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Isolation and characterization of site-specific endonucleases from oral bacteria Yarrow, Colin Fraser 1981

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ISOLATION AND CHARACTERIZATION OF SITE-SPECIFIC ENDONUCLEASES FROM ORAL BACTERIA by COLIN FRASER YARROW B.Sc , M c G i l l University, 1978 A Thesis Submitted In P a r t i a l Fulfilment For the Degree of Master of Science i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY THE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1981 ® C o l i n F. Yarrow, 1981 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 of the requirements f o r an advanced degree a t the University-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 and study. I f u r t h e r agree 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 copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of 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 not be allowed without my w r i t t e n p e r m i s s i o n . o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 DE-6 (2/79) Abstract Twelve s t r a i n s of o r a l bacteria were screened for the presence of s i t e - s p e c i f i c endonucleases. These screenings yielded the following r e s u l t s : recognition s p e c i f i c i t y , 5'-GATC-3', was i d e n t i f i e d from the enzymes FnuF793 I> FnuF79-4 I, and FnuBC6 I of the respective s t r a i n s of Fusobacterium nucleatum F793> F79-4 and BC6, and, i t was also i d e n t i f i e d i n the Val3 I enzyme from V e i l l o n e l l a alcalescens 3; recognition sequence 5'-GATC-3' (both modified and unmodified recognized) was i d e n t i f i e d i n the Val8 I enzyme of s t r a i n V e i l l o n e l l a  alcalescens 8; recognition sequence 5'-GGCC-3' was i d e n t i f i e d i n the FnuBC5 I enzyme of s t r a i n F. nucleatum BC5 and i n the Smu I enzyme of s t r a i n Streptococcus mutans; recognition sequence 5'-GCGC-3'was i d e n t i f i e d i n the FnuBC5 II enzyme of s t r a i n F. nucleatum BC5. spite of Ruth iv. Table of Contents Page Abstract H Dedication • • Table of Contents iv L i s t of Tables vi L i s t of Figures V%% Acknowledgements z x Abbreviations x Introduction 1 Nomenclature 8 Methods and Materials 9 The bacteria 9 (a) Growth conditions f o r ba c t e r i a 9 P u r i f i c a t i o n of s i t e - s p e c i f i c endonucleases 9 1. Crude Extracts 9 2. Biogel column 10 3. Phosphocellulose column 10 4. DEAE-Sephacel column 10 5. Concentration 11 6. Assay 11 7. S p e c i f i c A c t i v i t y 12 Characterization of s i t e - s p e c i f i c endonucleases 12 Results 14 The ba c t e r i a 14 FnuBC5 15 Page FnuF79*< I 22 FnuF793 I, FnuBC6 I, Val3 I 27 Val8 I 31 Smu I 31* Anaerobes with no apparent s i t e - s p e c i f i c endonucleases 38 Discussion 39 References 44 vi. L i s t of Tables Table Page 1 L i s t of s i t e - s p e c i f i c endonucleases i s o l a t e d HO L i s t of Figures Gel electrophoretic patterns of methylated xDNA ... digested by the fractions from the Biogel A 0.5 m column for the assay of endonucleolytic a c t i v i t y from F. nucleatum BC5 Gel electrophoretic patterns of methylated xDNA ... digested by the fractions from the phosphocellulose column for the assay of FnuBC5 I and FnuBC5 I I endonucleolytic a c t i v i t y Gel electrophoretic patterns of methylated xDNA ., digested by the fractions from the DEAE-Sephacel column for the assay of FnuBC5 I endonucleolytic a c t i v i t y Gel electrophoretic patterns of methylated xDNA . digested by the assayed fractions from the DEAE-Sephacel column for the assay of FnuBC5 I and FnuBC5 I I endonucleolytic a c t i v i t y Acrylamide gel patterns of XDNA, <t>X174 RF DNA, .. SVHO DNA incubated with s p e c i f i c endonucleases Acrylamide gel patterns of coX174 RF DNA incubated with FnuBC5 I I Gel electrophoretic patterns of XDNA digested by . the fractions from the phosphocellulose column for FnuF794 I endonucleolytic a c t i v i t y Gel electrophoretic patterns of XDNA digested by . the f r a c t i o n s from the DEAE-Sephacel column for FnuF794 I endonucleolytic a c t i v i t y T i t r a t i o n assay of FnuF794 I on SV40 DNA Endonucleolytic cleavage patterns on various DNA's with Mbo I and FnuF79M I Gel electrophoretic patterns of XDNA digested .... by the fractions from the phosphocellulose column for FnuF793 I endonucleolytic a c t i v i t y Gel electrophoretic patterns of XDNA digested .... by the fractions from the Biogel A 0.5 m column for FnuBC6 I endonucleolytic a c t i v i t y viii. F i gu re Page 13 Ge l e l e c t r o p h o r e t i c pa t te rn s of XDNA d iges ted 30 by the f r a c t i o n s from the phosphoce l lu lo se column f o r Val3 I e ndonuc l eo l y t i c a c t i v i t y 14 Ge l e l e c t r o p h o r e t i c pa t te rn s o f XDNA d iges ted 32 by the f r a c t i o n s from the phosphoce l lu lo se column f o r Val8 I endonuc l eo l y t i c a c t i v i t y 15 Endonuc l eo l y t i c c leavage pa t te rn s o f Val8 I on XDNA . . 