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Functional cooperation between exonucleases and endonucleases—basis for the evolution of restriction enzymes

Functional cooperation between exonucleases and endonucleases—basis for the evolution of... 1888±1896 Nucleic Acids Research, 2003, Vol. 31, No. 7 DOI: 10.1093/nar/gkg275 Functional cooperation between exonucleases and endonucleasesÐbasis for the evolution of restriction enzymes Nidhanapathi K. Raghavendra and Desirazu N. Rao* Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India Received December 20, 2002; Revised and Accepted January 30, 2003 ABSTRACT have been characterised (4). Type IIE restriction enzymes such as NaeI and EcoRII interact with two copies of their Many types of restriction enzymes cleave DNA away recognition sequence, one being the target for cleavage and from their recognition site. Using the type III restric- the other serving as an allosteric effector. Type IIF restriction tion enzyme, EcoP15I, which cleaves DNA 25±27 bp endonucleases such as NgoMIV also interact with two copies away from its recognition site, we provide evidence of their recognition sequence but cleave both sequences in a to show that an intact recognition site on the concerted reaction. Both types IIE and IIF cleave DNA within cleaved DNA sequesters the restriction enzyme and the recognition site. Type IIG enzymes cleave 14±16 bp away and type IIS enzymes cleave 9±13 bp away from the decreases the effective concentration of the recognition site while type IIB enzymes cleave 10±12 bp enzyme. EcoP15I restriction enzyme is shown here away on both sides of the recognition site. This contrasts with to perform only a single round of DNA cleavage. the orthodox type II restriction enzymes that cleave DNA Signi®cantly, we show that an exonuclease activity within the recognition site. is essential for EcoP15I restriction enzyme to per- The consequences of the presence of an intact recognition form multiple rounds of DNA cleavage. This observ- site upon restriction enzyme activity after cleavage of the ation may hold true for all restriction enzymes DNA by the enzyme have not yet been addressed. While cleaving DNA suf®ciently far away from their recog- essentially all restriction enzymes require the divalent metal 2+ nition site. Our results highlight the importance of ion, Mg , for DNA cleavage, types I and III in addition functional cooperation in the modulation of enzyme require ATP and AdoMet. It has also been shown that AdoMet activity. Based on results presented here and other stimulates DNA cleavage by type IIB and type IIG restriction data on well-characterised restriction enzymes, a endonucleases. Restriction enzymes bear little sequence similarity, yet all functional evolutionary hierarchy of restriction of them contain functionally conserved motifs and structurally enzymes is discussed. conserved domains (4). Lack of sequence similarity hinders sequence based phylogenetic analysis and computational modelling. As more R-M systems are found and characterised, INTRODUCTION new ones with novel subunit structures, cofactor requirements and cleavage patterns are still being identi®ed. Although A wide variety of restriction±modi®cation (R-M) systems several investigators have proposed possible evolutionary have been discovered and characterised. They are classi®ed links among various types of restriction enzymes based on based on their subunit composition, cofactor requirement and structural and sequence data (5±7), a clear evolutionary link mode of DNA cleavage (1). Table 1 summarises the charac- teristic features of the main R-M systems. Restriction enzymes among the well-characterised types of restriction enzymes has cleaving DNA in both the type I or type III classes are not been established. dependent upon ATP and S-adenosyl-L-methionine (AdoMet) EcoP15I restriction enzyme (R.EcoP15I), a member of the (2,3). Type III enzymes require two inversely oriented, type III R-M system, is composed of two subunits, restriction asymmetric, unmethylated recognition sites on the same (Res, R) and modi®cation (Mod, M) and recognises an DNA molecule to introduce one double strand break between asymmetric sequence, 5¢-CAGCAG-3¢ (8). The DNA binding the two sites. Type I enzymes are able to cleave supercoiled domain and methyl donor AdoMet binding domain are present DNA containing only one site but, in general, restrict DNA only in the Mod subunit (75 kDa) (9). The ATPase, helicase containing multiple sites more effectively. Type I enzymes and endonuclease domains are present in the Res subunit cleave many thousands of base pairs away from the recogni- (106 kDa) (10). Res cannot bind DNA independent of Mod tion site, while type III enzymes cleave 25±27 bp away from and is degraded when expressed alone (11). The Mod subunit the recognition site. ATP-independent restriction enzymes do alone can methylate DNA at the N position of the second not perform DNA translocation and include all types other adenine in the recognition site (12) and is a dimer, Mod ,in than types I and III. A variety of subtypes belonging to type II solution (13). Two inversely oriented (®¬) unmethylated *To whom correspondence should be addressed. Tel: +91 80 3942538; Fax: +91 80 3600814; Email: dnrao@biochem.iisc.ernet.in Nucleic Acids Research, Vol. 31 No. 7 ã Oxford University Press 2003; all rights reserved Nucleic Acids Research, 2003, Vol. 31, No. 7 1889 Table 1. Characteristic features of R-M systems MTase ENase Site requirement Site of cleavage subunits subunits for cleavage 2+ ATP, AdoMet and Mg -dependent restriction enzymes Type I S, M S, M, R Two 1000±7000 bp Type III M M, R Two 25±27 bp 2+ AdoMet and Mg -dependent restriction enzymes Type IIG M M + R One 14±16 bp Type IIB A, B A, B One 10±12 bp on either side of site 2+ Mg -dependent restriction enzymes Type IIS M R One 9±13 bp Type II M R One Within site M and R refer to methyltransferse and endonuclease subunits, respectively. A and B refer to A chain and B chain, respectively. Distance of cleavage site with respect to recognition site is given. sites are the substrates for cleavage by type III restriction (pH 8.0), 0.25 mM EDTA, 6.4 mM MgCl ,12mM enzymes. The intervening sequence between these sites can be 2-mercaptoethanol] in the presence of 1 mM ATP, except anywhere between 30 and 3000 bp in length (14). Physical where indicated otherwise. This was followed by Proteinase K interaction of enzyme