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DNA synthesis on primed template by T4 polymerase with gene product 32 Lee, Donald Dah-Chen 1978

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DNA SYNTHESIS ON PRIMED TEMPLATE BY T4 POLYMERASE WITH GENE PRODUCT '32 by DONALD DAH-CHEN|LEE B. S c , U n i v e r s i t y of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Micro b i o l o g y We accept t h i s t h e s i s as conforming to the req u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia December, 1978 © Donald Lee, 1978 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Microbiology The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e March 10, 1979 ABSTRACT A new approach i n mapping r e s t r i c t i o n fragments by means of primed extension was proposed but was found to be u n f e a s i b l e a f t e r studying the extent of T4 polymerase mediated DNA s y n t h e s i s . The maximum len g t h of DNA r e p l i c a t i o n mediated by T4 polymerase was st u d i e d using 0X-174 DNA as template primed by a r e s t r i c t i o n fragment of the same DNA. Both n u c l e o t i d e i n c o r p o r a t i o n k i n e t i c s and a l k a l i n e g e l e l e c t r o p h o r e s i s were used to study the products of DNA s y n t h e s i s . Although the i n c o r p o r a t i o n k i n e t i c s suggested that the primer was extended by approximately 100 n u c l e o t i d e s , the e l e c t r o p h o r e t i c m o b i l i t i e s of the products suggested much l e s s extension. The e f f e c t of T4 gene 32 p r o t e i n (unwinding p r o t e i n ) was al s o st u d i e d . This p r o t e i n was p u r i f i e d by DNA c e l l u l o s e chromatography to near homogeneity and was shown to be nuclease f r e e . The p u r i f i e d p r o t e i n stimu-l a t e d n u c l e o t i d e i n c o r p o r a t i o n t h r e e - f o l d when added to the usual T4 polymerase r e a c t i o n mixture. Contrary to the k i n e t i c r e s u l t s , however, the gel m o b i l i t i e s of the products again showed only l i m i t e d extension of the primer. — i i i — TABLE OF CONTENTS page LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i MATERIAL AND METHODS 7 Media 7 B a c t e r i a l s t r a i n s 7 Bacteriophages 7 Enzymes 7 Radioactive chemicals 8 Polyacrylamide g e l e l e c t r o p h o r e s i s 8 ( i ) P r o t e i n gels 8 ( i i ) DNA gels 8 Agarose g e l e l e c t r o p h o r e s i s 9 ( i ) N e u t r a l gels 9 ( i i ) A l k a l i n e gels 9 ( i i i ) Gel drying 1 0 Bacteriophage p u r i f i c a t i o n 10 ( i ) 0X-174 1 0 ( i i ) 0X-174 RF 10 ( i i i ) 3 2P-T7 11 DNA p r e p a r a t i o n H Ethanol p r e c i p i t a t i o n of DNA 12 P u r i f i c a t i o n of gene 32 p r o t e i n 12 ( i ) 3 5 g _ m e t h £ o n i n e p U i s e l a b e l 12 ( i i ) Gel e l e c t r o p h o r e s i s of crude f r a c t i o n s 12 ( i i i ) DNA c e l l u l o s e chromatography 13 - i v -TABLE OF CONTENTS (continued) page ( i v ) Concentration and storage 13 B i o l o g i c a l assay f o r 32-protein 14 Assay f o r endonuclease a c t i v i t y i n 32-protein 14 ( i ) A l k a l i n e sucrose gradient 14 ( i i ) Agarose g e l e l e c t r o p h o r e s i s 14 Primed extension 15 ( i ) Hae I I I r e s t r i c t i o n fragments 15 (a) R e s t r i c t i o n enzyme d i g e s t i o n 15 (b) Gel e l e c t r o p h o r e s i s 15 (c) E l e c t r o p h o r e t i c e l u t i o n 15 ( i i ) Priming 16 ( i i i ) E xtension 16 ( i v ) K i n e t i c s of extension 16 (v) Optimum 32-protein 16 ( v i ) A l k a l i n e gel e l e c t r o p h o r e s i s 17 RESULTS I 8 P u r i f i c a t i o n of gene 32-protein 18 ( i ) I d e n t i f i c a t i o n of 32-protein on g e l 18 ( i i ) I d e n t i f y i n g 32-protein c o n t a i n i n g crude f r a c t i o n 18 ( i i i ) DNA c e l l u l o s e chromatography 20 ( i v ) B i o l o g i c a l assay 20 (v) DNasesassay 24 Primed extension 24 ( i ) K i n e t i c s of extension by T4 polymerase 24 ( i i ) K i n e t i c s of extension w i t h 32-protein 30 -v-TABLE OF CONTENTS (continued) page ( i i i ) Gel e l e c t r o p h o r e s i s on extended product 30 DISCUSSION 36 REFERENCES 39 - v i -LIST OF FIGURES page Figure 1. Scheme f o r studying maximum l e n g t h of sy n t h e s i s 3 Figure 2. Scheme f o r mapping r e s t r i c t i o n fragments 5 Figure 3. Autoradiograph of gel of pulse l a b e l l e d p o s t - i n f e c t i o n T4 gene products 19 Figure 4. Polyacrylamide g e l patte r n s of l y s a t e f r a c t i o n s 21 Figure 5. Template and polymerase a c t i v i t y of 32-protein 22 Figure 6. T4 polymerase k i n e t i c s w i t h and without 32-protein 23 Figure 7. A l k a l i n e sucrose sedimentation p r o f i l e s of T7 DNA 25 Figure 8. Detection of nuclease by agarose g e l 27 Figure 9. T4 polymerase k i n e t i c s on primer, template, and primed template 28 Figure 10. T4 polymerase k i n e t i c s on two d i f f e r e n t preparations of 0X-174 DNA templates 29 Figure 11. T4 polymerase k i n e t i c s of primed template w i t h and without 32-protein 31 Figure 12. Diagram of g e l e l e c t r o p h o r e s i s patterns of Hpa I fragments of T7 DNA and Hae I I I fragments of 0X DNA 32 Figure 13. Autoradiograph of g e l of extension products 33 Figure 14. Autoradiograph of gel of extension using template alone .. 34 Figure 15. Autoradiograph of g e l of other extension products 35 - v i i -ACKNOWLEDGEMENTS I am g r a t e f u l to Dr. R.C. M i l l e r J r . f o r h i s p a t i e n t guidance and constant encouragement throughout t h i s i n v e s t i g a t i o n . I a l s o thank Debbie Taylor and Helen Smith f o r t e c h n i c a l a s s i s t a n c e and t h e i r e f f o r t s i n m aintaining peace and order i n the l a b o r a t o r y . I am a l s o indebted to Dr. R.A.J. Warren f o r proofreading during the pr e p a r a t i o n of the t h e s i s . Last but not l e a s t , I thank f e l l o w graduate students Kathy Burke, Richard Smith, and Mark Spurr f o r c r e a t i n g the t o l e r a b l e atmosphere i n the lab o r a t o r y which otherwise would be i n t o l e r a b l e . -1-INTRODUCTION An e s s e n t i a l component o£ the T4 r e p l i c a t i o n complex i s "unwinding p r o t e i n " , coded f o r by gene 32 ( A l b e r t s et a l . , 1976). E a r l y genetic s t u d i e s i n d i c a t e d that t h i s p r o t e i n i s e s s e n t i a l f o r both r e p l i c a t i o n and recombination ( K o z i n s k i and Felgenhauer, 1967; Tomizawa ejt a l . , 1966). I t was f i r s t p u r i f i e d by A l b e r t s (1970), using DNA c e l l u l o s e chromatography, as one of more than 20 d i f f e r e n t p r o t e i n s capable of b i n d i n g onto denatured DNA ( A l b e r t s , 1970; A l b e r t s and Frey, 1970; A l b e r t s et a l . , 1968). SDS gel e l e c t r o p h o r e s i s and sedimentation studies showed i t to contain a s i n g l e polypeptide of molecular weight around 35,000 ( A l b e r t s , 1970). One 32-protein binds per 10 n u c l e o t i d e s on average, and the b i