33 16 Ge l e l e c t r o p h o r e t i c pa t te rn s o f XDNA d iges ted 35 by the f r a c t i o n s from the B i o g e l A 0.5 m column f o r Smu I endonuc l eo l y t i c a c t i v i t y 17 Ge l e l e c t r o p h o r e t i c pa t te rn s o f XDNA d iges ted 36 by the f r a c t i o n s from the phosphoce l lu lo se column f o r Smu I endonuc l eo l y t i c a c t i v i t y 18 Endonuc l eo l y t i c c leavage pa t te rn s o f Smu I 37 on methylated XDNA ix. Acknowledgements I would l i k e to extend s p e c i a l thanks to Carol f or her unending patience and help towards t h i s thesis; S h i r l e y f or her cakes, encouragement and advice; David for h i s midnight philosophy and discussions; Diane f o r the typing; Barry, Heather and Mary for a l l the good bugs; and most importantly to Mike Smith, without whom a l l of t h i s would not have been possible, f or h i s time, understanding and i n s p i r a t i o n . X. Abbreviations ATP 5'-triphosphate ribonucleoside of adenine •diATP 5'-triphosphate deoxyribonucleoside of adenine DEAE diethylaminoethyl DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetate g gram M, mM, UM molar (moles per l i t r e ) , m i l l i m o l a r , micromolar min minutes rpm revolutions per minute Tr i s tris(hydroxymethy1)aminomethane 1. Introduction In the l a s t 10 years our approach to modern genetics has been fundamentally a l t e r e d by the development of new approaches and methodologies for probing the structure and function of DNA. The basic components for these techniques are the s i t e - s p e c i f i c type I I r e s t r i c t i o n endonucleases. These b a c t e r i a l endonucleases recognize s p e c i f i c nucleotide sequences i n duplex DNA and produce defined double stranded fragments by cleavage at a defined s i t e i n , or near, the recognition sequence. A c o l l e c t i o n of these enzymes, each with i t s own p a r t i c u l a r sequence s p e c i f i c i t y , can be used to cleave DNA molecules i n t o unique sets of fragments for DNA sequencing, chromosome ana l y s i s , gene i s o l a t i o n , and for the construction of recombinant DNA. Observations leading to the discovery of r e s t r i c t i o n enzymes span a period of nearly two decades and constitute a prime example of how basic research on an apparently inconsequential b a c t e r i o l o g i c a l phenomenon has had far-reaching implications. In the early 1950's studies by L u r i a and Human (1) and Bertani and Weigle (2) concerning the behaviour of bacteriophage grown on two d i f f e r e n t s t r a i n s of bacteria marked the beginning of t h i s research. A bacteriophage propagated on one s t r a i n of E. c o l i was found to grow poorly (that i s to say, was ' r e s t r i c t e d ' ) on a second s t r a i n , and vice versa. However, a few bacteriophage, which had apparently acquired some type of h o s t - s p e c i f i c modification that protected them from the e f f e c t s of r e s t r i c t i o n , grew on the new host. The biochemical basis of t h i s phenomenon was elucidated i n the early 1960*s when Arber and co-workers demonstrated that host s p e c i f i c modification was c a r r i e d on 2. the bacteriophage DNA (3) and that r e s t r i c t i o n was associated with the degradation of the bacteriophage DNA (4). In 1965 Arber speculated about the existence of s i t e - s p e c i f i c r e s t r i c t i o n endonucleases and suggested that modification might be produced by host s p e c i f i c DNA methylases (5). Thus, the idea became established that each r e s t r i c t i o n and modification (R-M) system i n bacteria consisted of two enzymes with i d e n t i c a l s p e c i f i c i t y . S p e c i f i c a l l y , each (R-M) system co n s i s t s of a modification enzyme that recognizes the same DNA sequence that the r e s t r i c t i o n enzyme recognizes and thus allows protection against cleavage. In t h i s way, the host c e l l DNA was protected, but foreign DNA entering from outside without modification would be degraded. In 1968 Linn and Arber (6) found'in extracts of Escherichia c o l i B an a c t i v i t y which had properties of a r e s t r i c t i o n endonuclease conforming to those previously predicted by Arber. At the same time, Meselson and Yuan (7) reported experiments with a highly p u r i f i e d endonuclease from E. c o l i 'K. They demonstrated that the enzyme cleaved unmodified bacteriophage XDNA in t o large fragments while modified DNA remained undegraded. An unusual feature of the enzyme was i t s requirement for the co-factors s-adenosylmethione, ATP, and Mg + 2. From Arber's work i t was i n c o r r e c t l y assumed that the enzyme cleaved XDNA at fix e d s i t e s . I t i s now known that these endonucleases of E. c o l i B and K are examples of a cl a s s of r e s t r i c t i o n enzymes that do not cleave DNA at s p e c i f i c s i t e s . Such cl a s s I enzymes are complex, multimeric proteins and are non-specific i n t h e i r cleavage patterns. Because of t h i s property, they have not proved useful as enzymatic tools for DNA ana l y s i s . 3. By 1970, Wilcox and Smith (8) had succeeded i n obtaining a p u r i f i e d preparation of a new and highly active r e s t r i c t i o n enzyme from Haemophilus influenzae, s t r a i n Rd. The fact that t h i s r e s t r i c t i o n enzyme appeared to require only Mg + Z as a cofactor suggested that i t would prove to be a simpler enzyme than those from eit h e r E. c o l i B or K. Using sucrose gradient c e n t r i f u g a t i o n , the p u r i f i e d enzyme was shown to s e l e c t i v e l y degrade duplex, but not single-stranded bacteriophage T7 DNA to large fragments. No nucleotides were detected during the reaction, therefore, the enzyme was c l e a r l y an endonuclease that produced double-strand breaks and was s p e c i f i c for non-host DNA. Kel l y and Smith (9) were able to show that the cleavages were sequence-specific by i d e n t i f y i n g the termini of the cleavage fragments. Cl e a r l y , the most important r e s u l t of these studies was the i d e n t i f i c a t i o n of cleavage s p e c i f i c i t y . The cleavage s p e c i f i c i t y of t h i s enzyme distinguishes i t from the E. c o l i B and K enzymes, which are randomly cleaving c l a s s I enzymes. The Haemophilus enzyme belonged to a d i f f e r e n t , and more important cl a s s of r e s t r i c t i o n endonucleases. Such c l a s s II enzymes (10) are cleavage s i t e - s p e c i f i c and require only Mg + 2 as a cofactor. Studies revealed that they are r e l a t i v e l y simple proteins, e x i s t i n g t