Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA

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1 Volume 5 Number 8 August 1978 Nucleic Acids Research Substrate dependence of the mechanism of EcoRI endonuclease Robert A. Rubin and Paul Modrich* Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA Received 25 May 1978 ABSTRACT The mechanism of EcoRI endonuclease is substrate dependent. At 37, dissociation of the enzyme-form II DNA intermediates of ColEl DNA and bacteriophage G4 RFI DNA is negligible. Therefore, both DNA strands within the EcoRI sequence are cleaved during a single binding event. However, double strand cleavage of SV40 DNA occurs without dissociation of the enzyme in only 75% of the catalytic events. This mechanistic difference presumably reflects sequence differences about the EcoRI sites of these DNA's. INTRODUCTION The mechanism of cleavage of double stranded DNA by the Escherichia coli RI (EcoRI) restriction endonuclease has been the subject of several recent studies '. This work has demonstrated that double strand cleavage by the enzyme occurs via an intermediate containing a single strand break within the EcoRI sequence. However, conclusions concerning the fate of this intermediate in the reaction have been conflicting. Using colicin El (ColEl) Form I DNA at a substrate to endonuclease molar ratio of 12, we concluded that the Form II intermediate remained enzyme-bound at 37, although extensive dissociation was observed at low temperature. In contrast, using SV40 Form I DNA at much lower enzyme to substrate ratios, Ruben et_ al_. observed accumulation 'of Form II molecules in solution to a level of 50 mole per cent prior to the appearance of the linear product. Hence, they concluded that dissociation of the intermediate from the enzyme surface is favored, and suggested that this phenomenon might be masked at higher enzyme to substrate ratios. Since knowledge of the mechanism of DNA cleavage by the endonuclease is important to understanding the nature of the interaction between the protein Information Retrieval Limited 1 Falconberg Court London W1V 5FG England 299 '

2 and the recognition sequence, we have reinvestigated this question with ColEl and SV40 substrates at low enzyme to DNA ratios. Results with ColEl DNA confirm our previous conclusion. However, with the SV40 substrate we find that although the intermediate does not dissociate in the majority of catalytic events, significant dissociation can be observed. Thus, there appear to be differences in the nature of specific interactions between the enzyme and SV40 or ColEl substrates which are manifested at the level of the mechanism of double strand cleavage. MATERIALS AND METHODS Enzymes - EcoRI endonuclease and methylase were preparations described previously '. Both enzymes were free of significant exo- or endonuclease activity. DNA - Form I Colicin El (ColEl) DNA was isolated as described, except that a phenol extraction step was added after CsCl-ethidium bromide centrifugation. SV40 DNA, generously provided by Dr. M. DePamphilis (Harvard University), was purified by the method used for ColEl DNA. Bacteriophage G4 replicative form I (RFI) DNA was a generous gift of Dr. G. N. Godson (Yale University). DNA concentrations were calculated assuming an E. of 200 and molecular c fi Jem, weights for ColEl DNA and SV40 DNA of 4.2x10 and 3.45x10, respectively. DNA methylated in^ vitro at the EcoRI sequence was prepared as described 4 DNA's containing a single strand scission within the EcoRI site were prepared by incubation with EcoRI endonuclease at 0 as described, and purified by CsCl-ethidium bromide equilibrium centrifugation. Gel Electrophoresis - Electrophoresis of DNA on 0.3 cm-thick slabs of 1% agarose was performed as described. DNA content of bands visualized with ethidium was determined by microdensitometer tracing of photographic negatives 1 4 as previously described ', or by liquid scintillation counting of solubilized bands. In the latter procedure excised gel samples were solubilized in 1 ml of 1 N HC1 at 75 for 2 hr and radioactivity determined in 10 ml Aquasol-2 (New England Nuclear). The two methods yielded identical results and were used interchangeably. RESULTS Mode of Cleavage of ColEl DNA by EcoRI Endonuclease Form I[ 3 H]ColEl DNA was incubated at 37 with 1low levels of EcoRI endonuclease to allow observation of any accumulation of the Form II intermediate 2992