n d i n g i s n o n - s p e c i f i c and cooperative; i . e . the binding of one molecule w i l l cause others to bind adjacent to i t ( A l b e r t s , 1970; Delius et a l . , 1972). The t i g h t b i n d i n g increases the r e s i s t a n c e of DNA to nuclease a t t a c k , presumably by covering the separated strands, thus s t a b i l i z i n g the denatured form (Huang and Lehman, 1972) . Binding a l s o lowers the T m of the double stranded DNA ( A l b e r t s and Frey, 1970). Recent experiments ( C u r t i s and A l b e r t s , 1976) suggested that the a c t u a l m e l t i n g of the DNA i s not caused by 32-protein b i n d i n g but by the gene 49 product; however, 32-protein s t a b i l i z e s the s i n g l e stranded conformation against f o l d i n g . An i n t e r e s t i n g property of 32-protein i s i t s s t i m u l a t i o n of T4 polymerase a c t i v i t y ( A l b e r t s and Frey, 1970; A l b e r t s , 1970; Huberman et a l . , 1971) . The T4 polymerase i s coded f o r by gene 43 and has a molecular weight of 112,000 (Goulian £t a l . , 1967). I t i s s i m i l a r to E_. c o l i polymerase I i n r e q u i r i n g a free 3'OH terminated primer and having a 3'-5' exonuclease a c t i v i t y , and, t h e r e f o r e , can only r e p l i c a t e alone i n v i t r o w i t h a s i n g l e stranded tem-p l a t e (Goulian et a l . , 1967). -2-32-protein increases T4 polymerase a c t i v i t y 5 to 1 0 - f o l d , probably by s t a b i l i z i n g the s i n g l e stranded template. The enhancement i s s p e c i f i c f o r T4 polymerase since the E. c o l i polymerases were unaffected (Hubermaniiet a l . , 1971). The s t i m u l a t i o n i s s t o i c h i o m e t r i c r a t h e r than c a t a l y t i c arid t h e r e f o r e must be dependent on the number of i n i t i a t i o n s i t e s (Sinha and Snustad, 1971). At present, a l l r e s u l t s on the s t i m u l a t o r y e f f e c t s of 32-protein on T4 polymerases have been based on k i n e t i c s t u d i e s . P h y s i c a l s t u d i e s have been qu i t e l i m i t e d , e s p e c i a l l y on the products of the r e a c t i o n . I t i s the main o b j e c t i v e of t h i s t h e s i s to study the s i z e of the products of the T4 polymerase i n the presenceaand the absence of the gene 32 p r o t e i n . S i m i l a r s t u d i e s have been conducted b r i e f l y by other i n v e s t i g a t o r s by means of k i n e t i c s and e l e c t r o n microscopy (Nossal, 1974). My approach uses b a c t e r i o -phage 0X-174 s i n g l e stranded DNA as template and a denatured r e s t r i c t i o n f r a g -ment of 0X-174 RF DNA as primer; the product of the T4 polymerase r e a c t i o n then can be analyzed by a l k a l i n e gel e l e c t r o p h o r e s i s since the r e l a t i v e m o b i l i t y of DNA on g e l i s a f u n c t i o n of i t s molecular weight (Figure 1). Both small DNA priming and a l k a l i n e g e l e l e c t r o p h o r e s i s have been employed e x t e n s i v e l y i n DNA sequencing (Sanger and Coulson, 1975) and r e s t r i c t i o n fragment mapping (McDonnell et a l . , 1977), therefore they have been s e l e c t e d as the major t o o l s f o r the s t u d i e s presented below. R e s t r i c t i o n enzymes are very u s e f u l t o o l s i n p r o v i d i n g s p e c i f i c DNA fragments f o r genetic and biochemical studies (Roberts, 1976). However, c h a r a c t e r i z i n g the r e s t r i c t i o n fragments o f t e n i n v o l v e s tedious procedures which i n most cases are u n s a t i s f a c t o r y . Current methods f o r o r d e r i n g r e s t r i c t i o n fragments are the f o l l o w i n g : 1) E l e c t r o n microscopy (Mulder et a l . , 1974). -3-Figure 1 Scheme f o r studying maximum l e n g t h of extension by T4 polymerase and 32-protein. -4-2) Genetic d e l e t i o n mapping ( A l l e t e t a l . , 1973). 3) A n a l y s i s of p a r t i a l r e s t r i c t i o n products (Danna e_t a l . , 1973). 4) Redigestion by other r e s t r i c t i o n enzymes (Lee and Sinsheimer, 1974). 5) Two dimensional gel e l e c t r o p h o r e s i s (C. Hutchison I I I , 1977 personal communication). 6) Pulse chase extension and r e c u t t i n g (Jeppesen e_t al _ . , 1976). A l l these s t r a t e g i e s have been used e x t e n s i v e l y . However, each has i t s l i m i t a t i o n s , due e i t h e r to the s i z e s of the l o c a t i o n s of the fragments. And most, i f not a l l , of them use at l e a s t one known r e s t r i c t i o n fragment map as a reference. A f u r t h e r o b j e c t i v e of t h i s t h e s i s was the use of the primer dependent r e a c t i o n to l o c a l i z e and order new r e s t r i c t i o n f r a g -ments of s m a l l DNA by determining the maximum length of DNA produced by T4 DNA polymerase w i t h a r e s t r i c t i o n f f r a g m e h t p p r i m e r on a l i n e a r template such as T7 DNA (Figure 2). A l i n e a r s i n g l e stranded DNA, prepared by strand separation of duplex DNA (Summers and S z y b a l s k i , 1968), i s primed by a r e s t r i c t i o n fragment and extended i n the presence of T4 polymerase, 32-protein and l a b e l l e d deoxynucleotides (see Figure 2). The newly synthesized, l a b e l l e d DNA i s then analyzed by a l k a l i n e gel e l e c t r o p h o r e s i s . Some template molecules w i l l not be r e p l i c a t e d f u l l y due e i t h e r to s t r a n d breakages or enzyme i n s t a b i l i t y , so some products w i l l be s h o r t e r than o t h e r s . By over-exposing the f i l m to the g e l , the l a r g e s t DNA product should be e a s i l y detected. Since agarose g e l e l e c t r o p h o r e s i s i s r o u t i n e l y used f o r molecular weight e s t i m a t i o n (Fangman, 1978), the s i z e of the product w i l l allow one to estimate the o r i g i n of DNA synthesis and t h e r e f o r e the p o s i t i o n of the primer. This technique should provide not only a r e s t r i c t i o n map but a l s o - 5 -l i n e a r DNA r e s t r i c t i o n fragments priming extension with 3 2 P dNTP extension (cont'd) T 5' * 3' a l k a l i n e denaturation 5* — 3' a l k a l i n e gel electrophoresis and autoradiography Figure 2 Scheme for mapping r e s t r i c -t i o n fragments by repair ^synthesis. -6-enable one to l o c a l i z e DNA fragments on any DNA. The advantages of t h i s method over others are two-fold. F i r s t , i t can serve as a r a p i d method fo r c o n s t r u c t i n g new r e s t r i c t i o n maps w i t h reasonable accuracy; second, t h i s technique enables one to c h a r a c t e r i z e a s i n g l e DNA fragment of i n t e r e s t . This i s important e s p e c i a l l y when one i s i n v o l v e d w i t h genetic mapping by using r e s t r i c t i o n enzymes that can generate a l a r g e number of fragments. The above method may be modified by d i g e s t i n g the extended DNA w i t h a s i n g l e strand s p e c i f i c nuclease (Vogt, 1973), then a n a l y z i n g the product on a n e u t r a