y p i c a l l y as dimers or tetramers of a sing l e polypeptide chain (11,12). The modification methylases which correspond to these c l a s s II enzymes are separate proteins that e x i s t as monomers (13). More recently, Nathans and colleagues conducted experiments applying cleavage s i t e - s p e c i f i c r e s t r i c t i o n endonucleases i n t h e i r ii. analysis of the Simian tumour virus 40 (SV40) genome. The success of these experiments provided further evidence of the value of cleavage s i t e - s p e c i f i c enzymes as tools for the physical analysis of DNA. The sequence s p e c i f i c property of these enzymes enables them to cleave a p a r t i c u l a r DNA into a unique subset of DNA fragments which can be analyzed i n d i v i d u a l l y . This fragmentation of the DNA enables chromosomes, which are l a r g e l y i n a c c e s s i b l e to molecular genetic methods to be analyzed piece by piece i n chemical d e t a i l . The c e n t r a l r o l e of r e s t r i c t i o n endonucleases i n the development of recombinant DNA technology has stimulated a search for new enzymes of d i f f e r i n g s p e c i f i c i t i e s . A consequence of the work with the Haemophilus s i t e - s p e c i f i c endonuclease was the r e a l i z a t i o n that such enzymes could be r e a d i l y detected i n bacteria through purely biochemical procedures. The e f f i c a c y of these biochemical procedures was enhanced by the introduction of g e l electrophoresis, which f a c i l i t a t e d the analysis of DNA r e s t r i c t i o n cleavage fragments (14), and by the introduction of ethidium bromide as a fluorescent s t a i n f o r DNA (15). Using these simple and s p e c i f i c assays, many laboratories have reported i s o l a t i o n of new s i t e - s p e c i f i c endonucleases. On the current l i s t , known s i t e - s p e c i f i c endonuclease cleavage s p e c i f i c i t i e s are grouped according to recognition sequence (16). In many cases, several enzymes are known which recognize the same sequence. These enzymes have been c a l l e d isoschizomers by Roberts (16). Further, the s i t e within the recognition sequence at which isoschizomers cleave may vary. For example, Sma I cleaves (5')-C-C-C-G-G-G-(3') while Xma I cleaves (5')-C-C-C-G-G-G-(3'). 5. Nucleotide sequences of recognition s i t e s have, i n most cases, been determined by analysis of the oligonucleotides which are released from the 3' or 5' l a b e l l e d termini of cleavage fragments. A simple method for detecting recognition sequences (very often palindromic sequences) has been devised. Recognition sequences can now be detected by comparing digest fragments of $X17h and SV40 DNA produced by a given s i t e - s p e c i f i c endonuclease, with a table of possible fragments which are predicted by computer analysis of a l l t e t r a , penta and hexanucleotide sequences i n these DNA's (18). Unique assignment of s p e c i f i c i t y i s usually possible. S i t e s are c l a s s i f i e d according to whether they show two-fold r o t a t i o n a l symmetry (palindromes) such as Hae I I I , which recognizes the sequence (5')-GGCC-(3'), or are asymmetric as i n Mbo I I , which (3')-CCGG-(5') recognizes the sequence (5')-GAAGANNNNNNNN-(3') (3')-GTTCTNNNNNNN-(5') . Duplex structure i s necessary for recognition i n most s i t e - s p e c i f i c endonucleases. However, i n several instances cleavages are found i n s i n g l e stranded DNA. Although the existence of cleavage suggests d i r e c t recognition of single-stranded sequences (19), these cleavages may, i n f a c t , be due to the presence of duplex regions generated by secondary structures i n the single stranded DNA's (20). Three main cleavage modes are observed i n enzymes with symmetric recognition s i t e s . These modes include even breaks ( f l u s h ends), staggered breaks which generate 3'-single-stranded cohesive termini (3'-sticky ends), and staggered breaks which generate 5'-single-stranded cohesive termini (5'-sticky ends). Each of these 6. types of terminus has found s p e c i a l use i n recombinant DNA work. So f a r , a l l of the enzymes examined cleave to produce 3'-hydroxyl, and 5'-phosphory1 termini. Many aerobic bacteria have been surveyed f o r r e s t r i c t i o n endonuclease a c t i v i t i e s , whereas r e l a t i v e l y few anaerobes have been examined. This t h e s i s describes studies on various i s o l a t e s of the anaerobes Fusobacterium nucleatum, V e i l l o n e l l a e alcalescens, Streptococcus f a e c a l i s , and the f a c u l t a t i v e anaerobe Streptococcus  mutans. These anaerobes were a l l i s o l a t e d from the human o r a l c a v i t y , and only the genus Streptococcus f a e c a l i s f a i l e d to demonstrate the existence of any s i t e - s p e c i f i c endonucleases. For t h i s t h e s i s , the screening of o r a l bacteria f o r s i t e - s p e c i f i c endonucleases conforms to the screening procedure used by Smith and Wilcox (21) i n t h e i r p u r i f i c a t i o n of the Hind enzyme from Haemophilus  in f l u e n z a . Enzymes that are used as tools f o r studying DNA must be free from non-specific endo- and exo-nucleases, from phosphatases and from nucleic acids. Further, these enzymes must be separated from other s i t e - s p e c i f i c endonuclease a c t i v i t i e s . The screening procedure involves the removal of nucleic acids from the c e l l extract. E i t h e r of the following methods can be used: gel f i l t r a t i o n chromatography, or p r e c i p i t a t i o n with ammonium s u l f a t e , streptomycin s u l f a t e , or polyethyleneimine (PEI) (22-25). In the experiments reported i n t h i s t h e s i s , a Biogel A 0.5 m column was used to remove nucleic acids and to e f f e c t some s i z e