molecules bound to both sites is treatment at 56°C for 1 h [20 mg/ml Proteinase K in 20 mM essential for DNA cleavage to occur. It is proposed that ATP EDTA, 0.5% (w/v) SDS]. The digests were analysed by hydrolysis allows the interaction of the enzyme molecules electrophoresis in TAE at 100 V on 1% (w/v) agarose gels for during the cleavage reaction (15). More recently, the presence 1 h. All gels were documented and DNA bands were of the methyl donor AdoMet has been shown to be mandatory quantitated using UVI Tech gel documentation system. All for restriction (16). assays used a 25 ml reaction volume, except for those with In general, phage DNA after endonuclease restriction is non-speci®c DNA (Fig. 3C), which used a 60 ml reaction further degraded by bacterial exonucleases (17,18). The role volume and 2 h of electrophoresis. of exonucleases in processes such as DNA replication, Puri®cation of EcoP15I restriction enzyme recombination and repair has been well documented in various organisms (19). Although restriction by EcoKI was R.EcoP15I enzyme was puri®ed according to the protocol observed to be reduced in the absence of the RecBCD described previously (16). Enzyme purity was monitored by exonuclease (20), there has been no study demonstrating SDS±polyacrylamide gel electrophoresis (21) and found to be essentiality of exonucleases for the activity of restriction homogeneous. Puri®ed enzyme was checked for ATP- enzymes. In the present study, we have explored possible dependent and AdoMet-independent cleavage of DNA, indi- functional cooperation between exonucleases and endo- cative of enzyme-bound AdoMet. Titration of the enzyme nucleases. The effect of intact recognition sites on cleaved with DNA was accomplished by incubating increasing DNA on restriction enzyme activity has been investigated. amounts of the enzyme with supercoiled pUC19 DNA (2 mg/ Based on our results and the biochemical, structural and 3.37 pmol EcoP15I sites) for 1 h at 37°C in the reaction buffer sequence data of well characterised R-M systems, we propose and analysed by electrophoresis. The minimum amount of a model by which restriction enzymes might have evolved enzyme yielding complete DNA cleavage was considered to through domain duplication, deletion and shuf¯ing to acquire represent a 1:1 ratio of enzyme to EcoP15I site on DNA. the ability to cleave DNA within their recognition site. DNA preparations pUC19 DNA was prepared as described (22) and found to be 95% supercoiled. pUC19 DNA linearised by R.EcoP15I was MATERIALS AND METHODS prepared by incubating supercoiled pUC19 DNA with Chemicals used R.EcoP15I in the presence of 1 mM ATP for 1 h at 37°Cin AdoMet, S-adenosyl-L-homocysteine (AdoHcy), sinefungin, reaction buffer. Linearised DNA was puri®ed using the gel bovine serum albumin, adenosine-5¢-triphosphate (sodium elution kit. R.EcoP15I acts as methyltransferase in the absence salt), ampicillin, HEPES, polyethyleneimine, Coomassie of ATP and in the presence of AdoMet (23). DNA methylated Brilliant Blue R-250 and RNase A were from Sigma by R.EcoP15I was prepared by incubating the supercoiled Chemical Company, USA. Restriction enzymes and exo- pUC19 DNA with excess enzyme in the absence of ATP for 3 h nuclease III were from New England Biolabs, USA. The DNA at 37°C. DNA was checked for complete methylation by gel elution kit was from Amersham Pharmacia Biotech, Asia subjecting it to R.EcoP15I restriction digestion in the presence Paci®c Ltd, Hong Kong. All other reagents used were of the of ATP at 37°C for 1 h. The absence of any linearisation of highest grade. supercoiled DNA was considered as indicative of complete EcoP15I methylation. EcoP15I methylated DNA was further DNA cleavage assay linearised with PstI to yield a linear EcoP15I methylated DNA was incubated with puri®ed R.EcoP15I at 37°C for pUC19 DNA. Non-speci®c DNA was prepared by restriction 60 min in the HEPES reaction buffer [100 mM HEPES digestion of supercoiled pUC19 DNA with HinfI restriction 1890 Nucleic Acids Research, 2003, Vol. 31, No. 7 of cleavage could be limitation of one or other of the reaction component(s). Such a limitation might become more apparent at higher concentrations of the enzyme leading to the observed sub-stoichiometric cleavage seen in phase B. Sub- stoichiometric cleavage could be a consequence of oligomer- isation of enzyme at higher concentrations or shortage of ATP or AdoMet. The concentration of ATP and/or time of incubation could affect the amount of DNA cleaved. Increasing the concentra- tion of ATP in the restriction assay (1±5 mM) or increasing the incubation period up to 3 h, did not seem to have any signi®cant effect on DNA cleavage, at any enzyme concentration used in Figure 1 (data not shown). Recently, it has been shown that the cofactor AdoMet is required for cleavage by type III restriction enzymes and that Figure 1. DNA cleavage as a function of R.EcoP15I concentration in the R.EcoP15I copuri®es with bound AdoMet (16). The molecular absence and presence of exogenous AdoMet. The amount of supercoiled details of the role of AdoMet in cleavage are not yet clear, but pUC19 DNA (7.2 mg) in each assay corresponds to 12.14 pmol EcoP15I it has been suggested that AdoMet binding causes conforma- sites. The DNA cleavage assays were carried out in the HEPES reaction buffer at 37°C for 1 h as described. The amount of cleaved DNA was tional changes in the restriction enzyme which are essential for quantitated from intensities of the product and substrate bands after staining cleavage (16). AdoMet copuri®ed with R.EcoP15I might be the agarose gel with ethidium bromide. The linear phase, indicating suf®cient to support only one round of cleavage (Fig. 1, phase stoichiometric cleavage by R.EcoP15I, is labelled as A, the non-linear phase A). Therefore, the above restriction assays were repeated in of cleavage is labelled as B. The dashed line indicates the assay done with the presence of 20 mM exogenous AdoMet. The progress of supercoiled pUC19 DNA containing 24.28 pmol EcoP15I sites as described in the text. DNA cleavage as a function of R.EcoP15I concentration in the presence of 20 mM exogenous AdoMet is shown in Figure 1 (open circles). As there was no detectable increase in DNA enzyme followed by agarose gel electrophoresis and cleavage in phase A, it would imply that exogenously added puri®cation of the 1419 bp pUC19 DNA HinfI fragment. AdoMet did not increase the catalytic activity of the enzyme. DNA