l r a t h e r than an a l k a l i n e g e l , thereby av o i d i n g the un-c e r t a i n e f f e c t s of denaturing gels on l a r g e r DNA. Another m o d i f i c a t i o n would be to use e n d - l a b e l l e d substrate i n the r e a c t i o n , then SI ( s i n g l e strand s p e c i f i c ) nuclease d i g e s t i o n . Since only DNA that i s extended to completion could render the end l a b e l l e d n u c l e o t i d e SI r e s i s t a n t , t h i s should be the only product on the gel that i s l a b e l l e d . In order to achieve t h i s o b j e c t i v e , one must c h a r a c t e r i z e the length of DNA synthesized by T4 polymerase under various c o n d i t i o n s . E a r l i e r studies i n d i c a t e d that T4 DNA polymerase does not r e p l i c a t e the template f u l l y (Nossal, 1974), and the extent of r e p l i c a t i o n i s s t i l l i n que s t i o n . The experiments described i n t h i s t h e s i s should help us under-stand t h i s enzyme more f u l l y . -7-MATERIALS AND METHODS Media H-broth contained .5% peptone ( D i f c o L a b o r a t o r i e s ) , .1% glucose, .5% NaCl, and .8% n u t r i e n t broth ( D i f c o L a b o r a t o r i e s ) . The pH was adjusted to 7.0 by adding 1 M NaOH. B-broth contained 1% tryptone ( D i f c o L a b o r a t o r i e s ) , .8% NaCl, and .1% glucose. pH was adjusted to 7.4. TAG medium contained the f o l l o w i n g : 100 mM T r i s (pH 7.5), 10 mM Na 2S04, 100 mM MgS04, 2.5% NaCl, 1% glucose, 1 mM FeCl3, 50 mM CaCl2, 10 mM KH2PO4, and 1 u g amino acids per ml. TCG medium contained the f o l l o w i n g : 100 mM T r i s (pH 7.5), .16 mM Na 2S04, 1 mM MgS04, -1% NaCl, .1% glucose, .003 mM FeCl3, .05% Casamino a c i d s , 1.4 mM CaCl2 and .32 mM KH2PO4. B a c t e r i a l s t r a i n s E. c o l i s t r a i n ' B23wwas used i n the p u r i f i c a t i o n of both T4 gene 32 p r o t e i n and T7 DNA. E. c o l i C ( s u - ) was used f o r preparing 0X-174 phages and 0X-174 RF DNA. Bacteriophages T4 D + tA3 (w.t.) and am453 (p32~) were from R.H. Epstei n ' s c o l l e c t i o n . 0X-174 am3 ( l y s i s d e f e c t i v e ) was from Clyde Hutchison I l l ' s c o l l e c t i o n . Enzymes R e s t r i c t i o n enzymes Hae I I I and Hpa I were purchased from New England B i o l a b s . T4 polymerase was a generous g i f t from C a r o l i n e A s t e l l (Department o f Biochemistry, U.B.C.). I t was prepared by the method of - 8 -Goulian et_ a l . (1967) and had an a c t i v i t y of 150 u n i t s / m l . Radioactive chemicals 3 5S-methionine, 3 2P-orthophosphate, [3H]-dTTP, and [a- 3 2P]-dATP were a l l purchased from NEN. Polyacrylamide gel e l e c t r o p h o r e s i s The v e r t i c a l slab g e l e l e c t r o p h o r e s i s apparatus was s i m i l a r to those a v a i l a b l e from Hoefer S c i e n t i f i c Instruments. The g e l dimensions were .75 mm x 10 cm x 14 cm, formed from p l a t e s (Hoefer S c i e n t i f i c ) 5.5" x 7". P r e p a r a t i v e separations were performed on slabs of dimensions 28 cm x 14 cm x 1.5 mm formed from p l a t e s 12.75" x 7". A l l s l o t s were formed using t e f l o n combs. ( i ) P r o t e i n gels 10% polyacrylamide running gels were used f r e q u e n t l y . They con-ta i n e d 0.375 M T r i s (pH 8.8), 0.1% SDS, 10% acrylamide, 0.25% B i s , 0.1% Temed, and 0.07%, N H ^ p e r s u l f ate . The s t a c k i n g gel contained 0.125 M T r i s - H C l (pH 6.8), 0.1% SDS, 3% acrylamide, 0.08% B i s , 0.1% Temed and 0.07% NH4-p e r s u l f a t e . Samples were prepared by b o i l i n g i n e l e c t r o p h o r e t i c sample b u f f e r (0.125 M T r i s - H C l pH 6.8, 4% SDS, 20% g l y c e r o l , 0.01% bromophenol blue, and 10% 2-mercaptoethanol) f o r 2 min before l o a d i n g . The running b u f f e r was 25 mM T r i s - H C l pH 8.3, 0.192 M g l y c i n e , and 0.1% SDS. Separation was continued at 40 mA, w i t h c o o l i n g , u n t i l the t r a c k i n g dye reached the bottom of the s l a b . Gels were s t a i n e d (50% TCA, 0.1% comasie blue) f o r 15 min w i t h a g i t a t i o n , r i n s e d overnight w i t h 3 changes of 5% methanol - 7% A c e t i c a c i d , then d r i e d w i t h heat under vacuum onto a sheet of Whatman 3MM f i l t e r paper. ( i i ) DNA gels Gels (5% acrylamide and .13% B i s ) were made i n running b u f f e r (36 -9-mM T r i s , 30 mM Na^PO^, and 1 mM EDTA, pH 7.5) as described p r e v i o u s l y . Samples were made at l e a s t 8% i n sucrose, 50 mM i n EDTA, and 0.004% i n bromophenol blue before l o a d i n g . Gels were run w i t h c o o l i n g under constant voltage (40 V) f o r 5.5 h r s , s t a i n e d w i t h ethidium bromide (4 ug/ml) f o r se v e r a l minutes, and DNA bands v i s u a l i z e d under UV. Agarose gel e l e c t r o p h o r e s i s The S t u d i e r h o r i z o n t a l s l a b gel apparatus (McDonnell et a l . , 1977) was used throughout t h i s study w i t h gels 12 cm x 13 cm x 3 mm. The appara-tus was made of l u c i t e , the s l o t formers of nylon. ( i ) N e u t r a l gels The running b u f f e r used i n n e u t r a l agarose g e l e l e c t r o p h o r e s i s was e i t h e r TBE (.89 mM T r i s , 89 mM borate, 2.5 mM EDTA, pH 8.3) or Tris-phosphate-EDTA (described above). Agarose (Biorad) was weighed (0.5% or 1.0%) and melted i n the appropriate volume of b u f f e r . The g e l was cooled to 60°C before ethidium bromide was added to a f i n a l c o n c e n t r a t i o n of 1 ug/ml. A f t e r pouring, the gel was allowed to set at room temperature f o r 1 hr. Samples were prepared as described f o r DNA polyacrylamide gels and loaded a f t e r sample w e l l s were flooded w i t h running b u f f e r . The gels were electrophoresed at 50 V u n t i l samples migrated i n t o g e l . A piece of Saran wrap then was placed on top of the g e l to prevent evaporation, and the voltage was readjusted to the appropriate s e t t i n g f o r running. DNA bands could be detected under UV during and a f t e r e l e c t r o p h o r e s i s . ( i i ) A l k a l i n e gels Agarose (1%) was weighed and melted i n de i o n i z e d dH.20 w i t h 30 mM NaOH, 2 mM EDTA and 1 Mg ethidium bromide/ml before pouring (McDonnell e_t a l . , 1977). Samples were denatured by adding NaOH to a f i n a l c o n c e n t r a t i o n of 0.2 M, incubated at room temperature f o r 30 min, and n e u t r a l i z e d w i t h an -10-equivalent amount of HCl. The samples were made 8% i n g l y c e r o l , 50 mM i n EDTA, and 0.004% i n bromophenol blue before l o a d i n g . 