Gene, 4 (1978) 329--336
329
© Elsevier/North-HollandBiomedicalPress, Amsterdam -- Printed in The Netherlands
A NEW SEQUENCE-SPECIFIC ENDONUCLEASE FROM faecalis SUBSP. z y m o g e n e s
Streptococcus
(Restriction endonucleases, SfaI; HaeIII isoschizomer; DNA sequence analysis, recognition sequence )
RAY WU, CHARLES T. KING and ERNEST JAY* Section of Biochemistry, Molecular and Cell Biology, CorneU University, Ithaca, NY 14853 (U.S.A.) (Received September 5th, 1978) (Accepted September 26th, 1978)
SUMMARY
A new sequence-specific endonuclease, 8faI, has been partially purified from Streptococcus faecalis subsp, zymogenes. 8faI recognizes the tetranucleotide sequence 5' G - G - - C - C 3' 3' C - C - C r - G 5' t and cleaves it at the sites indicated by the arrows. INTRODUCTION Restriction endonucleases are valuable new tools for physical mapping of genes, DNA sequence analysis, gene isolation, and recombinant DNA research. A number of site-specific endonucleases have been discovered (for reviews see Nathans and Smith, 1975; Roberts, 1976, 1978). These restriction endonucleases recognize specific nucleotide sequences and cleave double-stranded DNA at specific sites. Out o f 21 species of streptococci (Deibel and Seeley, 1974) we have screened for site-specific endonucleases in 15 strains from 8 non-pathogenic *Department of Che~stry, University of New Brunswick, Fredericton, N.B., Canada. Abbreviations: B ~ F , bacterial alkaline phosphatase; dNMP, monodeoxynueleotides.
330
:
"
species..Among these, only ce~sin ~ o f the species8. faecaUssubsp. zymogenes, 8. faecaUs Uquefaciens, 8. agalactiae and 8. zooepidemicus showed a site-specific endonuclease activity. We report in detail the isolation and analysis of the ~ o g n i t i o n sequence of a s i t e - s p e c ~ endonuclease, 8faI, from a strain of~treptococcus f~calis subsp, z y m o g e n e s ( ~ T R ) , MATERIALS ,
r
The following strains of streptococci from the culture collection of the Laboratory of Microbiology, Cornell University were kindly provided by T.W. Ritchey and H.W. Seeley, Jz. (1974): S. faecalis subsp, zymogenes (strains TR and 31), S. faecalis subsp, liquefaciens ( ~ s 827 and 9116), S. agalactiae (strains 55617 and Ii), S. faecium durans (strains 9136 and 9217), S. equinus, 8. faecalis (strains 19-1 and 10C1), S. lactis (strains TR and 14), S. saliuarius (strain TR), S. sanguis and 8. zooepidemicus (~ 20350). Bacteria were grown at 37°C in a medium containing (g/l): tryptone, 10; yeast extract, 5; glucose, 5; and potassium phosphate buffer (pH 7.2), 2. After approx. 9 h when A60o reached 1--1.5 (Zeiss spectrophotometer) and the pH of the medium dropped to approx. 6.0, the cells were harvested by centrifugation at 9000 g for 20 rain and washed once in 10 mM Tris. HCI (pH 7.5)--10 mM 2-mercaptoethanol. The yield of cells from 6 1 of medium was approx. 16 g (wet weight). METHODS
Assay for site-specific endonuclease activity Enzyme assay was carried out after each step of purification. An aliquot (5 to 10 ~1) of each column fraction was incubated at 37°C in a mixture (20/~1 volume) containing 1/~g bacteriophage ), DNA, 6.6 mM Tris- HCI (pH 7.5), 6.6 mM MgCI2 and 6.6 mM 2-mercaptoethanol. The reaction was terminated after 3 h by addition of 5/d of a 5X loading dye mixture containing 50% sucrose, 100 mM EDTA, 200 mM Tris- HCI (pH 7.5), 25 mM sodium acetate and 0.2% bromphenol blue. The cleavage products were subjected to electrophoresis in a 1.4% agarose gel (20 X 20 cm) using a buffer containing 40 mM Tris- HCI (pH 7.5), 5mM sodium acetate, l mM EDTAand ethidium bromide (0.5/~g/ml). Eiectrophoresis was carried o u t a t 60 mA and about 100 V for 5--6 h. The DNA banding pattern on the gel was visualized under a shortwave ultraviolet light and photographed using the Polaroid ASA 3000 film. Digestion of DNA with Sfa/endonuclease and labeling with 3~p )~ DNA (80 ~g) was digested by 8faI (320/d) in afinal volume of 800/d which contained 6.6 mM Tris, HCI (pH 7.5), 6.6 mM MgCI2 and 6.6 mM 2-mercaptoethanol. After 10 min of digestion at 37°C, the DNA fragments were extracted w i ~ phenol and e~er, and pr~ipitated by ethanol. The 5, phosphate Was removed ~ t ~ t B...... .... polynucleotide ~ F and rep!ac~ with 321~'using kinase and [7-32P]ATP as described earlier (Wuet al., 1976).