was quanti®ed by absorbance at 260 nm assuming an These results clearly demonstrate that R.EcoP15I does not OD of 1.0 corresponds to 50 mg/ml of double stranded DNA. perform multiple rounds of catalysis in vitro. However, a 10% DNA cleavage in the presence of exonuclease III increase in the amount of DNA cleaved was observed in the non-linear phase B in the presence of 20 mM exogenous These reactions were carried out in Lac buffer (10 mM AdoMet (open circles) relative to assays without exogenous Tris±HCl, pH 8.0, 10 mM KCl, 10 mM MgCl , 0.1 mM AdoMet (closed circles). This 10% increase in cleavage at all EDTA, 0.1 mM dithiothreitol) instead of HEPES reaction enzyme concentrations in phase B indicates that 10% of the buffer for 3 or 6 h as indicated in the text. Fifty units of R.EcoP15I had lost AdoMet and became re-activated upon the exonuclease III (NEB) was added to each reaction at time addition of exogenous AdoMet. zero. We next studied the effect of an increased DNA substrate concentration. Incubation of double the amount of supercoiled pUC19 DNA (24.28 pmol sites) with 11.0 pmol of R.EcoP15I RESULTS AND DISCUSSION for 1 h at 37°C resulted in stoichiometric cleavage of half of DNA cleavage versus R.EcoP15I concentration the pUC19 DNA (11.8 pmol sites) (closed inverted triangle on dashed line in Fig. 1). This indicates that although excess The amount of DNA cleaved by increasing amounts of substrate DNA alleviated the decrease in enzyme activity seen R.EcoP15I was studied to establish the relationship between DNA cleavage activity and enzyme concentration. Super- in phase B of Figure 1, there was still only a single round of coiled pUC19 DNA containing a pair of recognition sites in cleavage by the enzyme. This rules out the possibility of inverse orientation (®¬) was the substrate. Increasing oligomerisation of enzyme at higher concentrations, as the amounts of R.EcoP15I enzyme (1.82±21.84 pmol) were added activity of oligomers would not have been affected by to supercoiled pUC19 DNA (7.2 mg/12.14 pmol EcoP15I increasing substrate DNA concentration. sites) in a standard restriction assay and incubated for1hat To study the effect of DNA concentration upon cleavage by 37°C. The result is shown in Figure 1 (®lled circles). Initially, R.EcoP15I enzyme in phase A of Figure 1, 4.2 pmol of the amount of DNA cleaved is directly proportional to the R.EcoP15I was incubated with supercoiled pUC19 DNA enzyme concentrations (Fig. 1, phase A), indicating that the (4.6±13.9 pmol EcoP15I sites) for 1 h at 37°C. When the R.EcoP15I catalysed reaction is stoichiometric with respect to amount of substrate DNA (4.6 pmol EcoP15I sites) was enzyme concentration and that it performs a single round of slightly more than that of enzyme (4.2 pmol of R.EcoP15I) catalysis in vitro. However, there is a non-linear relationship (Fig. 2A, lane 1), a detectable amount of supercoiled DNA between the amount of DNA cleaved and higher concentra- (~8%) was left uncleaved. Even when the total amount of tions of R.EcoP15I (Fig. 1, phase B). Phase B shows that substrate DNA was in great excess over the enzyme, DNA at concentrations >6.3 pmol of enzyme, there is sub- corresponding to only 4.2 pmol EcoP15I sites was cleaved stoichiometric cleavage. One possibility for the stoichiometric (Fig. 2A, lanes 2 and 3). When an assay containing 4.2 pmol of cleavage observed in phase A as opposed to multiple rounds R.EcoP15I and supercoiled pUC19 DNA containing 4.6 pmol Nucleic Acids Research, 2003, Vol. 31, No. 7 1891 by ATP hydrolysis. ATP hydrolysis by R.EcoPI was shown to continue even after DNA cleavage (S.Saha and D.N.Rao, unpublished results). It has been reported that R.EcoP15I binds both methylated and unmethylated recognition sites and hydrolyses comparable amounts of ATP in a manner inde- pendent of DNA length and restriction (15). One interpretation of these ®ndings could be that continued ATP hydrolysis after cleavage leads to a low dissociation or `off' rate of the enzyme from the cleaved DNA. The lack of increase in DNA cleavage in our assays carried out over 3 h supports this interpretation. In addition, binding of any free restriction enzyme to cleaved DNA, thus decreasing the concentration of free enzyme in the assay, could elicit site-speci®c ATPase activity and a low off rate of enzyme from DNA. To test if cleaved DNA was responsible for the decrease in R.EcoP15I concentration and thus its restriction activity, supercoiled pUC19 substrate DNA (4.6 pmol EcoP15I sites) was incubated with R.EcoP15I enzyme (4.2 pmol) in the presence of either increasing amounts of R.EcoP15I cleaved pUC19 DNA or R.EcoP15I methylated pUC19 DNA or non- speci®c DNA (1419 bp HinfI fragment of pUC19). DNA cleavage by R.EcoP15I decreased (as evidenced by the increase in uncleaved supercoiled DNA) in a dose-dependent manner in the presence of R.EcoP15I cleaved DNA (Fig. 3A) or R.EcoP15I methylated DNA (Fig. 3B) but not in the presence of non-speci®c DNA (Fig. 3C). Hence, the seques- tration of enzyme by DNA is dependent upon there being a Figure 2. DNA cleavage by R.EcoP15I as a function of supercoiled pUC19 recognition site on the DNA. These results demonstrate that DNA concentration. (A) In the absence of exogenous AdoMet. Increasing binding of enzyme molecules to cleaved DNA or methylated amounts of supercoiled pUC19 DNA were incubated with R.EcoP15I DNA decreases the restriction activity of the enzyme. As (4.2 pmol) and DNA cleavage assays were carried out as described. Lane 1, demonstrated previously with methylated and unmethylated 4.63 pmol EcoP15I sites; lane 2, 9.27 pmol EcoP15I sites and lane 3, 13.9 pmol EcoP15I sites. (B) Time course of cleavage. Supercoiled pUC19 DNA (15), DNA cleaved by EcoP15I restriction enzyme and DNA (4.63 pmol EcoP15I sites) was incubated with R.EcoP15I (4.2 pmol) methylated DNA (Fig. 3A and B) seem to elicit ATPase in the absence of exogenous AdoMet for different periods of time and the activity of enzyme and result in a low off rate of enzyme from assay performed as described. Lane 1, 20 min; lane 2, 40 min; lane 3, DNA. Sequestration of enzyme by cleaved DNA thus leads to 60 min and lane 4, 90 min. (C) In the presence of exogenous AdoMet a decrease in the effective number of enzyme molecules. (20 mM). Increasing amounts of supercoiled pUC19 DNA were incubated with R.EcoP15I (4.2 pmol) and DNA cleavage assays performed as Non-linearity in the amount of DNA cleaved at high described. Lane 