0X-174 RF DNA, n a t i v e and denatured, were used as i n d i c a t o r s s s i n c e they could be s t a i n e d by the ethidium bromide under the a l k a l i n e c o n d i t i o n s while others d i d not. E l e c t r o p h o r e s i s was as f o r n e u t r a l g e l s . Loading w e l l s were r e p l e n i s h e d w i t h running b u f f e r whenever evaporation occured. ( i i i ) Gel drying Agarose gels were d r i e d f o r autoradiography. The gel was placed on a piece of Saran wrap, then a m i l l i p o r e f i l t e r membrane, three sheets of Whatmann 3MM f i l t e r paper, agl'asis p l a t e , and a 600 gram weight was added on top of the g e l . F i l t e r papers were replaced when thoroughly wet, and an a d d i t i o n a l 600 gram weight was added on top w i t h each change of f i l t e r paper u n t i l the g e l was d r i e d . Bacteriophage p u r i f i c a t i o n ( i ) 0X-174 0X-174 am3 was prepared according to Clyde Hutchison I l l ' s proce-dure (personal communication). E. c o l i C were grown at 37°C to 3 x 10^ c e l l s / m l i n B-broth, and CaC^ was added to a f i n a l c o n c e n t r a t i o n of 1 mM before i n f e c t i o n (moi=4). Incubation was continued f o r 4 hrs at 37°C. C e l l s then were sedimented, resuspended i n 25 ml of sodium t e t r a b o r a t e (2.5 mM, pH 9), t r e a t e d w i t h egg white lysozyme (.5 ug/ml) f o r 10 min at room temper-ature. EDTA was added to 10 mM before s o n i c a t i o n f o r phage r e l e a s e . Phage was banded twice i n CsCl density gradients 6p=1.4) before DNA e x t r a c t i o n . ( i i ) 0X-174 am3 RF 0X-174 RF was prepared as described by Clyde Hutchison I I I (personal communication). C e l l s were i n f e c t e d as described p r e v i o u s l y f o r 0X-174. Chloremphenicol (30 ug/ml) was added 10 min a f t e r i n f e c t i o n . C e l l s then were -11-sedimented, resuspended, t r e a t e d w i t h egg white lysozyme (10 ug/ml) f o r 5 min at 0°C. DNA was p r e c i p i t a t e d by adding .5 volume of 30% polyethylene g l y c o l i n 1.5 M NaCl and standing at room temperature f o r 2 h r s . RF DNA was f i n a l l y p u r i f i e d by CsCl e q u i l i b r i u m d e n s i t y gradient c e n t r i f u g a t i o n i n the presence of ethidium bromide. 0X RF DNA was the lower of two bands i n the CsCl gradient v i s i b l e i n UV l i g h t . I t was c o l l e c t e d by puncturing the side of the tube w i t h a s y r i n g e . Ethidium bromide and CsCl were removed by e x t r a c t i o n w i t h i s o b u t a n o l 3 times and d i a l y s i s against 20 mM T r i s pH 7.5, 1 mM EDTA. ( i i i ) 3 2P-T7 3 2P-T7 phage were prepared as f o l l o w s . E. c o l i B23 were grown two generations i n 40 ml of 1/5 phosphate TCG medium w i t h 3 2 p _ o r t ^ 0 p j 1 0 S p ] i a ^ e of s p e c i f i c a c t i v i t y =1 mCi/mg. Then T7 was added at moi=5 and the c u l t u r e incubated at 37°C u n t i l l y s i s . Then NaCl to 1 M, chloroform and DNase were added and i n c u b a t i o n continued f o r 1=5 min. A f t e r d i f f e r e n t i a l c e n t r i f u g a -t i o n the phage were banded i n a CsCl density gradient. The phage band was c o l l e c t e d by puncturing the side of the c e n t r i f u g e tube w i t h a s y r i n g e , then d i a l y s e d against 20 mM T r i s - H C l (pH 7.5), 1 mM EDTA. DNA p r e p a r a t i o n DNAs from 0X-174 and T7 phages were p u r i f i e d by phenol e x t r a c t i o n . An equal volume of R^ O saturated phenol was added to the phage suspension and the mixture r o l l e d gently i n a t e s t tube r o l l e r f o r 30 min at room tempera-t u r e . The phenol l a y e r and i n t e r f a c e were c a r e f u l l y removed and any r e s i d u a l phenol e l i m i n a t e d by e x t r a c t i o n s e v e r a l times w i t h water satura t e d ether. F i n a l l y , r e s i d u a l ether was evaporated. -12-Ethanol p r e c i p i t a t i o n of DNA DNA was p r e c i p i t a t e d by adding 0.1 v o l of 3 M sodium acetate (pH 7.8) and 3 v o l of c h i l l e d (-20°C) 95% ethanol, mixing and s t o r i n g overnight at -20°C. The DNA p e l l e t was c o l l e c t e d by spinning e i t h e r 5 min at 15,000 rpm i n an Eppendof microfuge or 1 hr at 30,000 rpm i n a Beckman SW50.1 r o t o r , depending on the volume. The supernatant was decanted, and the p e l l e t d r i e d under vacuum. 98% r e c o v e r i e s were e a s i l y obtained without c a r r i e r from as l i t t l e as 0.5 ug DNA, as determined by c o n t r o l experiments. 0.48 u g of 3 2P-0X-174 RF DNA ( n i c k t r a n s l a t e d ) was p r e c i p i t a t e d , c e n t r i f u g e d and the recovery determined by measuring r a d i o a c t i v i t i e s i n the p e l l e t and the supernatant. P u r i f i c a t i o n of gene 32 p r o t e i n ( i ) -^S-methionine pulse l a b e l E. c o l i B23 were grown i n H€-broth at 37°C to 2.5 x 10 8 c e l l s / m l . The c e l l s were sedimented and resuspended i n TAG medium and then i n f e c t e d w i t h e i t h e r T4 D + or T4 am453 (p32 _) at moi=8. 3 5S-methionine (5 uCi/ml) was added at times 5', 16', and 26' a f t e r i n f e c t i o n . 4 min a f t e r 3^S-methionine a d d i t i o n , the samples were chased by u n l a b e l l e d methionine (100 pg/ml) f o r another 4 min and then placed on i c e . The c e l l s i n the samples were sedimented, resuspended i n sample b u f f e r and b o i l e d f o r 3 min before e l e c t r o p h o r e s i s . ( i i ) Gel e l e c t r o p h o r e s i s of crude f r a c t i o n s Small samples of crude f r a c t i o n s were d i a l y z e d against 2..x 21 of 0.001 M T r i s - H C l (pH 7.5) overnight. Then 0.1 ml samples were mixed w i t h 2X sample b u f f e r and b o i l e d before e l e c t r o p h o r e s i s on 10% polyacrylamide g e l s . Approximately 100 u g of p r o t e i n were loaded per sample per s l o t . -13-( i i i ) DNA c e l l u l o s e chromatography DNA c e l l u l o s e was prepared as described by A l b e r t s ^ t al_. (1968; 1971). Ethanol washed c e l l u l o s e was r i n s e d s u c c e s s i v e l y w i t h 0.1 M NaOH, 1 mM EDTA, 10 mM HCl, and dH20, and then a i r d r i e d . C a l f thymus DNA (3 mg/ml) i n 10 mM K2HPO4 and 1 mM EDTA was denatured by heating 100°C f o r 15 min and quick cooled. C e l l u l o s e (1 gram per ml of DNA) was added to the denatured DNA s o l u t i o n w i t h s t i r r i n g and then spread over a t h i n glass p l a t e . The mixture was a i r d r i e d at 45°C overnight and l y o p h i l i z e d . The d r i e d powder was resuspended i n 20 mM T r i s - H C l (pH 7.5) and 1 mM EDTA, washed and stored i n the same b u f f e r as a frozen s l u r r y . In order to determine the amount of DNA attached, a small, sample of c e l l u l o s e was taken out, washed w i t h Tris-EDTA s e v e r a l times, resuspended i n 1 ml Tris-EDTA, b o i l e d f o r 20 min, quick cooled, and then sedimented. The UV spectrum of the supernatant was obtained; the p r e c i p i t a t e was d r i e d and weighed. U s u a l l y , the DNA c e l l u l o s e p r e p a r a t i o n contained approximately 2.7 mg DNA/gm c e l l u l o s e . DNA c e l l u l o s e was packed onto a 1" x 6" column and washed s e v e r a l times under s u c t i o n . Crude f r a c t i o n (30% ammonium s u l f a t e ppt) was d i a l y z e d overnight (10 mM T r i s , 50 mM NaCl, 5 mM EDTA, and 0.1 mM d i t h i o t h r e i t o l , pH 7.5) before chromatography. 