331
5' end analysis 8faI-cleaved )~D N A , labeled at the 5' ends with 32p, was completely digested with an excess of pancreatic DNase and venom phosphodiesterase at 37°C for 2 ~ produce5 labeled monon~cleotldes. After the addition of four carrierd ~ P , the ~ p l e was applied onto Whatman No. I filterpaper and chromatographed for 10 h in 3.2 M (NH4)2SO4--0.2 M sodium acetate (pH 7,2). The four.carrier dNMP spots were visualized under an ultraviolet light, cut out, and counted in a scintillation counter (Wu, 1970). h
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f
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•
•
Sequence analysis from the 5' termini 5' [s2P]-labeled SfaI-cleaved )~ DNA was partially digested with pancreatic DNase and venom phosphodiesterase to produce labeled oligonucleotides of different lengths (Tu et al., 1976). The partial digest was fractionated by twodimensional chromatography electrophoresis (Brownlee and Sanger, 1969) using a 40 X 40 cm DEAE-cellulose thin-layer plate and homomixture VI (Jay et al., 1974). DNA polymerase-catalyzed repair synthesis In order to lower the background incorporation, circular SV40 DNA instead of )k DNA was used. The SfaI-cleaved SV40 DNA (10 ~g) was incubated in a solution containing 70 mM potassium phosphate buffer (pH 6.9), 10 mM MgSO4, 70 mM NaCI, 10 mM dithiothreitol, 1 ~m each of [3H]dGTP and [SH]dCTP, and 2 units of Eseherichia coli polymerase I. Incubation was catTied out at 4°C for 5 h, and the SH counts incorporated into the DNA were determined after precipitation with trichloroacetic acid (Wu, 1970). RESULTS AND DISCUSSION
Detection and purification of a site-specific endonuclease 8. faecalis subsp, zymogenes(strain TR) was grown at 37°C to late log phase and harvested as described under METHODS. The cells (16 g) were suspended in 4 8 m l of 10 mM Tris. HCI (pH 7.5) containing 10 mM 2-mercaptoethanol and disrupted by sonication ~n an Ultrasonics Sonifier Cell Disruptor. During sonication (30 sec bursts, total 5 rain) the cell suspension was maintained at 2--6°C. Cell debris was removed by centrifu~ation at 28 000 rev./min for 90 rain in a Beckman No. 30 rotor. The supematant solution was adjusted to 1 M NaCI and applied to a Bio-Gel A 0.5 m column (2.5 × 85 cm). Fractions 42--48 (6 m! per fraction), containing restriction endonuclease activity (with distinct banding patterns ofDNA fragments), were pooled and concentrated by dialysis against * 100% glycer oL The sample was then dialyzed against a buffer c o n t m i n g 10raM potassium phosphate (pH 7.4), 10 mM 2-mercaptoethanoli 011 mM EDTA ~ d 10% glyceroi (phosphocellulose buffer of Roberts, 1976), and applied to a column of DEAE-cellulose (1 × 15 cm). ElutionW~ carried outwith a linear gradient from 0 to 0.5 M KCI in phosphocellUlose buffer. Endonuclease SfaI, which eluted between 0.12 and 0.30 M
332 KCI, was dialyzed as just described and further purified on a column of phosphocellulose (0.5 X 15 cm). Elution was carried out with a linear gradient (140 ml total) from 0 to 1.0 M KCI in phosphocellulose buffer. Endonuclease 8faI, which eluted between 0.23 and 0.47 M KCI, was pooled (20 m|, approx. 1000 units/ml) and used for all the expe~ments. The enzyme was essentially free of contaminating nucleolytic activity because ~ DNA samples digested with a 5-fold excess of enzyme gave sharp bands on gel electrophoresis. However, some contaminating activity was detected when a 50-fold excess of enzyme was used. Attempts to further purify SfaI on a hydroxylapatite column (6 ml volume) using a gradient of 0.05 to 0.5 M phosphate buffer (pH 7.4) were unsuccessful.