1, 4.63 pmol EcoP15I sites; lane 2, 9.27 pmol EcoP15I concentrations of R.EcoP15I (Fig. 1, phase B) could, there- sites and lane 3, 13.9 pmol EcoP15I sites. Lanes U and M, uncut super- fore, be a consequence of the accumulation of cleaved DNA coiled pUC19 DNA and marker DNA fragments (sizes given to the right in sequestering free enzyme molecules. This would require that base pairs). I and II represent supercoiled and linearised pUC19 DNA, respectively. the recognition site be exposed on the accumulating cleaved DNA to allow site-speci®c sequestration of free enzyme molecules. In the absence of multiple rounds of catalysis, as of EcoP15I sites was carried out, the kinetics of cleavage evident in Figure 1 (phase A), it can be postutated that the showed that most of the DNA was cleaved within 20 min enzyme molecules once they cleave stay bound to DNA but (Fig. 2B, lane 1). The effect of exogenous AdoMet was tested not necessarily at the recognition site. Absence of turnover by by repeating the above assays in the presence of 20 mM R.EcoP15I enzyme, demonstrated here, combined with an exogenous AdoMet. As can be seen in Figure 2C, the extent of earlier observation of a weak footprint in the presence of both cleavage was not signi®cantly different compared with that in ATP and AdoMet (28) support this interpretation. Doubling Figure 2A, indicating that addition of exogenous AdoMet had the amount of supercoiled pUC19 DNA in an assay would no appreciable effect on the extent of DNA cleavage. decrease the proportion of accumulating cleaved DNA and also enhance the binding of enzyme molecules to supercoiled Sequestration of the enzyme DNA. As expected, stoichiometric cleavage was restored in R.EcoP15I has been shown to hydrolyse ATP in the presence the assay when double the amount of supercoiled DNA was of a DNA recognition site (15). Similar observations were used (Fig. 1, closed inverted triangle on dashed line). obtained with the closely related type III enzyme R.EcoPI Sequestration of the enzyme by cleaved DNA resulting in a (24), whose Res subunit is almost identical to that of single round of cleavage (Fig. 3A and Fig. 1, phase A) with R.EcoP15I (25). Mutations in the helicase motifs of R.EcoPI R.EcoP15I may also explain the single round of cleavage affecting ATP hydrolysis abolished DNA cleaving ability observed with the functionally related type I restriction (26,27). DNA translocation during the cleavage reaction enzymes (29). It is possible that other types of restriction catalysed by these enzymes has been proposed to be assisted enzymes that cleave DNA suf®ciently far away from their 1892 Nucleic Acids Research, 2003, Vol. 31, No. 7 resistant to cleavage by cognate restriction enzymes. Sequestration of restriction enzyme by cleaved DNA per se provides the phage with a restriction alleviation mechanism. A somewhat similar kind of sequestration phenomenon has been reported when bacteriophage T7 infects Escherichia coli and bacteriophages PBS-1 and -2 infect Bacillus subtilis. The gene 0.3 protein of phage T7, also known as ocr (overcome classical restriction) (31) competitively inhibits type I DNA restriction enzymes by preventing them from binding to their DNA target (32). Biochemical observations that ocr is a competitive inhibitor of DNA binding (33) and that it contains a large excess of negatively charged amino acids, prompted the suggestion that ocr was not only a polyanionic inhibitor but that it could also be a mimic of an extended DNA structure (34). The atomic structure of ocr reveals remarkable molecular mimicry of B-form DNA (35). Ugi (UDG inhibitor) is an early gene product of B.subtilis bacteriophage PBS-1 and -2 and a protein mimic of DNA. These phages have genomes contain- ing uracil and UGI protects the genome by forming an extremely speci®c and physiologically irreversible complex with the host uracil DNA glycosylase (UDG) (36). Clearly, bacteriophages have a number of ways of regulating the activity of cellular nucleases. Exonuclease activity is essential for multiple rounds of DNA cleavage by EcoP15I restriction enzyme Type I restriction enzymes have been shown to perform only one round of cleavage reaction in vitro, i.e. each enzyme molecule cuts the DNA once only. No turnover of the enzyme Figure 3. Sequestration of EcoP15I restriction enzyme. (A) Supercoiled can be measured with respect to its cleavage function or with pUC19 DNA (4.63 pmol EcoP15I sites) was incubated with R.EcoP15I respect to its capacity to bind unmodi®ed DNA to nitrocellu- (4.2 pmol) in the presence of increasing amounts of R.EcoP15I cleaved lose membrane (29). Therefore, it has been an interesting pUC19 DNA. Lane 1, 1.375 mg; lane 2, 2.75 mg; lane 3, 5.5 mg and lane 4, 11 mg. (B) Supercoiled pUC19 DNA (4.63 pmol EcoP15I sites) was incu- question, whether one is justi®ed calling these proteins bated with R.EcoP15I (4.2 pmol) in the presence of increasing amounts of `enzymes' (37). The results presented in Figures 1 and 2 R.EcoP15I methylated pUC19 DNA. Lane 1, 2.75 mg and lane 2, 5.5 mg. indicate that type III restriction enzymes also show single (C) Supercoiled pUC19 DNA (4.63 pmol EcoP15I sites) was incubated with turnover kinetics in vitro. The ability of R.EcoP15I to perform R.EcoP15I (4.2 pmol) in the presence of increasing quantities of non- multiple rounds of catalysis was tested as follows. When the speci®c DNA. Lane 1, 2.75 mg; lane 2, 5.5 mg; lane 3, 11 mg and lane 4, 22 mg. I, II and Ns represent supercoiled pUC19 DNA, linearised pUC19 restriction assay is performed in the presence of exonuclease DNA and 1419 bp pUC19 DNA HinfI fragment, respectively. Lane C in all III, DNA cleaved by R.EcoP15I would be further digested by three panels: supercoiled pUC 19 DNA (4.63 pmol EcoP15I sites) incubated the exonuclease. The removal of cleaved DNA should release with R.EcoP15I (4.2 pmol). Lanes U and M, uncut supercoiled pUC19 R.EcoP15I to perform a further round of catalysis. DNA and marker DNA fragments, respectively. Supercoiled pUC19 DNA (4.5 mg/7.6 pmol EcoP15I sites) was incubated with R.EcoP15I (3.8 pmol) and 50 U of recognition site are also sequestered by cleaved DNA and may exonuclease III (where indicated) for 3 h at 37°C. Under exhibit slower cleavage kinetics. stoichiometric conditions, R.EcoP15I should cleave only Phage restriction is not generally absolute and phage 2.25 mg (3.8 pmol EcoP15I sites) of supercoiled pUC19 genomes that do escape cleavage normally