25 ml samples were a p p l i e d onto the column and r i n s e d w i t h 30 ml of e l u t i o n b u f f e r (20 mM T r i s - H C l pH 7.5, 50 mM NaCl, 1 mM EDTA, .1 mM d i t h i o t h r e i t o l , and 10% g l y c e r o l ) . The column was e l u t e d i n 25 ml steps of e l u t i o n b u f f e r c o n t a i n i n g , s u c c e s s i v e l y , .5, 1.0, 210, 3.0 M NaCl. F r a c t i o n s were d i a l y z e d against 20 mM T r i s - H C l (pH 7.5) before a n a l y s i s by e l e c t r o p h o r e s i s . ( i v ) Concentration and storage" F r a c t i o n s c o n t a i n i n g 32-protein were pooled and concentrated by -14-d i a l y s i s against e l u t i o n b u f f e r c o n t a i n i n g 50% g l y c e r o l , then stored at -20°C. Pr o t e i n s were a c t i v e a f t e r storage f o r at l e a s t several months. B i o l o g i c a l assay f o r 32-protein Assay co n d i t i o n s were based on the T4 polymerase assay (Goulian e t a l . , 1967). 15 y 1 r e a c t i o n mixtures contained 20 mM T r i s - H C l (pH 8.0), 6 mM MgCl 2, 12 mM 2-mereaptoethanol, 150 y g BSA/ml, 30 yM dNTP, [ 3 2P]-dATP (90,000 cpm/yl), 0.015 u n i t s polymerase, l y l 32-protein, and T r i s - a c t i v a t e d DNA ( . 3 y g ) . The r e a c t i o n was s t a r t e d by the a d d i t i o n of T4 polymerase, l y l samples were t r a n s f e r r e d i n t o c o l d 10% TCA, .5% sodium pyrophosphate at times 0', 5', 10', 20', 30', and 60'. A f t e r 1 hr, TCA p r e c i p i t a b l e m a t e r i a l was c o l l e c t e d by f i l t r a t i o n through a piece of Whatman GF/A f i l t e r , and the f i l t e r s d r i e d at 85°C f o r 2 h r . Counts were obtained by l i q u i d s c i n t i l l a t i o n . Assay f o r endonuclease a c t i v i t y i n 32-protein ( i ) A l k a l i n e sucrose gradient T7 3H-DNA (1 yg) was incubated i n the T4 polymerase assay c o c k t a i l (described p r e v i o u s l y ) w i t h e i t h e r 32-protein, T4 polymerase or no other a d d i t i o n . Incubation was at 37°C and terminated a f t e r 30 min by the a d d i t i o n of 15 mM EDTA. The mixture was loaded onto a 0-20% a l k a l i sucrose gradient (0.2 M NaOH, 1 M NaCl, and 1 mM EDTA) and c e n t r i f u g e d at 30,000 rpm (Beckman SW50.1 r o t o r ) f o r 3.5 h r s . 10 drop f r a c t i o n s were c o l l e c t e d onto f i l t e r s . R a d i o a c t i v i t y was determined by l i q u i d s c i n t i l l a t i o n a f t e r d r y i n g the f i l t e r s i n an oven. ( i i ) Agarose g e l e l e c t r o p h o r e s i s 0.5 y g 0X-174 v i r a l DNA was incubated w i t h l y l 32-protein i n 10 mM MgCl 2 f o r 1 hr at 37°C. The r e a c t i o n was stopped by the a d d i t i o n of stop mix (agarose g e l l o a d i n g b u f f e r ) , and the DNA was loaded onto a 5% agarose g e l . -15-A p o s i t i v e and negative c o n t r o l a l s o was in c l u d e d using a nuclease con-taminated p r e p a r a t i o n and T r i s (pH 8.0) r e s p e c t i v e l y . Gel e l e c t r o p h o r e s i s was as described p r e v i o u s l y . Primed extension ( i ) Hae^ I I I r e s t r i c t i o n fragments (a) R e s t r i c t i o n enzyme d i g e s t i o n 200 pg DNA was digested w i t h 35 u n i t s of Hae I I I r e s t r i c t i o n enzyme at 37°C f o r 5 hr i n 6 mM T r i s - H C l (pH 7.4), 6 mM NaCl, 6.6 mM 2-mercaptoethanol, 100 u g BSA/ml. The r e a c t i o n was terminated by adding 200u1 stop mix (agarose g e l loa d i n g b u f f e r ) . (b) Gel e l e c t r o p h o r e s i s Digested DNA was loaded onto a 5% polyacrylamide p r e p a r a t i v e g e l and electrophoresed at 100 V f o r 24 h r s . DNA bands were detected under UV and cut out using r a z o r . (c) E l e c t r o p h o r e t i c e l u t i o n DNA fragments were e l e c t r o p h o r e t i c a l l y e l u t e d from the cut out agarose g e l s l i c e s as described by G a l i b e r t t e t t a l 1 : (1974). B r i e f l y , a p l a s t i c p i p e t t e was plugged at one end w i t h a small amount of s i l i c o n i z e d glass wool and a d i a l y s i s bag t i e d onto i t . Gels were extruded through a 5 ml syringe i n t o the p i p e t t e and r i n s e d w i t h a small volume of b u f f e r (20 mM T r i s - H C l pH 8.0, 1 mM EDTA). S p e c i a l care was taken to minimize the formation of a i r bubbles i n s i d e the p i p e t t e . DNA was e l e c t r o p h o r e t i c a l l y e l u t e d overnight i n t o the d i a l y s i s bag using a tube gel e l e c t r o p h o r e s i s apparatus. Re t r i e v e d DNA fragments were d i a l y s e d against the same b u f f e r , ethanol concentrated and stored at 4°C. Fragments then were analyzed by gel e l e c t r o p h o r e s i s to ensure homogeneity. -16-( i i ) Priming The template (0X-174 v i r a l DNA) was primed by r e s t r i c t i o n fragments j u s t p r i o r to extension. The procedure was s i m i l a r to that of Brown and Smith (1977) and of Jeppessen et a l . (1976). 0.2pg 0X-174 DNA and primers (6 molar equivalents of r e s t r i c t i o n fragments) i n dR^O (5 u1) were -mixed and b o i l e d f o r 3 min i n sealed glass c a p i l l a r y tubes. A f t e r quick c o o l i n g , the tubes were opened, 0 . 5 y l 200 mM T r i s - H C l (pH 8.0), 1 M NaCl added, and the tubes r e s e a l e d . A f t e r reannealing at 65°C f o r 2 h r s , the tubes were opened and the mixture used immediately. ( i i i ) Extension The template was extended by i n c u b a t i n g the r e a c t i o n mixture (10 u l 20 mM T r i s pH 8.0, 6 mM MgCl 2, 12 mM 2-mercaptoethanol, 150 ug BSA/ml, 30 uM dNTP, and [ 3 2P]-dATP (approximately 1,000,000 cpm t o t a l ) , 0.7 u n i t s T4 p o l y -merase, 0.5 u l 3 2 - p r o t e i n , and primed template) at 37°C f o r 60 min i n a s i l i -conized glass tube. The r e a c t i o n was terminated by the a d d i t i o n of an equal volume of H 20-saturated phenol. Then 20 u1 of 20 mM Tris-100 mM EDTA, and 0.5ug 0X-174 RF DNA were added to serve as c a r r i e r . The mixture was ether/' washed, and the DNA ethanol p r e c i p i t a t e d to separate from the unincorporated n u c l e o t i d e s . ( i v ) K i n e t i c s of extension The usual extension c o n d i t i o n s were modified by using l a b e l l e d dNTP at lower s p e c i f i c a c t i v i t y to minimize background. Samples were taken at 0', 10', 20', 30', and 60' a f t e r the a d d i t i o n of T4 polymerase, made 10% i n TCA, and the i n s o l u b l e r a d i o a c t i v i t y determined. (v) Optimum 32-protein s t i m u l a t i o n A small amount of primed template was incubated under extension c o n d i t i o n (above) with T4 polymerase and v a r y i n g amounts of 32-protein f o r -17-30 min. TCA p r e c i p i t a b l e counts were obtained. The l e v e l o f p r o t e i n that gave maximum i n c o r p o r a t i o n was used i n the extension studies w i t h 32-protein. ( v i ) A l k a l i n e g e l e l e c t r o p h o r e s i s The products of the extension r e a c t i o n s were analyzed by e l e c t r o -phoresis on a l k a l i n e gels which were pre-run w i t h 10 mM ATP f o r 15 min before l o a d i n g to e l i m i n a t e 32p background. The gels were d r i e d and autoradio-graphed. The