Recognition sequence of Sfa/ Digestion of SV40 DNA and ~ DNA with SfaI gave more than 6 and more than 12 fragments, respectively. The DNA banding patterns of SfaI digests, as analyzed on a 1.4% agarose gel, were very similar to those produced by digesting SV40 DNA and ~ DNA with endonuclease HaeIII. A direct comparison of the digestion pattern of ), DNA with SfaI and HaeIII is shown in Fig. 1. The digestion of ;~ DNA with two different levels of SfaI (lanes 2 and 3), with HaeIII (lane 4), and with SfaI and HaeIII together (lane 5) gave identical banding patterns. This result suggests that SfaI and HaeIII share the same recognition sequence 5' @C--C 3' G C--C-G--G" However, with this method of analysis, one can not distinguish the exact cleavage sites within this recognition sequence. In principle, at least three sites for cleavage are possible within this tetranucleo. tide sequence. As shown in Figure 2, cleavage at sites (A) produces a DNA molecule with 5'-protruding end~, and sequence analysis from the 5'-labeled end should give 32pG--C--C--N. Cleavage at sites (B) produces flush-ended molecules, and sequence analysis from the 5' end should give 32pC--C--N--, where N is any one of the four deoxynucleosides. Cleavage at sites (C) produces 3'-protruding ends and sequence analysis from the 5' end should give 32pC--N. Furthermore, cleavage at sites (A) should produce a molecule which can serve as a template for the incorporation of dGMP and dCMP during repair synthesis (Wu, 1970). Attempts to incorporate [3H]dCMP and [3H]dGMP with K coil DNA polymerase into SfaI-digested SV40 DNA gave less than 0.1 molecule per DNA fragment. This rules out sites (A) for the cleavage of DNA by SfaI.
Analysis of the cleavage site of Sfa/ 5'-end analysis of 32P-labeled SfaI digested XDNA identified pdC as the 5'-terminal mononucleotide (Table I). 5 ~terminal di- and trinucleotides were determined after partial digestion of the DNA with pancreatic DNase and venom phosphodiesterase followed by two-dimensional electrophoresishomochromatography. The map shown in Fig. 3 is analyzed and the deduced sequence of each spot is listed in Table II. Since it has been established (Table I) that the 32p was attached to pdC at the 5' nucleotide, the first spot in Fig. 3
333
1
2
Z
%
i
"
Fig. 1. Eleetrophoretic banding patterns of X DNA digested with SfaI and HaelIl. Lane 1, no enzymes. Lane 2 and 3, 2 and 4 units of SfaI, respectively. Lane 4, 2 units of HaeIII. Lane 5, 2 units of SfaI and 2 units of HaeIII. Digestions were carried out at 3 7°C for 1 h. One unit of enzyme is defined as the amount required to cleave 1 ~g of ~, DNA per hour. The cleavage products were analyzed by eleetrophoresis in a 1.4% agarose gel.
(A)
(B)
(C)
5' - - N - - ~ - - C - - N - -
5'
G--C--C--N--
3'
>
3'
G--N--
5' - - N - - C c - G k C - - C - - N 3' - - N - - C - - C t - ~ - - G - - N - -
>
5' 3'
C-C--N-G--G--N--
>
5'
C--N--
5' --N--O-G--C--~C--N 3' --N--Ct--C--G--G--N
3'
C--G--Cf-N--
Fig. 2. Some possible modes of cleavage of DNA by Sfai Three possibilities (A, B, C) are shown and the cleavage sites marked with arrows. Other sites at the end or outside the recognition sequence are also possible but not considered here.
334
TABLE I IDENTIFICATION O F
M
5' MONONUCLEOTIDE
Mononueleotide
Rf
dpA
0.20
8
dpG
0.36
6
apT dpC
0.51
5
.
-
.
of total
%
0.63
.
~~ /
~
81
-
.
i , .
,
:~ - ....."
.
.
-.
. -..
.
L
.
..
. . - ~ -
Fig. 3. Twc~dimensional homoehromatogram of a partial pancreatic DNue and venom phosphodiesterase digest of the 5'-s~P-labeled8faI-digested ~, DNA. Dimension I, electrophoresis on a cellulose acetate strip in pyridine acetate buffer at pH 3.5. Dimension II, homochromatogTaphy on a DEAE-cellulose plate using homomixture VL must be 32pdC. The low observed mobility (UT °bs) of the nucleotide in spot 1 is also consistent with t h a t of 32pdC, b u t n o t with that o f other nucleotides. The mobility of nucleotides in spots 2, 3, 4, 5 and 6 is consistent with the sequence of pCpC, pCpCpC, pCpCpA, pCpCpG and p c P C p T , respectively. The sequence of nucleotides in spot 4 was f u r t h e r co .nfirmed, a f t e r e l u t i o n , b y . . :.... l o d i e ~ r ~ : f6 H0w~ i b y e!e~tr0ph0res]s partial digestion with venom. . .phospl " and h o m o c h r o m a t o ~ a p h y , i f this0!igonUc!e0tide W~ib~ifact t h e trim er [32P]d(CpCpA), one would expect to" find only " sp o t s I (pC), 2 (pCpC~,. and