separation, thus allowing detection of s i t e - s p e c i f i c endonuclease a c t i v i t y i n crude extracts a f t e r a sing l e chromatographic step. For most s i t e - s p e c i f i c r 7. endonucleases further p u r i f i c a t i o n was achieved by one or more steps of column chromatography. A phosphocellulose column, followed by a diethylaminoethyl (DEAE)-Sephacel column, was used f or preparations i n which the s i t e - s p e c i f i c endonuclease was d i f f i c u l t to free from non-specific nuclease contamination. Eight i s o l a t e s of Fusobacterium nucleatum were screened for the presence of s i t e - s p e c i f i c endonucleases i n Biogel A 0.5 m column eluents. Endonucleolytic a c t i v i t i e s were found i n four of these s t r a i n s . Two i s o l a t e s of V e i l o n e l l a e alcalescens and an i s o l a t e of Streptococcus mutans were also screened, and yielded presence of endonucleolytic enzyme a c t i v i t y . Further, a s t r a i n of Streptococcus  f a e c a l i s H.B. was screened but f a i l e d to demonstrate the presence of any such endonucleolytic a c t i v i t y . 8. Nomenclature Restriction endonucleases derive th e i r names from a restriction-modification (R-M) system nomenclature (17). An i t a l i c i z e d three-letter abbreviation i s used for the host organism followed by a fourth l e t t e r for s t r a i n when necessary. For example, the enzyme from Haemophilus influenzae, s t r a i n D, i s called Hind. Roman numerals are used when more than one enzyme i s found i n the same organism. Thus, there are Hind I, Hind I I , and Hind I I I enzymes. Sim i l a r l y , an enzyme from Streptococcus mutans i s called Smu I. Methods and Materials The bacteria A l l the b a c t e r i a l s t r a i n s used i n t h i s thesis were i s o l a t e d and characterized i n the laboratory of Dr. B.C. McBride (Department of Microbiology, U n i v e r s i t y of B r i t i s h Columbia) as described by L u i et a l . (26). (a) Growth conditions f o r bacteria Fusobacterium nucleatum i s o l a t e d were grown at 37° on 1.7% (w/v) Trypticase (BBL, Becton, Dickinson and Co.), 0.3% (w/v) yeast extract (Difco), 0.5% (w/v) NaCl, 0.25% B^HPO^, 0.25% (w/v) glucose, 5 ug/ml hemin, pH 7.0 i n an anaerobic chamber f i l l e d with 87% N,,, 5% CO2 and 8% H^. Streptococcus mutans, Streptococcus f a e c a l i s and V e i l o n e l l a e alcalescens i s o l a t e s were grown at 37° on Trypticase Soy Broth (BBL, Becton, Dickinson and Co.). The c e l l s were grown to stationary phase and were harvested by ce n t r i f u g a t i o n and stored at -20°. Between 2-3 g of c e l l s per l i t r e of media were obtained. P u r i f i c a t i o n of s i t e - s p e c i f i c endonucleases 1. Crude extracts Between 4 to 10 g of frozen c e l l s were used for the i s o a t i o n of s i t e - s p e c i f i c endonucleases. A l l p u r i f i c a t i o n procedures were done at 4°. C e l l s were allowed to thaw i n 10 ml of buffer A (0.01 M T r i s - H C l , pH 7.4, 0.01 M 2-mercaptoethanol) and sonicated (10 x 20 s) u n t i l most c e l l s were broken, as determined by microscopy. In cases where disruption of c e l l s was poor, the c e l l s were ground with HCl-washed glass beads (149-210 uM, Potter Industries) i n a m i n i - m i l l homogenizer (Gifford-Wood Inc.) at 3/4 speed for 60 min. The broken c e l l v preparation was centrifuged at 30,000 rpm for 90 min i n a Beckman Type 30 rotor to remove c e l l u l a r debris. 10. 2. Biogel A 0.5 m column The supernatant was adjusted to 1 M NaCl and applied to a 24 x 500 mm Biogel A 0.5 m (BioRad, 100-200 mesh) column pre-equilibrated with buffer B (1.0 M NaCl, 0.1 M Tr i s - H C l , pH 7.4, and 0.01 M 2-mercaptoethanol). The column was washed with buffer B at a flow rate of approximately 0.6 ml/min. Si x t y 5 ml f r a c t i o n s were c o l l e c t e d and 5 vii of selected f r a c t i o n s were assayed for s i t e - s p e c i f i c endonuclease a c t i v i t y (see section on assays). Fractions containing p o t e n t i a l endonuclease a c t i v i t y were pooled and dialyzed against buffer C (10% (v/v) g l y c e r o l , 0.01 M KHPO^, pH 7-4, 0.01 M 2-mercaptoethanol, IO"" M K^EDTA). 3. Phosphocellulose column The dialyzed enzyme f r a c t i o n from the Biogel column was applied to a 25 x 150 mm phosphocellulose (Whatman P l l ) column pre-equilibrated with buffer C. The column was washed with 50 ml of buffer C, and eluted with 200 ml of buffer C, which contained a l i n e a r gradient of KC1 (0 to 1.0 M). Five ml f r a c t i o n s were c o l l e c t e d at a flow rate of approximately 2 ml/min, and 5 u l of selected f r a c t i o n s were assayed as above. Fractions containing a c t i v i t y were pooled and dialyzed against buffer C. 4. DEAE-Sephacel column The dialyzed enzyme f r a c t i o n from the phosphocellulose column was applied to a 12 x 100 mm DEAE-Sephacel (Pharmacia) column pre-equilibrated with buffer C. I t was then eluted with 200 ml of buffer C which contained a l i n e a r gradient of KC1 (0 to 0.4 M) set at a flow rate of approximately 0.3 ml/min. Five ml f r a c t i o n s were c o l l e c t e d and 5 u l aliquots were assayed. 11. 5. Concentration The p u r i f i e d fractions were concentrated either by d i a l y s i s i n a buffer C solution containing 50% glycerol (v/v), or by adsorption onto an ion exchange column aTter being eluted from either the phosphocellulose or DEAE-Sephacel column. Concentration using the chromatography method involves elution with M KC1 i n buffer C from a 8 x 50 mm DEAE-Sephacel column pre-equilibrated with buffer C. This eluent was then dialyzed against 50% glycerol (v/v) i n buffer C. The f i n a l enzyme preparation was then stored at -20° where i t remained active for 6 months. 