3 < s c 0) Perc ColEl DNA v ~ ^N^ i Z ^ ^ TL lv^ S* i i i, SV40 DNA 100, L tl \l ^ * 30 - ^ ^ ^ 20 a 10 _/?O -^ ' I fr^\ I Time (min) Figure I. Upper Panel: Kinetics of EcoRI endonuclease digestion of ColEl DNA. The reaction (200 pi) contained 0.1 M Tris-HCl fph 7.6), 0.05 M NaCl, 5 mm MgCl 2, 50 vig/ml bovine serum albumin, 29.7 nm [ 3 H]ColEl DNA molecules and 56 pm EcoRI endonuclease (as dimer, the active form at this concentration ). A control reaction was incubated without enzyme. Incubation was at 37. At indicated times, 15 yl samples were removed and added to 135 pi of 5 mm EDTA (ph 8), 0.8% w/v SDS, 10% w/v sucrose, 0.025% bromphenol blue. Samples were subjected to electrophoresis on agarose gels at 9.1 v/cm for 4 hr. (METHODS). Data are not corrected for 7% Form II DNA present in the ColEl preparation used. The single strand break in such molecules is presumably not in the EcoRI site. Figure I. Lower Panel: Kinetics of EcoRI endonuclease digestion of SV40 DNA. The reaction contained 29.8 nm [ H]SV40 DNA molecules and 56 pm EcoRI endonuclease dimers. Other conditions were as above, except that electrophoresis was at 2.7 v/cm for 15 hr. Data are uncorrected for 2% Form II present in the SV40 preparation used. o-o Form I; - Form III; A A Form II; A 4 Form II levels in a parallel incubation without EcoRI endonuclease. DNA methylated at the EcoRI site (METHODS) was unaffected by enzyme (not shown), indicating that the single strand break in the accumulated Form II species was located within the EcoRI site. 2993

4 2 as described by Ruben et al. (Fig. 1, upper panel). However, the fraction of Form II increased linearly from the zero-time value of 7% to only 10% throughout the course of the reaction and a parallel increase was observed in control incubations lacking enzyme. This failure to detect the Form II intermediate free in solution implies either that it remains enzyme bound under these conditions, or alternatively, that upon dissociation it is subject to preferential attack by other endonuclease molecules in solution. To distinguish between these possibilities we compared the rate of hydrolysis of Form I ColEl DNA with that of Form II molecules containing a single strand break in the EcoRI site. The latter substrate was prepared by incubation with the endonuclease at 0. As shown in Table I (lines 1 and 2), Form I molecules and the isolated intermediate were attacked at equivalent rates. The Form II substrate employed in these experiments was contaminated by a significant level of Form III (linears) resulting from complete cleavage by the endonuclease at 0. To control for possible end product inhibition by such Form III molecules, the rate of cleavage of Form I circles was also determined in the presence of an equivalent concentration of the linear species (Table I, line 3). As can be seen, the presence of the end product was without effect, thus establishing that Form I circles and the intermediate are attacked at equivalent rates. These results, together with those described previously, therefore prove that under these conditions the Form II intermediate of ColEl DNA remains enzyme bound at 37. Hence the endonuclease interacts with both strands of the recognition sequence during a single DNA binding event. Table I. Rates of Cleavage of ColEl Strand Break in the EcoRI Site. Reaction Substrate DNA and ColEl DNA containing a Single Initial Rate of Hydrolysis pm min ColEl DNA EgoRI-Nicked ColEl DNA ColEl DNA (plus linear) (plus linear) Reaction conditions and gel electrophoresis were as in Fig. 1 except that circular DNA species were present at 18 nm and EcoRI endonuclease at 32 pm. At least 90% of the Form II circles in reaction 2 contained a single strand break in the EcoRI site. Reaction 2 also contained 8 nm Form III linears resulting from complete cleavage by the endonuclease during the 0 nicking reaction. To control for the presence of such linears, reaction 3 containing Form I ColEl DNA was supplemented with EcoRI-generated linear molecules to a concentration of 8 nm. Rates were determined by least square fit of linear portions of time courses analogous to those shown in Fig

5 Mode of Cleavage of SV40 DNA by EcoRI Endonuclease. To determine if the mechanism of endonuclease cleavage is substrate-dependent, the experiments described above were repeated with SV40 DNA. contrast to the mode of cleavage observed with ColEl DNA, Form II SV40 DNA containing a single strand break in the EcoRI site accumulated in solution under steady state conditions (Fig. 1, lower panel). In Since both Form II and Form III accumulated linearly from zero time, it seemed unlikely that free Form II molecules were necessary intermediates in the production of Form III. This assumption was confirmed by comparison of rates of hydrolysis of Form I SV40 DNA and Form II intermediate. As shown in Table II, the initial rates of hydrolysis are comparable for these two DNA species. Therefore, we have concluded that the free Form II molecules observed under steady state conditions arise by dissociation of a fraction of the enzyme-form II intermediates, and that dissociation per se_ is not a necessary condition for double strand cleavage. Since Form II molecules are not preferentially attacked, we can calculate from the initial rates of formation of Forms II and III (Fig. 1, lower panel) that approximately 75% of the SV40 Form II intermediates remain enzymebound during double strand cleavage under these conditions. Table II. Rates of Cleavage of SV40 DNA and SV40 DNA containing a Single Strand Break in the EcoRI Site. Substrate Initial Rate of Hydrolysis pm min SV40 DNA 24 EcoRI-Nicked SV40 DNA 20 Procedures were as in Table I except SV40 DNA was present at 10.4 nm, EcoRInicked SV40 DNA at 9.9 nm, and the concentration of EcoRI endonuclease was 26 pm. The reaction with EcoRI-nicked SV40 DNA also contained 0.8 nm linears. DISCUSSION A reaction mechanism for double strand cleavage consistent with previous results and those presented here is: K,, where I, II, and III represent Forms I, II and III DNA, respectively; K a and 2995