335 TABLE II THE USE OF OBSERVED AND CALCULATED ELECTROPHORETIC MOBILITIES IN DETERMINING THE SfaI RECOGNITION SEQUENCE a Nucleotide
Distance from origin (in mm)
U~bs
UT care
Sequence deduced
1 2 3
16 37 50
0.08 0.18 0.24
0.14 b 0.21 0.24
~C "CppC ~pC CpC
4 5 6
82 135 160
0.39 0.65 0.77
0.39 0.63 0.73
~CpCpA ~CpCpG ~CpCpT
7 (dpT)
62 207
0.30 (1.0)
0.27
~CpCpCpC
aExperimental data presented here ~ taken from Figure 3. U~bs is the observed electrophoretic mobility relative to pT; U~al is the calculated electrophoretic mobility according to Tu et al. (1976). bA q value of 0.14 for pC was used. * d e n o t e s 32p. p
4 (pCpCpA) in the new two-dimensional map. This result was in fact observed (data not shown), and the electrophoretic mobility of new spots 1, 2 and 4 are fully consistent with the sequence of spot 4 being [s2P]d(CpCpA). Thus, the 5' trinucleotide sequence at the SfaI cleavage site is identified as 5' pCpCpN where N is dC, dA, dG or dT. This sequence is consistent only with cleavage at site (B) shown in Fig. 2. Therefore, we conclude that SfaI recognizes a sequence with 2-fold rotational symmetry, 51G-G--C-C--c_c._G_G_' and cleaves •
t
between the central dG and dC residues to produce flush-ended frag~,q.lts (Kelly and Smith, 1970). The recognition sequence and the cleavage sit~ of SfaI is, therefore, identical to that of HaeIII and BsuRI endonuc;eases (Bron and Murray, 1975). We have also screened several other major species of the genus Streptococcus for site-specific endonucleases. Fractions of the agarose column and phosphocellulose column chromatography were assayed for nuclease activity as described under Methods. Low levels of a site-specific endonuclease activity were detected inS. faecalis subsp, liquefaciens (strain 9116) and 8. zooepidemicus. Thef;e endonuclea~es produced DNA banding patterns identical to that found for SfaI, and they are probably identical to SfaI. A small amount of sitespecific endonudease which produced a differen~ DNA banding pattern was found in 8. agalactiae. No site-specific endonuclease was detected in the strains of 8. faecalis, 8. faecium, 8. equinus, S. lactis or S. salivarius we have
336
analyzed. Since not all strains of S. faecaUs subsp, zymogenes and a parent strain of S. faecaUs showed detectable site-specific endonuclease, it is likely that the enzyme 8faIis coded for by a plasmid in ~the bacteri~ The pin,mid probably was absent in those strains in which no site-specific endonuclease was detected. ACKNOWLEDGE~.qENTS
We thank Sue Blodgett for valuable assistance. This work was supported by a research grant, 77-20313, from the National Science Foundation and in part by funds l~rovided by the Cornell University Experiment Station. REFERENCES Bron, S. and Murray, K., Restriction and modification in 13. subtilis, Mol. Gen. Genet., 143 (1975) 25--33. Brownlee, G.G. and Sanger, F., Chromatography of 32P-labelled oligonucleotides on thin layers of DEAE-cellulose, Eur. J. Biochem., 11 (1969) 395--399. Deibel, R.H. and Seeley, H.W., Jr., Streptococcus, in R.E. Buchanan and N.E. Gibbons (Eds.), Bergey's Manual of Determinative Bacteriology, 8th ed., Williams and Wilkins, Baltimore, 1974, pp. 490--509. Jay, E., Bambara, R., Padmanabhan, R. and Wu, R., DNA Sequence analysis; A general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping, NucL Acid Res., 1 (1974) 331--353. Kelly, T.J. and Smith, H.O., A restriction enzyme from Haemophilus influenzae, IL Base sequence of the recognition site, J. MoL Biol., 51 (1970) 393--409. Nathans, D. and Smith, H.O., Restriction endonucleases in the analysis and restructuring of DNA molecules, Annu. Rev. Biochem., 44 (1975) 273--293. Ritchey, T.W. and Seeley, Jr., H.W., Cytochromes in Streptococcus faecalis var. zymogenes grown in a haematin-containing medium, J. Gen. Microbiol., 85 (1974)220--228. Roberts, R.J., Restriction endonucleases, CRC Crit. Rev. Biochem., 4 (1976) 123-164. Roberts, R.J., Restriction and modification enzymes and their recognition sequences, Gene, 4 (1978) 183--193. Tu, C.D., Jay, E., Bahl, C.P. and Wu, R., A reliable mapping method for sequence determination of oligodeoxyribonucleotides by mobility shift analysis, Anal. Biochem., 74 (1976) 73--93. Wu, R., Nucleotide sequence analysis of DNA, J. Mol. Biol., 51 (1970) 501--521. Wu, R., Jay, E. and Roychoudhury, R., Nucleotide sequence analysis of DNA, Methods in Cancer Res., 12 (1976) 88--176. Communicated by H.O. Smith.