6. Assay Column eluents were assayed for s i t e - s p e c i f i c endonuclease a c t i v i t y using both methylated x DNA (a g i f t from Dr. P. Dennis) and t unmethylated x DNA (New England Biolabs) as the substrate. The assay method was es s e n t i a l l y that described by Sharp et a l . (15). Aliquots of the column fractions were incubated i n a 20 u l reaction mixture containing buffer D ( f i n a l concentration; 6 mM Tris-HCl, pH 7.4, 6 mM 2-mercaptoethanol, 6 mM MgCl^, 1-0 mg/ml gelatin) and either 0.6 ug of methylated or 0.25 ug of unmethylated xDNA at 37° for one hour. The incubation period of some samples were as long as 16 hours, and were set at 37° either to check for non-specific nuclease a c t i v i t y , or to determine whether weak endonuclease a c t i v i t y was present. The reaction was stopped by the addition of 5 u l of stop mix A (40% sucrose (w/v), 25 mM *C>EDTA, pH 7.4, 0.02% bromophenol blue, and 0.02% xylene cyanol). The resulting mixture was loaded onto a 1.4% agarose (Sigma type 1) horizontal slab gel (100 mm long and approximately 3 mm thick) i n TBE buffer (90 mM Tris-borate, 12. pH 8.3, 90 mM boric acid, 25 mM K^EDTA) (27) containing 1.0 ug/ml ethidium bromide. The mixture was then electrophoresed at 400 v o l t s for two hours. The gel was then photographed under u l t r a v i o l e t l i g h t using Polaroid (Type 57) f i l m and an orange f i l t e r . 7. S p e c i f i c A c t i v i t y The concentrated enzyme preparations were assayed, as above, using varying amounts of enzyme with 1.0 ug of xDNA to determine the s p e c i f i c a c t i v i t y of the preparation. One unit of enzyme a c t i v i t y was defined as the amount of enzyme required to completely digest 1.0 ug of XDNA i n one hour at 37°. Characterization of s i t e - s p e c i f i c endonucleases One ug of methylated X, unmethylated x, <bX174 am 3 r e p l i c a t i v e form (courtesy of Miss A.CP. L u i ) , and SV40 ( g i f t from Dr. J. Colter) DNA was separately digested, as before, with one unit of a p u r i f i e d enzyme, and with one unit of Fnu DI (A. L u i ) , Fnu DII (A. L u i ) , Fnu D i l i (A. L u i ) , Fnu EI (A. Lui) and Mbo I (M. Smith). Digestion of the X DNAs was conducted for two hours at 37°, while digestion of the *X174 am 3 and SV40 DNAs ent a i l e d incubation for 16 hours i n order to check f or complete cleavages, and for the presence of non-specific nucleases. The reaction was stopped as before, and the mixtures were loaded onto a 5% acrylamide (Eastman) v e r t i c a l slab g e l , 1.5 x 200 x 200 mm. The gel was prepared from a deionized stock s o l u t i o n of acrylamide (43.5%) and from bis-acrylamide (1.5%, w/v i n H^O) and 0.05% ammonium persulfate i n TBE buffer. The g e l was polymerized by adding N'N'N'N'-Tetramethylethylenediamine (TEMED) ( f i n a l concentration; 1 ul/ml s o l u t i o n ) . After 70 min of 13. electrophoresis at 300 v o l t s , the g e l was stained f o r approximately 30 min i n an aqueous s o l u t i o n of ethidium bromide (1.0 ug/ml). The stained g e l was then photographed i n the manner described previously. The recognition sequence of the p u r i f i e d enzyme was determined by comparing the d i f f e r e n t fragment patterns which were produced. 14. Results The Bacteria The bacteria used i n t h i s thesis were i s o l a t e d from the human o r a l c a v i t y . The species that was most productive i n s i t e - s p e c i f i c endonucleases was the Fusobacterium nucleatum, a gram negative obligate anaerobe that was e a s i l y i d e n t i f i e d by i t s t y p i c a l needle-shaped morphology. The F. nucleatum species i s o l a t e d from various i n d i v i d u a l s were designated as d i f f e r e n t s t r a i n s although they were s i m i l a r i n properties (McBride, B.C., personal communication). The anaerobe V e i l l o n e l l a alcalescens ATCC 17744 was obtained from the American Type Culture C o l l e c t i o n . 15. FnuBC5 P u r i f i c a t i o n and I d e n t i f i c a t i o n of FnuBC5 I and FnuBC5 I I The endonuclease a c t i v i t y i n the extract from F. nucleatum BC5 was eluted from the Biogel A 0.5 m column as shown i n Fig. 1. Fractions that degraded XDNA to discrete fragments were pooled, dialyzed and applied to a phosphocellulose column. The FnuBC5 I enzyme was eluted by 0.05 M KC1, and the FnuBC5 I I enzyme by 0.35 M KC1 (Fig. 2). The FnuBC5 I and FnuBC5 I I enzymes were further p u r i f i e d , separately, by eluting the enzyme from a DEAE-Sephacel column with a li n e a r gradient of KC1 (0 to 0.4 M) and fractions were assayed at 37° for 16 hours to check for non-specific nuclease a c t i v i t y (Fig. 3). Both enzymes contained very l i t t l e non-specific nuclease contamination, as evidenced by the constancy of the band pattern which remained after the xDNA was digested with the enzyme for 10 hours at 37° (Fig. 4). The FhuBC5 I and FnuBC5 I I enzymes were concentrated by d i a l y s i s against 50% glycerol (v/v) i n buffer C and both exhibited l i t t l e loss i n s p e c i f i c endonucleolytic a c t i v i t y . Comparison of the observed banding patterns with catalogued banding (29), and with FnuD I, FnuD I I , and FnuD I I I controls, showed that the banding pattern for FnuBC5 I was sim i l a r to FnuD I (Fig. 5), and that FnuBC5 I I had si m i l a r banding patterns to those of FnuD I I I (Fig. 6). Studies by Lui (28) have shown that FnuD I and FnuD I I I recognize respecitvely, 5'GGCC-3' and 5'-GCGC-3' sequences for cleavage s p e c i f i c i t y , and that these enzymes are isoschizomers of the prototype enzymes Hae I I I and Hha I (29,30). 16. F i g . 