become modi®ed. DNA. However, the cleavage of 4.5 mg of DNA (7.6 pmol Progeny of such modi®ed phages are protected against EcoP15I sites) would indicate a second round of catalysis by restriction by bacteria with R-M system of the same speci®city the enzyme. In this assay, linearisation of 2.25 mg DNA was (30). The molecular basis of this phenomenon has so far observed both in the absence and presence of 20 mM AdoMet remained obscure. The sequestration phenomenon described and in the absence of exonuclease III (Fig. 4A, lanes 1 and 2). here provides a clue to understanding how non-resistant In the assays performed in the presence of exonuclease III, phages acquire resistance to a given R-M system. Infection of DNA linearised by R.EcoP15I will be subjected to complete a bacterium with an `overwhelming number' of non-resistant degradation by exonuclease III, hence the amount of super- phages might result in a scenario where all the intracellular coiled pUC19 DNA left at the end of the assay would indicate restriction enzyme is involved in stoichiometric cleavage of the amount of DNA cleaved by R.EcoP15I. Complete infecting phage and gets sequestered by the cleaved phage disappearance of DNA would indicate cleavage of 4.5 mg DNA. Such a situation provides the remaining phages with an supercoiled pUC19 DNA by 3.8 pmol of R.EcoP15I. As can oppurtunity to be completely methylated by the cognate be seen from Figure 4A (lane 3) the enzyme showed complete methyltransferase (MTase) before the endonuclease acts on cleavage of 4.5 mg supercoiled pUC19 DNA only in the them. The phages with methylated recognition sites are presence of exonuclease III. As a control, we show that the Nucleic Acids Research, 2003, Vol. 31, No. 7 1893 that cleave DNA suf®ciently far outside their recognition site. An earlier report demonstrating reduction in restriction by EcoKI in the absence of the RecBCD exonuclease supports this suggestion (20). Assistance of multiple rounds of catalysis by the restriction enzyme in the presence of exonuclease is characterised by a time lag between DNA cleavage and dissociation of the restriction enzyme from the cleaved DNA. The time lag provides an opportunity for the infecting phage to acquire methylation by the cognate methyltransferase before the restriction enzyme acts on them under situations discussed in the previous section. Implications for the evolution of R-M enzymes Sequence comparisons and biochemical experiments in con- junction with site-directed mutagenesis studies have estab- lished the fact that the Mod subunit of type III R-M systems contains the target recognition domain (TRD) (9,13,23). The Res subunit contains the characteristic endonuclease motif (PD¼D/E-X-K) involved in phosphodiester bond cleavage (27), and the helicase motifs responsible for DNA trans- Figure 4. Multiple rounds of DNA cleavage by EcoP15I restriction enzyme location (driven by ATP hydrolysis) (26). Consequently, the in the presence of exonuclease III. (A) Supercoiled pUC19 DNA (7.6 pmol amino acid residues involved in target recognition and the EcoP15I sites) was incubated with EcoP15I restriction enzyme (3.8 pmol) active site residues involved in DNA cleavage are likely to be in the absence or presence of 20 mM exogenous AdoMet or exonuclease III spatially distant in the holoenzyme and thus lead to cleavage or both as indicated on top of each lane. (B) Supercoiled pUC19 DNA was incubated with R.EcoP15I (3.8 pmol) for 3 or 6 h at 37°C in the presence of some distance from the recognition site. This is probably true exonuclease III. Lane 1, 11.4 pmol EcoP15I sites; lane 2, 11.4 pmol for other restriction enzymes cleaving DNA outside their EcoP15I sites; lane 3, 15.2 pmol EcoP15I sites; lanes U and M, uncut recognition site (39). In contrast, these sequence recognition supercoiled pUC19 DNA and marker DNA fragments, respectively. and cleavage residues are in close proximity in the tertiary structures of orthodox type II enzymes, allowing cleavage to occur within the recognition site (40). supercoiled DNA was not affected by exonuclease III in the Since the amino acid sequences of R-M enzymes do not absence of EcoP15I restriction enzyme (Fig. 4A, lane 4). This share signi®cant similarity and are not easily amendable to demonstrated that removal of DNA, once cleaved by standard alignment procedures, it has been dif®cult to ®nd an R.EcoP15I, allowed a second round of catalysis by the evolutionary link among R-M systems through sequence restriction enzyme. analysis alone. Despite limited sequence similarities, from the To check for multiple rounds of catalysis, supercoiled crystal structures of restriction enzymes and methyltrans- pUC19 DNA (11.4 pmol EcoP15I sites) was incubated with ferases and predicted secondary structures of other R-M R.EcoP15I (3.8 pmol) and 50 U of exonuclease III for 3 h at enzymes, the presence of common folds responsible for 37°C. As evident from Figure 4B (lane 1) 2.25 mg supercoiled enzyme activity has been shown (41±47). It has been DNA was left uncleaved, indicating only two rounds of suggested that spatial conservation of side-chain locations cleavage by the enzyme. The above assay was repeated by plays a dominant role and that the primary sequence of increasing the incubation period from 3 to 6 h at 37°C. conserved active site residues is less important when searching Disappearance of the entire supercoiled DNA in Figure 4B for similarities among restriction enzymes (5). This would (lane 2) clearly shows that a third round of DNA cleavage is suggest that an evolutionary link among these enzymes could possible. A fourth round of catalysis was also demonstrated by be deduced from their catalytic ef®ciency (as they share the incubating supercoiled pUC19 DNA (15.2 pmol EcoP15I same folds) in conjunction with sequence analysis. Based on sites) for 6 h at 37°C with 3.8 pmol of R.EcoP15I in the the results described here, which indicate that the ef®ciency of presence of exonuclease III (Fig. 4B, lane 3). restriction enzymes increases with decreasing af®nity for These results indicate that the type III restriction enzyme cleaved DNA, we propose a functional evolutionary hierarchy R.EcoP15I, in the presence of an exonuclease, can perform for R-M systems illustrated schematically in Figure 5. Our multiple rounds of DNA cleavage. This is the ®rst report scheme suggests that restriction enzymes are under pressure to demonstrating functional cooperation between a