease and the enddnuclease, normal phage recombination i s not necessary f d r the formation df cdncatemers i n T7. Data presented here does not e l i m i n a t e the p o s s i b i l i t y t h a t some cdncatemers c o u l d be formed by recombination, although cdncat-emer sfdrmation i s very e f f i c i e n t i n the absence df molecular recdmbinatidn. 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