1. Gel electrophoretic patterns of methylated xDNA digested by the f r a c t i o n s from the Biogel A 0.5 m column for the assay of endonucleolytic a c t i v i t y from F. nucleatum BC5 Aliquots (5 ul) from column f r a c t i o n s (5 ml) were assayed as described i n methods and materials and subjected to electrophoresis on a 1.4% agarose g e l containing 1 ug/ml ethidium bromide. The r e s u l t i n g electrophoretic patterns were photographed under u l t r a v i o l e t l i g h t . The appearance of low molecular weight fragments i n f r a c t i o n s 35-38 are i n d i c a t i v e of endonucleolytic a c t i v i t y . Assay r e s u l t s of a l l column f r a c t i o n s i n subsequent figures were determined i n the same manner. F r a c t i o n number c was the c o n t r o l , no enzyme present i n the assay mix. Incubation conditions were 15 min at 37°. 17. F i g . 2. Gel electrophoretic patterns of methylated xDNA digested by the fractions from the phosphocellulose column for the assay of FnuBC5 I and FnuBC5 I I endonucleolytic a c t i v i t y Incubation conditions were 9 hours at 37°. fraction no. FNUBC 5 15 17 19 21 23 FNUBC 5 H 27 29 31 33 18. Fig. 3. Gel electrophoretic patterns of methylated xDNA digested by the fractions from the DEAE-Sephacel column for the assay of FnuBC5 I endonucleolytic activity Incubation conditions were 16 hours at 37°. fraction no. 18 20 22 24 26 28 30 32 34 19. F i g . 4. Gel electrophoretic patterns of methylated \DNA digested by the f r a c t i o n s from the DEAE-Sephacel column for the assay of FnuBC 5 I and FnuBC5 II endonucleolytic a c t i v i t y Low molecular weight DNA fragments were separated on a 5% acrylamide g e l as described i n methods and materials. Incubation conditions were 10 hours at 37°• The samples were as follows: s l o t 1, FnuD III standard; s l o t 2, FnuD II standard; s l o t 3, FnuD I standard; s l o t s 4-9, FnuBC5 I I ; s l o t s 10-13, FnuBC5 I. 19a CO CO O CO I J 20. F i g . 5. Endonucleolytic cleavage patterns of methylated xDNA and unmethylated XDNA, <t>X174 RF DNA, SV40 DNA Incubation conditions were 14 hours at 37°. Low molecular weight DNA fragments were separated on a 5% acrylamide g e l . S l o t s 1-8 were the FnuBC5 I enzyme digest samples, s l o t s 9, 10, and 11 were the FnuD I, FnuD II and FnuD I I I enzyme samples, r e s p e c t i v e l y . S l o t s 1, 2, 12 and 13 were the digest patterns of methylated XDNA, s l o t s 3, 4, 9, 10 and 11 were the digest patterns of unmethylated XDNA, s l o t s 5 and 6 were the digest patterns of <)>X174 RF DNA and s l o t s 7 and 8 were the digest patterns of SV40 DNA. 20a 21. F i g . 6. Gel electrophoretic patterns of digested <(>X174 DNA One unit of r e s t r i c t i o n enzymes were incubated with 1 ug <t>Xl74 RF DNA for 1 hour at 37°. Low molecular weight DNA fragments were separated on a 5% acrylamide g e l . The samples were as follows: s l o t 1, FnuBC5 I; s l o t s 2-5, FnuBC5 I I ; s l o t 6, FnuD I standard; s l o t 7, FnuD II standard; s l o t 8, FnuD I I I standard; s l o t 9, no enzyme. 2 1 a slot no. 1 3 5 7 9 22. FnuF794 I P u r i f i c a t i o n and I d e n t i f i c a t i o n of FnuF794 I Endonuclease a c t i v i t y was isolated from F. nucleatum F794. This a c t i v i t y , eluted at 0.4 M KC1 from the phosphocellulose column (Fig. 7) i n which the line a r gradient of buffer C was 0-1.0 M KC1. This enzyme eluted at 0.05 M KC1 from the DEAE-Sephacel column (Fig. 8 ) i n which the l i n e a r gradient of buffer C was 0-0.2 M KC1. The enzyme yielded about 600 units of the s i t e - s p e c i f i c endonuclease FnuF794 I from 4 g of frozen c e l l s (Fig. 9 ) . The f i n a l preparation had a small amount of contaminating non-specific nuclease. The FnuF794 I and Mbo I digestion patterns, which appeared on a series of different DNAs (methylated x, <t>X174 RF, SV40 and pBR322 DNAs, a l l incubated at 37° for 90 min), were compared to allow i d e n t i f i c a t i o n of the FnuF794 I endonucleolytic cleavage sequence. The FnuF794 I enzyme was i d e n t i f i e d as an isoschizomer of Mbo I recognizing the unmethylated sequence 5'-GATC-3' (Fig. 10) ( 3 D . 23. F i g . 7. Gel electrophoretic patterns of xDNA digested by the fractions from the phosphocellulose column for FnuF794 I endonucleolytic a c t i v i t y Slot 2 was the control, Fnu4H. o fraction no. 31 C 33 35 37 39 41 43 45 47 49 24. F i g . 8. Gel electrophoretic patterns of xDNA digested by the fractions from the DEAE-Sephacel column for FnuF794 I endonucleolytic a c t i v i t y 24 a 25. F i g . 9. Specific a c t i v i t y of FnuF794 I on SV40 DNA The assay was performed under standard conditions using 1.0 yg of SV40 DNA and varying amounts of FriuF79-< I . Incubation conditions were 1 hour at 37°. Digested DNA fragments were separated by gel electrophoresis on 5% acrylamide g e l . The control (C) was a FnuD I standard digest. t^l of enzyme used-F794I C10.08.06.04.0 2.0 1.0 0.5 26. F i g . 10. Endonucleolytic cleavage patterns of methylated \, <$,X17H, SV40, pBR322 DNAs digested with r e s t r i c t i o n enzymes Incubation conditions were 90 min at 37°. Low molecular weight DNA fragments were separated by ge l electrophoresis on a 5% acrylamide g e l . S l o t s 1, 3, 5 and 7 were the Mbo I enzyme digest samples, s l o t s 2, 4, 6 and 8 were the FnuF794 I enzyme digest samples and s l o t 9 the con t r o l enzyme sample, FnuD I . Slots 1, 2, 11 were the digest patterns of SV40 DNA (1 ug), s l o t s 3, 4, 9 and 10 were the digest patterns of <*>X174 DNA (1 ug), s l o t s 5, 6 and 12 were the digest patterns of pBR322 (1 n g ) and s l o t s 7, 8 and 13 were the digest patterns of methylated >DNA (1 n g ) . 26a l o t n o . 