restriction evolve the ability to cut within their recognition site rather endonuclease and an exonuclease and this may re¯ect the than cutting away from the site and, as a consequence, having situation in vivo. Such functional cooperation between DNA to rely upon other enzymes to release them from the DNA for transaction proteins has been reported recently (38). The further catalytic cycles. unexpected functional cooperation between an exonuclease Restriction enzymes have structural homology with and an endonuclease provides a highly ef®cient mechanism enzymes involved in DNA repair and recombination which for an increase in the ef®ciency of the R.EcoP15I restriction also perform phosphodiester bond cleavage (5,46,48±51). enzyme. Our observations make it attractive to suggest that Type I R-M systems (e.g. R.EcoKI) have high structural exonucleases might play a similar role in the in vivo activity of complexities and can be considered as the most suitable forms other restriction enzymes, such as type I restriction enzymes, for the evolution of new types of R-M systems through domain 1894 Nucleic Acids Research, 2003, Vol. 31, No. 7 enzymes. One such form would be a type IIB R-M enzyme. Type IIB enzymes (e.g. BcgI) have two polypeptides, one containing only TRDs, the other containing both MTase and ENase domains (56). The polypeptide with TRDs associates with the polypeptide having both ENase and MTase domains to form a functional type IIB restriction enzyme that can also methylate DNA. Alternatively, duplication of a domain such as TRD in a type IIG enzyme is probable. A polypeptide with a duplicated TRD allows simultaneous segregation of the two TRDs. Segregation of one TRD with MTase domains would produce a protein resembling the MTase of type IIS R-M enzymes, while segregation of other TRD with the ENase domain would produce a protein resembling a type IIS Figure 5. Schematic representation of a functional evolutionary hierarchy restriction enzyme. Based on the biochemical properties of the of R-M systems. Different colours indicate different domains. All domains prototype type IIG enzyme, Eco57I, it was suggested in single polypeptide are represented as fused domains. (Purple, target previously that the enzyme might be regarded as an inter- recognition domain; green, endonuclease domain; yellow, AdoMet binding mediate type re¯ecting evolutionary link between the enzymes domain; red, helicase domain; black, methyltransferase catalytic domain.) of types III and IIS (55). Later it was argued that type IIG R-M systems might have evolved from a progenitor formed by the fusion of a single strand-speci®c MTase of type IIS with a shuf¯ing, deletions, insertions and duplication (2,52). The DNA endonuclease (57). However, acquisition of MTase HsdS subunit determines DNA speci®city and it is composed domains, which are not related to restriction activity, by an of two separate DNA binding domains, each recognising one endonuclease to form the predecessor of type IIG seems rather speci®c part of a non-palindromic sequence. The HsdM unlikely. Type IIS enzymes (FokI) have DNA binding domain subunit contains the AdoMet binding site and the catalytic site in both the modi®cation and the restriction subunits. A type for DNA methylation (53). The HsdR subunit is essential for IIS system has two MTases, one for each strand, which could restriction and contains a set of seven amino acid sequence also be a consequence of these genetic rearrangements. An motifs typical of the superfamily of helicases, the so-called endonuclease capable of recognising DNA without requiring DEAD box proteins, involved in ATP binding and ATP- association with an MTase allows DNA cleavage independent dependent DNA translocation (54). Fusion of one DNA of AdoMet. However, both types IIB and IIS cleave outside binding domain of a type I HsdS subunit, recognising one their recognition sequence. FokI endonuclease supplemented speci®c part of a non-palindromic sequence, with a type I with a DNA binding-de®cient mutant of FokI endonuclease HsdM subunit, would, in conjunction with rearrangements of the tertiary structure, result in a subunit similar to the Mod containing an active catalytic domain was shown to increase the rate of DNA cleavage 10±20-fold relative to the wild-type subunits of type III R-M enzymes. This would be a step FokI alone (58). This increased cleavage is possibly a towards increasing the ef®ciency of the enzyme with now only consequence of an increased off rate from cleaved DNA for one subunit performing the functions of two subunits, HsdS the heterodimer (wild-type + mutant) compared with the and HsdM. However, both types I and III restriction enzymes homodimer (wild-type). Earlier quantitative evaluation of perform ATP and AdoMet-dependent DNA cleavage and cleave outside the recognition sequence (Table 1). In type III sequence and structure compatibility has shown that cleavage R-M enzymes, ATP hydrolysis and DNA translocation are domain of homologues of FokI (type IIS) have a higher degree of similarity to type I and type III R-M systems than to essential for oligomerisation of enzyme molecules involved in orthodox type II enzymes such as EcoRV and PvuII. DNA cleavage. Although type I restriction enzymes possess Furthermore, the cleavage domain of FokI is apparently ATPase activity and have been shown to translocate DNA, evolutionarily older than type II restriction enzymes (51). there has been evidence from atomic force microscopy Amino acid residues involved in site recognition and indicating that type I R-M complexes interact and oligomerise even in the absence of ATP-hydrolysis (54). phosphodiester bond cleavage are spatially separated in type Fusion of the Mod and Res subunits of type III enzymes, IIS enzymes. Any change(s) bringing these residues into close accompanied by deletion of the helicase domains, would proximity (in tertiary structure) might have resulted in probably result in type IIG enzymes (e.g. Eco57I) (53) that orthodox type II enzymes. Orthodox type II restriction enzymes have amino acid residues involved in recognition cleave DNA independent of ATP. Type IIG enzymes would and cleavage in close proximity in tertiary structure and cleave thus have four domains, namely TRD, AdoMet-binding DNA within the recognition site (4). These are the most domain, MTase catalytic domain and an endonuclease (ENase) domain in a single polypeptide chain. Type IIG ef®cient restriction enzymes