denatured RF DNA that was added along w i t h the extension products was detected under UV and served as an i n d i c a t o r . The reason ethidium bromide can s t a i n a l k a l i n e t r e a t e d 0X RF DNA i s not known. RESULTS P u r i f i c a t i o n of gene 32-protein ( i ) I d e n t i f i c a t i o n of 32-protein Gene 32 p r o t e i n could be i d e n t i f i e d only by i t s m o b i l i t y i n SDS polyacrylamide g e l e l e c t r o p h o r e s i s , so the r e l a t i v e p o s i t i o n of the 32-p r o t e i n band had to be determined. Although T4 gene products have been J. i d e n t i f i e d on polyacrylamide gels ( O ' F a r r e l l and Gold, 1973), there are u s u a l l y some d i f f e r e n c e s i n g e l patt e r n s between l a b o r a t o r i e s . This i s probably due to d i f f e r e n c e s i n g e l systems and p r e p a r a t i o n s . Therefore, the f i r s t step i n p u r i f y i n g the 32-protein was to as s i g n i t a p o s i t i o n on our gels . The str a t e g y employed i n l o c a t i n g the band was simply to compare the p r o t e i n s produced during i n f e c t i o n by two s t r a i n s of T4 bacteriophage: T4 w.t. (tA3) and a gene 32 mutant, am453. Since the mutant phage produces only a fragment of gp32, the p o s i t i o n of 32-protein could be determined as the band present i n the w i l d type but absent i n the mutant i n f e c t i o n . ^5g_ methionine pulse l a b e l l i n g a f t e r i n f e c t i o n was used since host p r o t e i n bands overlap the phage p r o t e i n s on gels s t a i n e d by comasie blue dye. The r e s u l t s are i l l u s t r a t e d i n Figure 3. Approximate l o c a t i o n s of other p r o t e i n bands were taken from O ' F a r r e l l and Gold (1973). ( i i ) I d e n t i f y i n g 32-protein c o n t a i n i n g crude f r a c t i o n Crude f r a c t i o n s were obtained from C a r o l i n e As t e l l ; they were mainly f r a c t i o n s remaining from the various steps of a T4 polymerase p u r i f i -c a t i o n . B r i e f l y , c e l l l y s a t e s were taken through a streptomycin p r e c i p i t a t i o n , an.aut-ollsy'ssi's,, a 0-30%, 30-50%, and 50% ammonium s u l f a t e p r e c i p i t a t i o n (Goulian et a l . , 1968). Gei e l e c t r o p h o r e s i s of samples of these preparations showed that the 0-30% ammonium s u l f a t e f r a c t i o n contained much p r o t e i n banding at -19-T4 D + (w.t.) T4 am453 (p32 _) T4 gene products 5' 16' 26' 5* 16' 26' Figure 3 Autoradiograph of gel of p u l s e - l a b e l l e d p o s t - i n f e c t i o n T4 gene products. C e l l s i n f e c t e d by D+ (w.t.) ( l e f t ) and am453 (p32 _) ( r i g h t ) were pulse l a b e l l e d w i t h S-methionine at 5', 16' and 26' a f t e r i n f e c -t i o n f o r 4 min. P o s i t i o n s of T4 pr o t e i n s were assigned according to O ' F a r r e l l and Gold (1973). 32-protein i s seen as the major band present i n the w.t. i n f e c t i o n but absent i n the mutant i n f e c t i o n . E. c o l i p r o t e i n s were l a b e l l e d at 5' of the mutant i n f e c t i o n probably due to a s l i g h t delay by the mutant i n the host s h u t - o f f . -20-the 32-protein p o s i t i o n but r e l a t i v e l y l i t t l e banding elsewhere (Figure 4) . This f r a c t i o n was then used to f u r t h e r p u r i f y f o r 32-protein^by DNA c e l l u -l o s e a f f i n i t y chromatography. ( i i i ) DNA c e l l u l o s e chromatography The DNA c e l l u l o s e was always checked f o r bound DNA before use. In a l l cases, i t s DNA content remained constant throughout the experiments. Pr.orteins were a p p l i e d and e l u t e d from the column by stepwise increases i n NaCl c o n c e n t r a t i o n , and the f r a c t i o n s analysed by SDS polyacrylamide gel e l e c t r o p h o r e s i s . 32-protein was found to be e l u t e d by 0.5 M NaCl. Since gel e l e c t r o p h o r e s i s showed i t to be homogenous, f u r t h e r p u r i f i c a t i o n was unnecessary. The 32-protein was concentrated to approximately 2 mg/ml by d i a l y s i s against 50% g l y c e r o l , and stored at -20°C. Small samples of t h i s p r o t e i n s tored i n l e s s than 10% g l y c e r o l at -70°C f o r over s e v e r a l months r e t a i n e d a c t i v i t y as reported by A l b e r t s and Frey (1970). ( i v ) B i o l o g i c a l assay The a c t i v i t y of 32-protein could not be q u a n t i f i e d . However, i t s s t i m u l a t i o n of T4 polymerase a c t i v i t y was used to detect i t (Huberman et a l . , 1971) . Nucleotide i n c o r p o r a t i o n k i n e t i c s of T4 polymerase w i t h and without the a d d i t i o n of gp32 were s t u d i e d f o r t h i s purpose. Figure 5 shows that the 32-protein p r e p a r a t i o n was free of polymer-ase a c t i v i t y and template DNA. Their absence-was e s s e n t i a l f o r the f o l l o w i n g assay. The 32-protein p r e p a r a t i o n s t i m u l a t e d T4 polymerase a c t i v i t y f i v e - . , f o l d (Figure 6), i n agreement w i t h the r e s u l t s of Huberman et a l . (1971) and Nossal (1974). S t i m u l a t i o n was maximal at lower temperature and higher s a l t c o n c e n t r a t i o n . Since T r i s - a c t i v a t e d T4 DNA (DNA b o i l e d i n T r i s pH 7.5 and -21-O r i g i n R e l a t i v e p o s i t i o n of 32-protein G F E D C B A Figure 4 10% SDS polyacrylamide gel e l e c t r o p h o r e s i s p a t t e r n s of d i f f e r e n t f r a c t i o n s of an i n f e c t e d c e l l l y s a t e (see t e x t ) . A) crude l y s a t e ; B) streptomycin p r e c i p i t a t i o n supernatant; C) streptomycin p r e c i p i t a t i o n p e l l e t ; D) a u t o l y s i s supernatant; E) a u t o l y s i s p e l l e t ; F) 0-30%; and G) 30-50% ammonium s u l f a t e p r e c i p i t a t i o n p e l l e t . -22-Figure 5 Template and polymerase a c t i v i t y of 32-protein as measured by [a- 3 2P]dATP i n c o r p o r a t i o n . (•) c o n t r o l r e a c t i o n w i t h T4 polymerase and T r i s - a c t i v a t e d T4 DNA; ( •) r e a c t i o n w i t h 32-protein and T4 p o l y -merase but not DNA; ( A ) r e a c t i o n w i t h 32-protein and DNA but no p o l y -merase . -23--24-r a p i d l y c h i l l e d ) was used, i t was impossible to estimate the number of i n i t i a t i o n s i t e s and therefore the number of 32-protein molecules present, (v) DNase assay The 32-protein p r e p a r a t i o n was assayed f o r nuclease a c t i v i t y since i t was c r u c i a l that no nuclease (endo- and exo-) be present to degrade the T4 polymerase end product. Both double stranded and s i n g l e stranded DNA were not cleaved by e i t h e r 3 2 - p r o t e i n or T4 polymerase (Fig u r e 7 and 8). Primed extension ( i ) K i n e t i c s of extension by T4 polymerase The k i n e t i c s of extension by T4 polymerase on primed and unprimed 0X-174 v i r a l DNA are shown i n Figure 9. Approximately 20 pmole of dNTP (or 5 pmole of [ 3H]dTTP) were in c o r p o r a t e d i n 60 min. Assuming