329
© Elsevier/North-HollandBiomedicalPress, Amsterdam -- Printed in The Netherlands
A NEW SEQUENCE-SPECIFIC ENDONUCLEASE FROM faecalis SUBSP. z y m o g e n e s
Streptococcus
(Restriction endonucleases, SfaI; HaeIII isoschizomer; DNA sequence analysis, recognition sequence )
RAY WU, CHARLES T. KING and ERNEST JAY* Section of Biochemistry, Molecular and Cell Biology, CorneU University, Ithaca, NY 14853 (U.S.A.) (Received September 5th, 1978) (Accepted September 26th, 1978)
SUMMARY
A new sequence-specific endonuclease, 8faI, has been partially purified from Streptococcus faecalis subsp, zymogenes. 8faI recognizes the tetranucleotide sequence 5' G - G - - C - C 3' 3' C - C - C r - G 5' t and cleaves it at the sites indicated by the arrows. INTRODUCTION Restriction endonucleases are valuable new tools for physical mapping of genes, DNA sequence analysis, gene isolation, and recombinant DNA research. A number of site-specific endonucleases have been discovered (for reviews see Nathans and Smith, 1975; Roberts, 1976, 1978). These restriction endonucleases recognize specific nucleotide sequences and cleave double-stranded DNA at specific sites. Out o f 21 species of streptococci (Deibel and Seeley, 1974) we have screened for site-specific endonucleases in 15 strains from 8 non-pathogenic *Department of Che~stry, University of New Brunswick, Fredericton, N.B., Canada. Abbreviations: B ~ F , bacterial alkaline phosphatase; dNMP, monodeoxynueleotides.
330
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species..Among these, only ce~sin ~ o f the species8. faecaUssubsp. zymogenes, 8. faecaUs Uquefaciens, 8. agalactiae and 8. zooepidemicus showed a site-specific endonuclease activity. We report in detail the isolation and analysis of the ~ o g n i t i o n sequence of a s i t e - s p e c ~ endonuclease, 8faI, from a strain of~treptococcus f~calis subsp, z y m o g e n e s ( ~ T R ) , MATERIALS ,
r
The following strains of streptococci from the culture collection of the Laboratory of Microbiology, Cornell University were kindly provided by T.W. Ritchey and H.W. Seeley, Jz. (1974): S. faecalis subsp, zymogenes (strains TR and 31), S. faecalis subsp, liquefaciens ( ~ s 827 and 9116), S. agalactiae (strains 55617 and Ii), S. faecium durans (strains 9136 and 9217), S. equinus, 8. faecalis (strains 19-1 and 10C1), S. lactis (strains TR and 14), S. saliuarius (strain TR), S. sanguis and 8. zooepidemicus (~ 20350). Bacteria were grown at 37°C in a medium containing (g/l): tryptone, 10; yeast extract, 5; glucose, 5; and potassium phosphate buffer (pH 7.2), 2. After approx. 9 h when A60o reached 1--1.5 (Zeiss spectrophotometer) and the pH of the medium dropped to approx. 6.0, the cells were harvested by centrifugation at 9000 g for 20 rain and washed once in 10 mM Tris. HCI (pH 7.5)--10 mM 2-mercaptoethanol. The yield of cells from 6 1 of medium was approx. 16 g (wet weight). METHODS
Assay for site-specific endonuclease activity Enzyme assay was carried out after each step of purification. An aliquot (5 to 10 ~1) of each column fraction was incubated at 37°C in a mixture (20/~1 volume) containing 1/~g bacteriophage ), DNA, 6.6 mM Tris- HCI (pH 7.5), 6.6 mM MgCI2 and 6.6 mM 2-mercaptoethanol. The reaction was terminated after 3 h by addition of 5/d of a 5X loading dye mixture containing 50% sucrose, 100 mM EDTA, 200 mM Tris- HCI (pH 7.5), 25 mM sodium acetate and 0.2% bromphenol blue. The cleavage products were subjected to electrophoresis in a 1.4% agarose gel (20 X 20 cm) using a buffer containing 40 mM Tris- HCI (pH 7.5), 5mM sodium acetate, l mM EDTAand ethidium bromide (0.5/~g/ml). Eiectrophoresis was carried o u t a t 60 mA and about 100 V for 5--6 h. The DNA banding pattern on the gel was visualized under a shortwave ultraviolet light and photographed using the Polaroid ASA 3000 film. Digestion of DNA with Sfa/endonuclease and labeling with 3~p )~ DNA (80 ~g) was digested by 8faI (320/d) in afinal volume of 800/d which contained 6.6 mM Tris, HCI (pH 7.5), 6.6 mM MgCI2 and 6.6 mM 2-mercaptoethanol. After 10 min of digestion at 37°C, the DNA fragments were extracted w i ~ phenol and e~er, and pr~ipitated by ethanol. The 5, phosphate Was removed ~ t ~ t B...... .... polynucleotide ~ F and rep!ac~ with 321~'using kinase and [7-32P]ATP as described earlier (Wuet al., 1976).