with respect to DNA cleavage. enzymes also cleave DNA in an AdoMet-dependent manner Dissociation of these enzymes from cleaved DNA can be (55). Interestingly, association of endonuclease domains with expected to be faster than that of restriction enzymes, which MTase catalytic domains (type I, type III, type IIG, type IIB) cleave DNA away from the recognition site. The evolutionary hierarchy described here re¯ects the fact that although the leads to requirement for AdoMet in DNA cleavage (3,16,56). enzymes need to maintain high af®nity for their substrates, A type IIG restriction enzyme could give rise to a number of variants. Deletion of one of the four domains with the fusion of they should have low af®nity for their products to be remaining three domains can result in four different forms of functionally ef®cient. Nucleic Acids Research, 2003, Vol. 31, No. 7 1895 12. Meisel,A., Kru È ger,D.H. and Bickle,T.A. (1991) M.EcoP15 methylates It must be mentioned that the above model does not take the second adenine in its recognition sequence. Nucleic Acids Res., 19, into consideration a lot of variants that might have appeared between and within each step of the functional evolutionary 13. Ahmad,I. and Rao,D.N. (1994) Interaction of EcoP15I DNA hierarchy. Identi®cation and characterisation of new restric- methyltransferase with oligonucleotides containing the asymmetric sequence 5¢-CAGCAG-3¢. J. Mol. Biol., 242, 378±388. tion enzymes such as HaeIV (59) and AloI (60) provide 14. Meisel,A., Bickle,T.A., Kruger,D.H. and Schroeder,C. (1992) Type III enzymes that share properties with more than one existing restriction enzymes need two inversely oriented recognition sites for type. As new R-M systems are characterised, it is likely that DNA cleavage. Nature, 355, 467±469. the biochemical properties of the various types of enzymes 15. Meisel,A., Mackeldanz,P., Bickle,T.A., Kru È ger,D.H. and Schroeder,C. will eventually represent a continuum. (1995) Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-speci®c ATP hydrolysis. EMBO J., 14, Collectively, our data demonstrate that the type III EcoP15I 2958±2966. restriction enzyme is sequestered by the intact recognition site 16. Bist,P., Srivani,S., Krishnamurthy,V., Asha,A., Chandrakala,B. and remaining after DNA cleavage. The presence of cleaved DNA Rao,D.N. (2001) S-adenosyl-L-methionine is required for DNA cleavage with an intact recognition site decreases the effective by type III restriction enzymes. J. Mol. Biol., 310, 93±109. concentration of the restriction enzymeÐan observation that 17. Simmon,V.F. and Lederberg,S. (1972) Degradation of bacteriophage lambda deoxyribonucleic acid after restriction by Escherichia coli K-12. has implications for the evolution of type III R-M systems and J. Bacteriol., 112, 161±169. also for all restriction enzymes which cleave at a distance from 18. Brammar,W.J., Murray,N.E. and Winton,S. (1974) Restriction of lambda their recognition site. The type III restriction enzymes depend trp bacteriophages by Escherichia coli K. J. Mol. Biol., 90, 633±647. on exonucleases to perform multiple rounds of catalysis and to 19. Shevelev,I.V. and Hu È bscher,U. (2002) The 3¢±5¢ exonucleases. Nature Rev. Mol. Cell Biol., 3, 364±376. act as ef®cient endonucleases. The functional evolutionary 20. Salaj-Smic,E., Marsic,N., Trgovcevic,Z. and Lloyd,R.G. (1997) hierarchy proposed here suggests that product release after Modulation of EcoKI restriction in vivo: role of the lambda Gam protein DNA cleavage, independent of exonuclease, has been the and plasmid metabolism. J. Bacteriol., 179, 1852±1856. driving force for the evolution of ef®cient restriction enzymes. 21. Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680±685. 22. Sambrook,J. and Russell,D.W. (2001) Molecular Cloning. A Laboratory Manual, 3rd Edn. Cold Spring Harbor Laboratory Press, New York, NY. ACKNOWLEDGEMENTS 23. Hadi,S.M., Ba Èchi,B., Iida,S. and Bickle,T.A. (1983) DNA restriction- modi®cation enzymes of phage P1 and plasmid p15B. Subunit functions N.K.R. acknowledges Shashi Bhusan Pandit, S. Varadarajan and structural homologies. J. Mol. Biol., 165, 19±34. and Surbhi Gupta for stimulating discussions, and Pradeep 24. Saha,S. and Rao,D.N. (1995) ATP hydrolysis is required for DNA Bist and S. 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E.coli mismatch repair and relationship of MutH to restriction 60. Cesnaviciene,E., Petrusyte,M., Kazlauskiene,R., Maneliene,Z., endonucleases EMBO J., 17, 1526±1534. Timinskas,A., Lubys,A. and Janulaitis,A. (2001) Characterization of 49. Aravind,L., Makarova,K.S. and Koonin,E.V. (2000) Holliday junction AloI, a restriction±modi®cation system of a new type. J. Mol. Biol., 314, resolvases and related nucleases: identi®cation of new families, 205±216. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Functional cooperation between exonucleases and endonucleases—basis for the evolution of restriction enzymes

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1888±1896 Nucleic Acids Research, 2003, Vol. 31, No. 7 DOI: 10.1093/nar/gkg275 Functional cooperation between exonucleases and endonucleasesÐbasis for the evolution of restriction enzymes Nidhanapathi K. Raghavendra and Desirazu N. Rao* Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India Received December 20, 2002; Revised and Accepted January 30, 2003 ABSTRACT have been characterised (4). Type IIE restriction enzymes such as NaeI and EcoRII interact with two copies of their Many types of restriction enzymes cleave DNA away recognition sequence, one being the target for cleavage and from their recognition site. Using the type III restric- the other serving as an allosteric effector. Type IIF restriction tion enzyme, EcoP15I, which cleaves DNA 25±27 bp endonucleases such as NgoMIV also interact with two copies away from its recognition site, we provide evidence of their recognition sequence but cleave both sequences in a to show that an intact recognition site on the concerted reaction. Both types IIE and IIF cleave DNA within cleaved DNA sequesters the restriction enzyme and the recognition site. Type IIG enzymes cleave 14±16 bp away and type IIS enzymes cleave 9±13 bp away from the decreases the effective concentration of the recognition site while type IIB enzymes cleave 10±12 bp enzyme. EcoP15I restriction enzyme is shown here away on both sides of the recognition site. This contrasts with to perform only a single round of DNA cleavage. the