1 3 5 7 9 11 13 27. FnuF793 I, FnuBC6 I, Val3 I The r e s t r i c t i o n endonucleases FnuF793 I, FnuBC6 I, and Val_3 I (isolated, respectively, from F. nucleatum F793» F. nucleatum BC6, and V e i l l o n e l l a alcalenscens 3 ATCC 17744) were also found to be isoschizomers of Mbo I. The FnuF793 I, FnuBC6 I, and Val3 I enzymes were eluted at approximately 0.24 M KC1 from the phosphocellulose column (Fig. 11, 12, 13). Overnight digests (16 hours) were performed on methylated XDNA with a l l three enzymes. Fractions containing enzyme a c t i v i t y and no detectable non-specific nuclease were concentrated by d i a l y s i s against 50% glycerol (v/v) i n buffer C. About 500 units of a c t i v i t y were obtained from 6 g of frozen c e l l s . By comparing the resultant banding patterns to both Mbo I controls and the Mbo I catalogued banding pattern (New England Biolabs), FnuF793 I, FnuBC6 I, and Val3 I were i d e n t i f i e d to be isoschizomers of Mbo I, recognizing the sequence 5'-GATC-3" i n unmethylated DNA (Fig. 11). 28. Fig. 11. Gel electrophoretic patterns of xDNA digested by the fractions from the phosphocellulose column for FnuF793 I endonucleolytic activity Incubation conditions were 60 min at 37°. Slots 1 and 2 were Mbo I standard and FnuF794 I standard, respectively. 28a F i g . 12. Gel electrophoretic patterns of xDNA digested by the fractions from the phosphocellulose column for FnuBC6 I endonucleolytic a c t i v i t y Incubation conditions were 60 min at 37°. fraction no. 21 23 25 27 29 31 33 35 27 39 41 30. F i g . 13. Gel electrophoretic patterns of xDNA digested by the fractions from the phosphocellulose column for Val3 I endonucleolytic a c t i v i t y Incubation conditions were 75 min at 37°. 30a Val8 I Endonucleolytic a c t i v i t y with a Mbo I - l i k e digestion pattern was i s o l a t e d from V e i l l o n e l l a alcalescens 8 and was eluted at O.36 M KC1 from the phosphocellulose column ( F i g . 14). Fractions with endonucleolytic a c t i v i t y were incubated f o r 10 hours i n order to check f o r non-specific nuclease contamination. Comparison of the observed digestion pattern of Val8 I with the digestion pattern of methylated and unmethylated XDNA indicated that Val8 I was able to recognize and cleave both, methylated and unmethylated DNA ( F i g . 15). This indiscriminate q u a l i t y i s not exhibited by Mbo I which can cleave only unmethylated DNA. The Val8 I enzyme was i d e n t i f i e d as an isoschizomer of Sau3a and FnuE I enzymes which recognize the sequence 5'-GATC-3', and which cleave methylated and unmethylated sequences (31>28). 32. F i g . 14. Gel electrophoretic patterns of xDNA digested by the fractions from the phosphocellulose column for Val8 I endonucleolytic a c t i v i t y Incubation conditions were 90 min at 37°• 32a F i g . 15. Endonucleolytic cleavage patterns of methylated xDNA and unmethylated XDNA digested with Val8 I enzyme Incubation conditions were 8 hours at 37°. Slots 1-6 were the methylated xDNA samples and sl o t s 7-12 were the unmethylated XDNA samples. 3 3 a 34. Smu I Endonucleolytic a c t i v i t y from Streptococcus mutans, was eluted from the Biogel A 0.5 m column (Fig. 16). The a c t i v i t y which followed the phosphocellulose (Fig. 17) was eluted at 0.67 M KC1 i n buffer C. Fractions were incubated 10 hours along side FnuD I, FnuD I I , and FnuD I I I controls to check for non-specific nuclease contaminants and to allow i d e n t i f i c a t i o n of Smu I's recognition s p e c i f i c i t y (Fig. 18). Fractions which exhibited endonucleolytic a c t i v i t y were concentrated by d i a l y s i s against 50% glycerol (v/v) i n buffer C. About 3500 units of a c t i v i t y were obtained from 8 g of c e l l s . By comparing the observed banding patterns of Smu I with those of the controls, Smu I was i d e n t i f i e d as an isoschizomer of FnuD I, recognizing the sequence 5'-GGCC-3'. F i g . 16. Gel electrophoretic patterns of xDNA digested by the f r a c t i o n s from the Biogel A 0.5 m column for Smu I endonucleolytic a c t i v i t y Incubation conditions were 60 min at 37°. fract ion no. 21 23 25 27 29 31 33 35 37 39 41 CO cn F i g . 17. Gel electrophoretic patterns of xDNA digested by the f r a c t i o n s from the phosphocellulose column for Smu I endonucleolyti a c t i v i t y Incubation conditions were 60 min at 37°. 36a 37. Fi g . 18. Endonucleolytic cleavage patterns of methylated xDNA digested with the enzyme, Smu I Incubation conditions were 10 hours at 37°• Slots 1-13 were the DNA samples digested with Smu I; sl o t s 14, 15 and 16 were the DNA samples digested with FnuD I, FnuD I I and FnuD I I I , respectively. 37a 38. Anaerobes With No Apparent S i t e - s p e c i f i c Endonuclease. A s e r i e s of s t r a i n s of F. nucleatum: including BC2, BC7, BC11, BC12, and a Streptococcus f a e c a l i s H.B., were examined for the presence of s i t e - s p e c i f i c endonucleases. No endonucleolytic a c t i v i t y was detected i n any of these b a c t e r i a . When each b a c t e r i a l s t r a i n was assayed, at le a s t two types of DNA (x and SV40) were used as substrates f o r di g e s t i o n . This strategy was used to investigate the p o s s i b i l i t y that an endonuclease was indeed present, despite the absence of a recognition s p e c i f i c i t y . 