6 Kjj represent equilibrium dissociation constants for E-I and E-II complexes; ki and k2 are first order rate constants for introduction of the first and the second single strand break, while k3 is that for dissociation of the E III complex. With ColEl DNA detectable dissociation of the E-II complex does not occur. Although not shown, the same result was obtained with bacteriophage G4 RFI DNA. Therefore, for these substrates k 3 >>k_ 5. On the other hand, the initial rate data of Fig. 1 (lower panel) indicate that in the case of SV40 DNA 25% of k, the E-II complexes dissociate, implying that _i. = 3 for this substrate. K -5 Furthermore,the demonstration that the endonuclease attacks Form I DNA and the Form II intermediate at equivalent rates (Tables I and II) indicates that k 3 or k 4 is rate limiting for both SV40 and ColEl substrates. This finding is consistent with our previous conclusion that k4 is the slow step in catalysis Introduction of the first single strand break into the EcoRI site of closed circular molecules would make the superhelical free energy available to do work. However, the failure of the E-II complex to dissociate in the case of ColEl and G4 substrates indicates either that the superhelical free energy is insufficient to drive release of enzyme from the intermediate, or alternatively, that relaxation of superhelices is slow relative to introduction of the second single strand break. Furthermore, the substrate dependence of endonuclease mechanism described here cannot be attributed to differing superhelical densities of the substrates since the ColEl and SV40 molecules employed in these experiments have similar superhelical densities It is therefore likely that the mechanistic difference we have observed is a consequence of sequence differences about the EcoRI sites of the substrates employed. We have inspected available sequence information ' ", and the only obvious differences among the substrates involve the amount of purine-pyrimidine 2-fold rotational symmetry extending beyond the EcoRI sites in question: G4 PuPuPuPu GAATTC PyPyPyPy ColEl PuPu GAATTC PyPy SV40 p u GAATTC Py It is possible that the results presented here reflect these varying degrees of extended symmetry. Substrate dependence of cleavage by EcoRI endonuclease was first demonstrated by Thomas and Davis, who observed that the several EcoRI sites of phage lambda DNA are subject to double strand cleavage at unequal rates. We have shown, in addition, that the mechanism of double strand cleavage is sub- 2996

7 strate dependent. Although both phenomena presumably reflect differing nucleotide sequences about the EcoRI site, it is not clear if they are related. However, if a relationship does exist, then dissociation of the E-II complex would be associated with double strand cleavage at "poor" sites. While we have observed free Form II intermediate in reactions involving SV40 DNA, the extent of accumulation of Form II molecules we observe is considerably less than that reported by Ruben et al.. Although the reason for this discrepancy is not apparent, it could reflect the different procedures used for isolation of the endonuclease employed in the two studies ACKNOWLEDGMENT This work was supported by grants from the National Institutes of Health and the National Science Foundation. * To whom correspondence should be sent. REFERENCES 1 Modrich, P. and Zabel, D. (1976) J. Biol. Chem. 2S1, Ruben, G., Spielman, P., Tu, C. D., Jay, E., Siegel, B. and Wu, R. (1977) Nucleic Acids Res._ 4, Abbreviations used:. Form I, superhelical DNA; Form II, DNA containing a single strand break located in the EcoRI site, unless specified otherwise; Form III, linear DNA. RFI, covalently closed circular replicative form I. 4 Rubin, R. A. and Modrich, P. (1977) J. Biol. Chem. 252, Bazaral, M. and Helinski, D. R. (1968) J. Mol. Biol. 36, Reddy, V. B., Thimmappaya, B., Dahr, R., Subramanian, K. N., Zain, B.S., Pan, J., Ghosh, P. K., Celma, M. L. and Weissman, S. M. (1978) Science 200, [ 32 P]ColEl DNA and [ 3 H]SV4O DNA containing Forms I and II molecules were cocentrifuged to equilibrium in a CsCl-ethidium density gradient. Results of the experiment indicated that the superhelical densities of the 2 DNA's were similar, with that of ColEl DNA being slightly greater. 8 Garfin, D. E., Boyer, H. W. and Goodman, H. M. (1975) Nucleic Acids Res. 2, Godson, G. N., Fiddes, J. C., Barrell, B. G. and Sanger, F. (1978) J. Mol. Biol. in press 10 Contreras, R., Volckaert, G., Thys, F., Van de Voorde, A. and Fiers, W. (1977) Nucleic Acids Res. 4, Thomas, M. and Davis, R. W. (1975) J. Mol. Biol. 91, The enzyme used by Ruben et al., was purified by the procedure of Greene et al., which employs the nonionic detergent NP40. We have found that presence of NP40 in reaction mixtures at the 0.02% (w/v) concentration anticipated from the data of Ruben et al., has no effect on the cleavage mechanism. 13 Greene, P. J., Betlach, M. C, Boyer, H. W. and Goodman, H. M. (1974) Methods Mol. Biol. 7,

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