t h a t the 0X-174 DNA templates were primed by r e s t r i c t i o n fragments at an e f f i c i e n c y of 100%, there would be 110 n u c l e o t i d e s incorporated per molecule of primer, which i s equivalent to 2% of the 0X-174 DNA molecule. [ H ]dTTP was incorporated at a slow ra t e i n the absence of added primer (Figure 9 ) . This was not due to contaminating o l i g o n u c l e o t i d e s from the 0X^174 phage p r e p a r a t i o n which could have acted as primers, since r e p u r i f i c a t i o n of the phage before DNA e x t r a c t i o n , or treatment of DNase before DNA e x t r a c t i o n d i d not a f f e c t the rate of n u c l e o t i d e i n c o r p o r a t i o n (Figure 10). [3H ]dTTP was incorporated a l s o i n r e a c t i o n mixtures w i t h primers but no template ( F i g u r e 9 ) . Someunicking of the fragments probably occurred during t h e i r p u r i f i c a t i o n , so that t h i s i n c o r p o r a t i o n was the r e s u l t of a r e p a i r r e a c t i o n a f t e r reannealing (see D i s c u s s i o n ) . -25-Figure 7 A l k a l i n e sucrose sedimentation p r o f i l e s of T7 DNA incubated w i t h T r i s b u f f e r (A); with, nuclease contaminated p r o t e i n prepara-t i o n ( B ) ; w i t h T4 polymerase ( C ) ; and w i t h p u r i f i e d 3 2-protein (D). Untreated 3 2 p - x 7 DNA was used as an i n t e r n a l c o n t r o l i n each gradient ( •) ; H-T7 DNA was used f o r i n c u b a t i o n ( ). -26-FRACTIONS FRACTIONS -27-O r i g i n 0X-174 v i r a l DNA C B A Figure 8 Absence of nuclease a c t i v i t y from 32-protein. 0X-174 v i r a l DNA was incubated w i t h T r i s b u f f e r ( A ) , nuclease contaminated p r o t e i n prep-a r a t i o n (B), and w i t h p u r i f i e d 32-protein p r e p a r a t i o n (C) before e l e c t r o -phoresis on a 0.5% agarose g e l . -28-Figure 9 T4 polymerase ac t i v i t y ' . a s measured by the i n c o r p o r a t i o n of [ H] dTTP . Reaction mixtures were supplemented w i t h primers only ( A ) ; w i t h templates only ( • ) ; and w i t h primed templates only ( o ) . Figure 10 3 T4 polymerase a c t i v i t y as measured by the i n c o r p o r a t i o n of [ H] dTTP using two d i f f e r e n t preparations of unprimed 0X-174 v i r a l DNA as template. Phages were r e p u r i f i e d by a second CsCl d e n s i t y gradient ( • ) , or pre-t r e a t e d w i t h DNase (•) before DNA e x t r a c t i o n . -30-( i i ) K i n e t i c s o f ext e n s i o n i n the presenceoof 32-protein 32-protein s t i m u l a t e d extension maximally about t h r e e f o l d (Figure 11). Approximately 60 pmole of deoxynucleotides were inc o r p o r a t e d . Assuming that templates were primed at 100% e f f i c i e n c y , each primer was extended approximately 300 n u c l e o t i d e s , or 5.5% of the le n g t h of the tem-p l a t e . ( i i i ) Gel e l e c t r o p h o r e s i s of the products of extension The ext e n s i o n r e a c t i o n s were run w i t h [a-32p ]dATP. Products were denatured and electrophoresed i n an a l k a l i n e agarose g e l . Since the Hpa I d i g e s t i o n of T7 DNA produces fragments of v a r y i n g lengths from 283 to 5956 base p a i r s long (McDonnell et a l . , 1977), these were used as markers f o r es t i m a t i n g the s i z e s of the extended fragments. Figure 12 i s a schematic diagram of agarose g e l e l e c t r o p h o r e t i c patterns of the Hpa I r e s t r i c t i o n fragments of T7 DNA. Also shown are the r e l a t i v e p o s i t i o n s of 0X-174 v i r a l DNA (template) and some of i t s Hae I I I r e s t r i c t i o n fragments ( p r i m e r s ) . When the products of the extension r e a c t i o n were electrophoresed and autoradiographed, the banding patterns shown i n Figure 13 were obtained. Reactions w i t h only primers or templates showed some i n c o r p o r a t i o n at t h e i r r e l a t i v e p o s i t i o n s (Figure 13 and 14) which seemed to be c o n s i s t e n t w i t h the k i n e t i c r e s u l t s . Although heavy bands were observed from the primed template extensions w i t h and without 32-protein, no s i g n i f i c a n t increase i n fragment s i z e was apparent. Despite t h i s , i t was evident from the autoradiogram that there was more i n c o r p o r a t i o n i n the presence of 32-protein than i n i t s absence, which was al s o c o n s i s t e n t w i t h the k i n e t i c data. S i m i l a r banding patterns were observed w i t h repeated experiments and w i t h d i f f e r e n t templates (Figure 15). Hae I I I fragments 1, 3 and 4 as primers were extended also to a l i m i t e d extent. -31-Figure 11 T4 polymerase a c t i v i t y of primed template, as measured by the i n c o r p o r a t i o n of [H]dTTP, i n the presence Co) and the absence, (o ) of 32-protein. -32-(Length i n b.p.) (5956) (4368) mm (2640) (2110) (1756) (1384) (996) (892) (840) (604) (440) (412) (284) D E+i f K M N 0X-174 v i r a l DNA Z l Z2 Z3 Z4 (Length i n b.p.) (5393) (1300) (1100) (870) (610) Figure 12 Diagram of g e l e l e c t r o p h o r e s i s patterns of Hpa I fragments of T7 DNA ( l e f t ) and some Hae I I I fragments of 0X-174 DNA ( r i g h t ) . Frag-ments lengths were from McDonnell et a l . (1977) and Jeppesen e_t a l . (1976). The scale i s approximate. -33-O r i g i n R e l a t i v e p o s i t i o n o f Hae I I I Fragment 2 of 0X-174 RF DNA A B C D Figure 13 Autoradiogram of extension products a f t e r a l k a l i n e agarose gel (1%) e l e c t r o p h o r e s i s . (A) Extension using primers alone; (B) T4 polymerase extension of primed template; (C) T4 polymerase extension o f primed template w i t h 32-protein; and (D) Hpa I f r a g -ments of T7 DNA. -34-R e l a t i v e p o s i t i o n of 0X-174 v i r a l DNA A B Figure 14 Autoradiogram of gel of the T4 polymerase product using template (0X-174 v i r a l DNA) alone (A). Hpa I fragments of T7 DNA were al s o i n c l u d e d as molecular weight markers ( B ) . The l e f t p a r t of the p i c t u r e was d e l i b e r a t e l y overexposed during p r i n t i n g to a l l o w the f a i n t band to appear. -35-R e l a t i v e p o s i t i o n s of: Fragment 1 Fragment 2 Fragment 4 A B C D Figure 15 Autoradiograph of g e l of the extension products of primed tem-p l a t e using Hae I I I fragments 1 (A), 2 ( B ) , and 4 (C) of 0X-174 RF DNA as primers. Hpa I fragments of T7 DNA were used as markers. -36-DISCUSSION Although T4 'DNA unwinding p r o t e i n ' (gene 32 product) s t i m u l a t e d T4 polymerase (gene 43 product) a c t i v i t y s e v e r a l f o l d as determined by k i n e t i c s t u d i e s , gel e l e c t r o p h o r e s i s a n a l y s i s of the products suggested only l i m i t e d e x t e n s i o n . Presumably the nu c l e o t i d e i n c o r p o r a t i o n observed i n r e a c t i o n mixtures c o n t a i n i n g unprimed templates was due to s e l f - p r i m i n g by nicked 0X DNA. Observations by Goulian et a l . (1967) and Romberg et a l . (1964) suggest a mechanism f o r such priming. The templates