39. Discussion The wide v a r i e t y of s i t e - s p e c i f i c endonucleases i n Fusobacterium, and the ease with which they were i s o l a t e d by Lui et a l . (26) warranted a more thorough search i n t h i s genus f or 'new' s i t e - s p e c i f i c endonucleases. These'new' s i t e - s p e c i f i c endonucleases could be enzymes with novel sequence recognition, or could act as substitute sources of the isoschizomer which i s found only i n small amounts i n other bacteria or pathogenic s t r a i n s . The i n i t i a l a n a lysis of Fusobacterium nucleatum suggested a high p r o b a b i l i t y of fi n d i n g d i f f e r e n t a c t i v i t i e s i n various i s o l a t e s i n t h i s species (28). This thesis reports the screening of eight new i s o l a t e s of F. nucleatum, four of which produced detectable s i t e - s p e c i f i c endonucleolytic a c t i v i t y . Other o r a l bacteria species were also screened for a c t i v i t y . A l i s t of the i d e n t i f i e d enzymes and t h e i r recognition sequences can be found i n Table 1. The detection of endonucleases i s dependent on many factors i n c l u d i n g : ( i ) the presence of at l e a s t 50 units of (stable) enzyme a c t i v i t y per g of c e l l s ; ( i i ) the adequate breakage of c e l l s and release of enzymes in t o the supernatant; ( i i i ) the presence of the NaCl (250 mM) i n the assays of the Biogel A 0.5 m column eluents which i n h i b i t endonucleolytic a c t i v i t y ; ( i v ) the enzyme being activated i n T r i s buffer (6 mM, pH 7.4) by the sing l e cofactor Mg (6 mM); (v) the use of substrate DNA which has a s u f f i c i e n t number of recognition sequences f or the detection of endonucleolytic a c t i v i t y on agarose gels, (for example, Xma I cuts XDNA three times but the DNA fragments are unresolvable as the assay i s run) and ( v i ) , the absence of highly active non-specific nucleases which obscure defined endonucleolytic cleavage patterns. 40. Table 1. L i s t of s i t e - s p e c i f i c endonucleases i s o l a t e d from a se r i e s of anaerobic bacteria Recognition Sequence GATC GATC a GGCC GCGC FnuF793 I Val8 I FnuBC5 I FnuBC5 II Enzyme FnuF7.94 I Smu I FnuBC6 I Val3 I a The recognition sequence may have a methyl group i n the N-6 p o s i t i o n of the adenine residue. 41. Of the reported four enzymes that yielded site-specific endonucleolytic activity from Fusobacterium nucleatum, three have been identified as isoschizomers of Mbo I (GATC). This identification was achieved through comparison of the cleavage patterns which they generated in their digestion of XDNA. Lui (28) and D. Leung (personal communication) have also reported isolates with similar activity. It i s possible that these isolates are the same strain. Further studies on screening F. nucleatum for site-specific endonucleases may require a better classification of isolates in order to enhance the probability that new strains of this bacterium w i l l be screened. This poor classification may also account for the high number of isolates (four of eight) screened from F. nucleatum that yielded no evidence of site-specific endonucleases. Like FnuF793 I, F794 I, and BC6 I, Val3 I has a recognition sequence identical to that of Mbo I. FnuBC5 I and Smu I recognize the same sequence as Hae III, (GGCC), while FnuBC5 II and Val8 I are identical in recognition to Hha I (GCGC) and FnuE I (GATC) respectively. This thesis has shown that in the screening of oral bacteria new isoschizomers can replace low producing, or pathogenic strains of bacteria as sources of site-specific endonucleases. Unfortunately, some isoschizomers are d i f f i c u l t to obtain in a nuclease-free form. One example i s FnuE I, which i s invaluable for the digestion of DNA's containing methylated adenine residues and also for the analysis of methylation patterns in eucaryotic genomes (32). 42. In t h i s laboratory, FnuE I has r o u t i n e l y been i s o l a t e d with trace amounts of non-specific nucleases. While Fusobacterium nucleatum s t r a i n s are generally low i n non-specific nucleases, and hence are good sources of s i t e - s p e c i f i c endonucleases, t h e i r trace contamination can be d i f f i c u l t to remove. The bacterium V e i l l o n e l l a alcalescens 8 y i e l d e d Val8 I, an izoschizomer of FnuE I which, under standard p u r i f i c a t i o n procedures gave a nuclease-free preparation. Detailed studies of r e s t r i c t i o n and modification enzymes require su b s t a n t i a l q u a n t i t i e s of these enzymes i n a p u r i f i e d s tate. However, pure enzymes are not e a s i l y obtained i n large amounts. Because of t h i s d e f i ciency, genetic engineers are exploring the p o s s i b i l i t y of cloning various R-M systems as a means to achieve enzyme overproduction. Mann et a l . (33) have cloned the Hha II system from H. haemolyticus i n the E. coli-pBR322 host-vector system using a 'shotgun' approach. After t r a n s f e c t i o n into an r m E. c o l i host (HB101), recombinant clones were tested, using bacteriophage x, for the presence of a new r e s t r i c t i o n phenotype. A recombinant plasmid was found that exhibited c l a s s i c a l r e s t r i c t i o n and modification behaviour with bacteriophage x. To increase plasmid copy number and consequent enzyme overproduction, a fragment of the recombinant plasmid DNA which contained the cloned gene was recloned i n t o a second plasmid vector. This vector was a hybrid of pBR322 and the thermally inducible X r e p l i c a t i o n region (XC^857 region) of bacteriophage x (34). The recloning yielded a new hybrid plasmid. This plasmid when incubated at 42° for 20 min, raised the plasmid copy number and the enzyme 0 43. y i e l d to several times the amount that was obtained from the o r i g i n a l plasmid. At the same time, the new plasmids' enzymes were found to be subject to simple one-step p u r i f i c a t i o n methods. In crude extracts, single-stranded DNA agarose a f f i n i t y chromatography was used to p u r i f y both the r e s t r i c t i o n and modification methylase. There i s a great advantage to having a R-M systems' genes on a small segment of DNA that can be propagated and expressed i n E_. c o l i . In the new host the genes are e a s i l y accessible for genetic studies. Also, the DNA segment i s now small enough to be r e a d i l y sequenced, thus providing d i r e c t information on gene arrangement, regulatory sequences, and protein amino acid sequences. 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