331
5' end analysis 8faI-cleaved )~D N A , labeled at the 5' ends with 32p, was completely digested with an excess of pancreatic DNase and venom phosphodiesterase at 37°C for 2 ~ produce5 labeled monon~cleotldes. After the addition of four carrierd ~ P , the ~ p l e was applied onto Whatman No. I filterpaper and chromatographed for 10 h in 3.2 M (NH4)2SO4--0.2 M sodium acetate (pH 7,2). The four.carrier dNMP spots were visualized under an ultraviolet light, cut out, and counted in a scintillation counter (Wu, 1970). h
.
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f
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•
•
Sequence analysis from the 5' termini 5' [s2P]-labeled SfaI-cleaved )~ DNA was partially digested with pancreatic DNase and venom phosphodiesterase to produce labeled oligonucleotides of different lengths (Tu et al., 1976). The partial digest was fractionated by twodimensional chromatography electrophoresis (Brownlee and Sanger, 1969) using a 40 X 40 cm DEAE-cellulose thin-layer plate and homomixture VI (Jay et al., 1974). DNA polymerase-catalyzed repair synthesis In order to lower the background incorporation, circular SV40 DNA instead of )k DNA was used. The SfaI-cleaved SV40 DNA (10 ~g) was incubated in a solution containing 70 mM potassium phosphate buffer (pH 6.9), 10 mM MgSO4, 70 mM NaCI, 10 mM dithiothreitol, 1 ~m each of [3H]dGTP and [SH]dCTP, and 2 units of Eseherichia coli polymerase I. Incubation was catTied out at 4°C for 5 h, and the SH counts incorporated into the DNA were determined after precipitation with trichloroacetic acid (Wu, 1970). RESULTS AND DISCUSSION
Detection and purification of a site-specific endonuclease 8. faecalis subsp, zymogenes(strain TR) was grown at 37°C to late log phase and harvested as described under METHODS. The cells (16 g) were suspended in 4 8 m l of 10 mM Tris. HCI (pH 7.5) containing 10 mM 2-mercaptoethanol and disrupted by sonication ~n an Ultrasonics Sonifier Cell Disruptor. During sonication (30 sec bursts, total 5 rain) the cell suspension was maintained at 2--6°C. Cell debris was removed by centrifu~ation at 28 000 rev./min for 90 rain in a Beckman No. 30 rotor. The supematant solution was adjusted to 1 M NaCI and applied to a Bio-Gel A 0.5 m column (2.5 × 85 cm). Fractions 42--48 (6 m! per fraction), containing restriction endonuclease activity (with distinct banding patterns ofDNA fragments), were pooled and concentrated by dialysis against * 100% glycer oL The sample was then dialyzed against a buffer c o n t m i n g 10raM potassium phosphate (pH 7.4), 10 mM 2-mercaptoethanoli 011 mM EDTA ~ d 10% glyceroi (phosphocellulose buffer of Roberts, 1976), and applied to a column of DEAE-cellulose (1 × 15 cm). ElutionW~ carried outwith a linear gradient from 0 to 0.5 M KCI in phosphocellUlose buffer. Endonuclease SfaI, which eluted between 0.12 and 0.30 M
332 KCI, was dialyzed as just described and further purified on a column of phosphocellulose (0.5 X 15 cm). Elution was carried out with a linear gradient (140 ml total) from 0 to 1.0 M KCI in phosphocellulose buffer. Endonuclease 8faI, which eluted between 0.23 and 0.47 M KCI, was pooled (20 m|, approx. 1000 units/ml) and used for all the expe~ments. The enzyme was essentially free of contaminating nucleolytic activity because ~ DNA samples digested with a 5-fold excess of enzyme gave sharp bands on gel electrophoresis. However, some contaminating activity was detected when a 50-fold excess of enzyme was used. Attempts to further purify SfaI on a hydroxylapatite column (6 ml volume) using a gradient of 0.05 to 0.5 M phosphate buffer (pH 7.4) were unsuccessful.