used were s i n g l e stranded c i r c u l a r DNA molecules and s e l f - p r i m i n g was impossible unless one or more nick s were introduced. Several h a i r p i n s of around twenty base p a i r s e x i s t i n the 0X-174 v i r a l DNA (Bartok e t a l . , 1975), and a n i c k w i t h i n such a h a i r p i n would allow the i n i t i a t i o n of r e p l i c a t i o n . I n i t i a t i o n would a l s o occur i f the nicked l i n e a r end could reanneal w i t h some other complementary region of the DNA molecule. A s i m i l a r e x p l a n a t i o n could a l s o apply to the n u c l e o t i d e i n c o r p o r a t i o n observed i n r e a c t i o n mixtures without template. However, a more l i k e l y e x p l a n a t i o n i s that some fragments were nic k e d during t h e i r p r e p a r a t i o n ; de-n a t u r a t i o n and reannealing would allow them to act as primers on DNA from i n t a c t fragments. Since Hae I I I r e s t r i c t i o n endonuclease gives f l u s h and cleavages at GG^CC, the [ 3 2P]dATP and [3H]dTTP i n c o r p o r a t i o n s were not due to end r e p a i r even though T4 polymerase does give l i m i t e d end h y d r o l y s i s (Englund, 1972) . R e p l i c a t i o n on primed template was followed by [3H]dTTP i n c o r p o r a -t i o n . The rate of 3H i n c o r p o r a t i o n was at l e a s t t h r e e f o l d that seen i n the absence of e i t h e r primer or template. The extent of i n c o r p o r a t i o n was al s o i n excess of the sum of both i n c o r p o r a t i o n s using only primer or template. R e p l i c a t i o n was increased a f u r t h e r t h r e e f o l d i n the presence of 32-protein which was c o n s i s t e n t w i t h published r e s u l t s (Huberman et a l . , 1971). C a l c u l a t i o n s based on the i n c o r p o r a t i o n a c t i v i t y suggested that a maximum extension of 300 nu c l e o t i d e s was expected i n the presence of 32-protein. A l k a l i n e g e l e l e c t r o p h o r e s i s a n a l y s i s on these extension products, however, produced unexpected r e s u l t s . Extensions w i t h only primer or tem-p l a t e i n the r e a c t i o n mixture showed small amounts of r a d i o a c t i v i t y at e i t h e r the r e s t r i c t i o n fragment or the 0X v i r a l DNA p o s i t i o n r e s p e c t i v e l y . T h i s , however, was c o n s i s t e n t w i t h the i n c o r p o r a t i o n k i n e t i c s data which- suggested that some s e l f - p r i m i n g d i d occur w i t h the primer and the template. Extension products of the primed template r e a c t i o n showed only a heavy band at only the fragment p o s i t i o n along w i t h some smearing below i t . This suggested that the product was not much longer than the primer despite the f a c t that n u c l e o t i d e i n c o r p o r a t i o n k i n e t i c s p r e d i c t e d that the product s i z e to be much l a r g e r (Figure 10 and 14). In the presence of 32-protein, a s i m i l a r but heavier banding p a t t e r n was observed, suggesting that the s i z e of the product was s i m i l a r to that from the r e a c t i o n without 32-protein. The f a c t that T4 polymerase extension of primed template gave a band at the primer p o s i t i o n i n i t i a l l y suggested that e i t h e r only primer DNA were synthesized or extension was short. The former e x p l a n a t i o n was not l i k e l y s i nce c o n t r o l experiments (gels and i n c o r p o r a t i o n k i n e t i c s ) w i t h primer DNA alone e x h i b i t e d much l e s s i n c o r p o r a t i o n than w i t h primed template. The l a t t e r was also u n l i k e l y because i t was i n c o n s i s t e n t w i t h the k i n e t i c data which suggested that a t l e a s t 100 n u c l e o t i d e s were incorporated (without 32-p r o t e i n ) which could e a s i l y be detected by gel e l e c t r o p h o r e s i s . I t , however, was p o s s i b l e that some primer molecules had longer extensions but escaped u d e t e c t i o n due to the i n s e n s i t i v i t y of the autoradiography whereas the m a j o r i t y of the primers had short extensions. -38-The above r e s u l t s suggested that the T4 polymerase was i n e f f i c i e n t i n extending primed template, and that most r e p l i c a t i v e products were s h o r t . Defective r e p l i c a t i o n by T4 polymerase were also observed by Nossal (1974) who reported observations of h i g h l y branching and r a p i d l y renaturable product s i m i l a r to those reported by Masamune and Richardson (1971) and Inman et a l . (1965). Subsequent experiments using nearest neighbor and sedimentation a n a l y s i s demonstrated that the new product was poly d(A-T) formed presumably by strand slippage of the template by which the mechanism i s s t i l l not known. However, the studies by Nossal (1974) d i f f e r e d from those presented here i n that the author there was studying the T4 polymerase r e p l i c a t i o n on nicked duplex T7 DNA, therefore comparison between the two s t u d i e s must be approached w i t h c a u t i o n . I t had been suggested t h a t the T4 polymerase could extend only p a r t i a l l y and pause before c o n t i n u i n g (Nossal, personal communica t i o n ) . I t was a l s o shown that r e p l i c a t i o n e f f i c i e n c y could be enhanced g r e a t l y by the presence of two other p r o t e i n s (gp 44, and gp 45) of the r e p l i c a t i o n complex (Nossal, personal communication). I t i s c l e a r , however, that more studies must be done to i n v e s t i g a t e the reason f o r the apparent s i z e s of the new products before any s p e c u l a t i o n can be drawn. The present r e s u l t s of the a l k a l i n e g e l a n a l y s i s i n d i c a t e that the use of r e p a i r synthesis w i t h T4 gene product 43 and 32 to map r e s t r i c t i o n fragments i s not f e a s i b l e due to an unusual property of the T4 DNA polymer-ase under c o n d i t i o n s of extended r e p a i r . The general scheme f o r l o c a l i z i n g DNA fragment i s s t i l l a t t r a c t i v e , and a more e f f i c i e n t i n v i t r o DNA s y n t h e s i s system could be developed using other enzymes such as the E. c o l i DNA p o l y -merase (enzyme A) (Klenow et a l . , 1971). -39-REFERENCES 1. A l b e r t s , B., 1970. Function of Gene 32-protein, a new p r o t e i n e s s e n t i a l f o r the genetic recombination and r e p l i c a t i o n of T4 bacteriophage DNA. Federation Proceedings 29: 1154-1163; 2. A l b e r t s , B., Amodio, F., Jenkins, M., Gutmann, E. and F e r r i s , F., 1968. Studies w i t h DNA-cellulose chromatography. Cold Spring Harbor  Symp. Quant. B i o l . 33: 289. 3. A l b e r t s , B., Barry, J . , B i t t n e r , M., Davies, M., Hama-Inaba, H., L i u , C , Mace, D., Moran, L. , M o r r i s , C , Piperno, J . , and Sinha, N., 1977 . I n v i t r o DNA r e p l i c a t i o n c a t a l y z e d by s i x p u r i f i e d T4 bacteriophage p r o t e i n s . 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