Recognition sequence of Sfa/ Digestion of SV40 DNA and ~ DNA with SfaI gave more than 6 and more than 12 fragments, respectively. The DNA banding patterns of SfaI digests, as analyzed on a 1.4% agarose gel, were very similar to those produced by digesting SV40 DNA and ~ DNA with endonuclease HaeIII. A direct comparison of the digestion pattern of ), DNA with SfaI and HaeIII is shown in Fig. 1. The digestion of ;~ DNA with two different levels of SfaI (lanes 2 and 3), with HaeIII (lane 4), and with SfaI and HaeIII together (lane 5) gave identical banding patterns. This result suggests that SfaI and HaeIII share the same recognition sequence 5' @C--C 3' G C--C-G--G" However, with this method of analysis, one can not distinguish the exact cleavage sites within this recognition sequence. In principle, at least three sites for cleavage are possible within this tetranucleo. tide sequence. As shown in Figure 2, cleavage at sites (A) produces a DNA molecule with 5'-protruding end~, and sequence analysis from the 5'-labeled end should give 32pG--C--C--N. Cleavage at sites (B) produces flush-ended molecules, and sequence analysis from the 5' end should give 32pC--C--N--, where N is any one of the four deoxynucleosides. Cleavage at sites (C) produces 3'-protruding ends and sequence analysis from the 5' end should give 32pC--N. Furthermore, cleavage at sites (A) should produce a molecule which can serve as a template for the incorporation of dGMP and dCMP during repair synthesis (Wu, 1970). Attempts to incorporate [3H]dCMP and [3H]dGMP with K coil DNA polymerase into SfaI-digested SV40 DNA gave less than 0.1 molecule per DNA fragment. This rules out sites (A) for the cleavage of DNA by SfaI.
Analysis of the cleavage site of Sfa/ 5'-end analysis of 32P-labeled SfaI digested XDNA identified pdC as the 5'-terminal mononucleotide (Table I). 5 ~terminal di- and trinucleotides were determined after partial digestion of the DNA with pancreatic DNase and venom phosphodiesterase followed by two-dimensional electrophoresishomochromatography. The map shown in Fig. 3 is analyzed and the deduced sequence of each spot is listed in Table II. Since it has been established (Table I) that the 32p was attached to pdC at the 5' nucleotide, the first spot in Fig. 3
333
1
2
Z
%
i
"
Fig. 1. Eleetrophoretic banding patterns of X DNA digested with SfaI and HaelIl. Lane 1, no enzymes. Lane 2 and 3, 2 and 4 units of SfaI, respectively. Lane 4, 2 units of HaeIII. Lane 5, 2 units of SfaI and 2 units of HaeIII. Digestions were carried out at 3 7°C for 1 h. One unit of enzyme is defined as the amount required to cleave 1 ~g of ~, DNA per hour. The cleavage products were analyzed by eleetrophoresis in a 1.4% agarose gel.
(A)
(B)
(C)
5' - - N - - ~ - - C - - N - -
5'
G--C--C--N--
3'
>
3'
G--N--
5' - - N - - C c - G k C - - C - - N 3' - - N - - C - - C t - ~ - - G - - N - -
>
5' 3'
C-C--N-G--G--N--
>
5'
C--N--
5' --N--O-G--C--~C--N 3' --N--Ct--C--G--G--N
3'
C--G--Cf-N--
Fig. 2. Some possible modes of cleavage of DNA by Sfai Three possibilities (A, B, C) are shown and the cleavage sites marked with arrows. Other sites at the end or outside the recognition sequence are also possible but not considered here.
334
TABLE I IDENTIFICATION O F
M
5' MONONUCLEOTIDE
Mononueleotide
Rf
dpA
0.20
8
dpG
0.36
6
apT dpC
0.51
5
.
-
.
of total
%
0.63
.
~~ /
~
81
-
.
i , .
,
:~ - ....."
.
.
-.
. -..
.
L
.
..
. . - ~ -
Fig. 3. Twc~dimensional homoehromatogram of a partial pancreatic DNue and venom phosphodiesterase digest of the 5'-s~P-labeled8faI-digested ~, DNA. Dimension I, electrophoresis on a cellulose acetate strip in pyridine acetate buffer at pH 3.5. Dimension II, homochromatogTaphy on a DEAE-cellulose plate using homomixture VL must be 32pdC. The low observed mobility (UT °bs) of the nucleotide in spot 1 is also consistent with t h a t of 32pdC, b u t n o t with that o f other nucleotides. The mobility of nucleotides in spots 2, 3, 4, 5 and 6 is consistent with the sequence of pCpC, pCpCpC, pCpCpA, pCpCpG and p c P C p T , respectively. The sequence of nucleotides in spot 4 was f u r t h e r co .nfirmed, a f t e r e l u t i o n , b y . . :.... l o d i e ~ r ~ : f6 H0w~ i b y e!e~tr0ph0res]s partial digestion with venom. . .phospl " and h o m o c h r o m a t o ~ a p h y , i f this0!igonUc!e0tide W~ib~ifact t h e trim er [32P]d(CpCpA), one would expect to" find only " sp o t s I (pC), 2 (pCpC~,. and
335 TABLE II THE USE OF OBSERVED AND CALCULATED ELECTROPHORETIC MOBILITIES IN DETERMINING THE SfaI RECOGNITION SEQUENCE a Nucleotide
Distance from origin (in mm)
U~bs
UT care
Sequence deduced
1 2 3
16 37 50
0.08 0.18 0.24
0.14 b 0.21 0.24
~C "CppC ~pC CpC
4 5 6
82 135 160
0.39 0.65 0.77
0.39 0.63 0.73
~CpCpA ~CpCpG ~CpCpT
7 (dpT)
62 207
0.30 (1.0)
0.27
~CpCpCpC
aExperimental data presented here ~ taken from Figure 3. U~bs is the observed electrophoretic mobility relative to pT; U~al is the calculated electrophoretic mobility according to Tu et al. (1976). bA q value of 0.14 for pC was used. * d e n o t e s 32p. p
4 (pCpCpA) in the new two-dimensional map. This result was in fact observed (data not shown), and the electrophoretic mobility of new spots 1, 2 and 4 are fully consistent with the sequence of spot 4 being [s2P]d(CpCpA). Thus, the 5' trinucleotide sequence at the SfaI cleavage site is identified as 5' pCpCpN where N is dC, dA, dG or dT. This sequence is consistent only with cleavage at site (B) shown in Fig. 2. Therefore, we conclude that SfaI recognizes a sequence with 2-fold rotational symmetry, 51G-G--C-C--c_c._G_G_' and cleaves •
t
between the central dG and dC residues to produce flush-ended frag~,q.lts (Kelly and Smith, 1970). The recognition sequence and the cleavage sit~ of SfaI is, therefore, identical to that of HaeIII and BsuRI endonuc;eases (Bron and Murray, 1975). We have also screened several other major species of the genus Streptococcus for site-specific endonucleases. Fractions of the agarose column and phosphocellulose column chromatography were assayed for nuclease activity as described under Methods. Low levels of a site-specific endonuclease activity were detected inS. faecalis subsp, liquefaciens (strain 9116) and 8. zooepidemicus. Thef;e endonuclea~es produced DNA banding patterns identical to that found for SfaI, and they are probably identical to SfaI. A small amount of sitespecific endonudease which produced a differen~ DNA banding pattern was found in 8. agalactiae. No site-specific endonuclease was detected in the strains of 8. faecalis, 8. faecium, 8. equinus, S. lactis or S. salivarius we have
336
analyzed. Since not all strains of S. faecaUs subsp, zymogenes and a parent strain of S. faecaUs showed detectable site-specific endonuclease, it is likely that the enzyme 8faIis coded for by a plasmid in ~the bacteri~ The pin,mid probably was absent in those strains in which no site-specific endonuclease was detected. ACKNOWLEDGE~.qENTS
We thank Sue Blodgett for valuable assistance. This work was supported by a research grant, 77-20313, from the National Science Foundation and in part by funds l~rovided by the Cornell University Experiment Station. REFERENCES Bron, S. and Murray, K., Restriction and modification in 13. subtilis, Mol. Gen. Genet., 143 (1975) 25--33. Brownlee, G.G. and Sanger, F., Chromatography of 32P-labelled oligonucleotides on thin layers of DEAE-cellulose, Eur. J. Biochem., 11 (1969) 395--399. Deibel, R.H. and Seeley, H.W., Jr., Streptococcus, in R.E. Buchanan and N.E. Gibbons (Eds.), Bergey's Manual of Determinative Bacteriology, 8th ed., Williams and Wilkins, Baltimore, 1974, pp. 490--509. Jay, E., Bambara, R., Padmanabhan, R. and Wu, R., DNA Sequence analysis; A general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping, NucL Acid Res., 1 (1974) 331--353. Kelly, T.J. and Smith, H.O., A restriction enzyme from Haemophilus influenzae, IL Base sequence of the recognition site, J. MoL Biol., 51 (1970) 393--409. Nathans, D. and Smith, H.O., Restriction endonucleases in the analysis and restructuring of DNA molecules, Annu. Rev. Biochem., 44 (1975) 273--293. Ritchey, T.W. and Seeley, Jr., H.W., Cytochromes in Streptococcus faecalis var. zymogenes grown in a haematin-containing medium, J. Gen. Microbiol., 85 (1974)220--228. Roberts, R.J., Restriction endonucleases, CRC Crit. Rev. Biochem., 4 (1976) 123-164. Roberts, R.J., Restriction and modification enzymes and their recognition sequences, Gene, 4 (1978) 183--193. Tu, C.D., Jay, E., Bahl, C.P. and Wu, R., A reliable mapping method for sequence determination of oligodeoxyribonucleotides by mobility shift analysis, Anal. Biochem., 74 (1976) 73--93. Wu, R., Nucleotide sequence analysis of DNA, J. Mol. Biol., 51 (1970) 501--521. Wu, R., Jay, E. and Roychoudhury, R., Nucleotide sequence analysis of DNA, Methods in Cancer Res., 12 (1976) 88--176. Communicated by H.O. Smith.