orthodox type II restriction enzymes that cleave DNA Signi®cantly, we show that an exonuclease activity within the recognition site. is essential for EcoP15I restriction enzyme to per- The consequences of the presence of an intact recognition form multiple rounds of DNA cleavage. This observ- site upon restriction enzyme activity after cleavage of the ation may hold true for all restriction enzymes DNA by the enzyme have not yet been addressed. While cleaving DNA suf®ciently far away from their recog- essentially all restriction enzymes require the divalent metal 2+ nition site. Our results highlight the importance of ion, Mg , for DNA cleavage, types I and III in addition functional cooperation in the modulation of enzyme require ATP and AdoMet. It has also been shown that AdoMet activity. Based on results presented here and other stimulates DNA cleavage by type IIB and type IIG restriction data on well-characterised restriction enzymes, a endonucleases. Restriction enzymes bear little sequence similarity, yet all functional evolutionary hierarchy of restriction of them contain functionally conserved motifs and structurally enzymes is discussed. conserved domains (4). Lack of sequence similarity hinders sequence based phylogenetic analysis and computational modelling. As more R-M systems are found and characterised, INTRODUCTION new ones with novel subunit structures, cofactor requirements and cleavage patterns are still being identi®ed. Although A wide variety of restriction±modi®cation (R-M) systems several investigators have proposed possible evolutionary have been discovered and characterised. They are classi®ed links among various types of restriction enzymes based on based on their subunit composition, cofactor requirement and structural and sequence data (5±7), a clear evolutionary link mode of DNA cleavage (1). Table 1 summarises the charac- teristic features of the main R-M systems. Restriction enzymes among the well-characterised types of restriction enzymes has cleaving DNA in both the type I or type III classes are not been established. dependent upon ATP and S-adenosyl-L-methionine (AdoMet) EcoP15I restriction enzyme (R.EcoP15I), a member of the (2,3). Type III enzymes require two inversely oriented, type III R-M system, is composed of two subunits, restriction asymmetric, unmethylated recognition sites on the same (Res, R) and modi®cation (Mod, M) and recognises an DNA molecule to introduce one double strand break between asymmetric sequence, 5¢-CAGCAG-3¢ (8). The DNA binding the two sites. Type I enzymes are able to cleave supercoiled domain and methyl donor AdoMet binding domain are present DNA containing only one site but, in general, restrict DNA only in the Mod subunit (75 kDa) (9). The ATPase, helicase containing multiple sites more effectively. Type I enzymes and endonuclease domains are present in the Res subunit cleave many thousands of base pairs away from the recogni- (106 kDa) (10). Res cannot bind DNA independent of Mod tion site, while type III enzymes cleave 25±27 bp away from and is degraded when expressed alone (11). The Mod subunit the recognition site. ATP-independent restriction enzymes do alone can methylate DNA at the N position of the second not perform DNA translocation and include all types other adenine in the recognition site (12) and is a dimer, Mod ,in than types I and III. A variety of subtypes belonging to type II solution (13). Two inversely oriented (®¬) unmethylated *To whom correspondence should be addressed. Tel: +91 80 3942538; Fax: +91 80 3600814; Email: dnrao@biochem.iisc.ernet.in Nucleic Acids Research, Vol. 31 No. 7 ã Oxford University Press 2003; all rights reserved Nucleic Acids Research, 2003, Vol. 31, No. 7 1889 Table 1. Characteristic features of R-M systems MTase ENase Site requirement Site of cleavage subunits subunits for cleavage 2+ ATP, AdoMet and Mg -dependent restriction enzymes Type I S, M S, M, R Two 1000±7000 bp Type III M M, R Two 25±27 bp 2+ AdoMet and Mg -dependent restriction enzymes Type IIG M M + R One 14±16 bp Type IIB A, B A, B One 10±12 bp on either side of site 2+ Mg -dependent restriction enzymes Type IIS M R One 9±13 bp Type II M R One Within site M and R refer to methyltransferse and endonuclease subunits, respectively. A and B refer to A chain and B chain, respectively. Distance of cleavage site with respect to recognition site is given. sites are the substrates for cleavage by type III restriction (pH 8.0), 0.25 mM EDTA, 6.4 mM MgCl ,12mM enzymes. The intervening sequence between these sites can be 2-mercaptoethanol] in the presence of 1 mM ATP, except anywhere between 30 and 3000 bp in length (14). Physical where indicated otherwise. This was followed by Proteinase K interaction of enzyme molecules bound to both sites is treatment at 56°C for 1 h [20 mg/ml Proteinase K in 20 mM essential for DNA cleavage to occur. It is proposed that ATP EDTA, 0.5% (w/v) SDS]. The digests were analysed by hydrolysis allows the interaction of the enzyme molecules electrophoresis in TAE at 100 V on 1% (w/v) agarose gels for during the cleavage reaction (15). More recently, the presence 1 h. All gels were documented and DNA bands were of the methyl donor AdoMet has been shown to be mandatory quantitated using UVI Tech gel documentation system. All for restriction (16). assays used a 25 ml reaction volume, except for those with In general, phage DNA after endonuclease restriction is non-speci®c DNA (Fig. 3C), which used a 60 ml reaction further degraded by bacterial exonucleases (17,18). The role volume and 2 h of electrophoresis. of exonucleases in processes such as DNA replication, Puri®cation of EcoP15I restriction enzyme recombination and repair has been well documented in various organisms (19). Although restriction by EcoKI was R.EcoP15I enzyme was puri®ed according to the protocol observed to be reduced in the absence of the RecBCD described previously (16). Enzyme purity was monitored by exonuclease (20), there has been no study demonstrating SDS±polyacrylamide gel electrophoresis (21) and found to be essentiality of exonucleases for the activity of restriction homogeneous. Puri®ed enzyme was checked for ATP- enzymes. In the present study, we have explored possible dependent and AdoMet-independent cleavage of DNA, indi- functional cooperation between exonucleases and endo- cative of enzyme-bound AdoMet. Titration of the enzyme nucleases. The effect of intact recognition sites on cleaved with DNA was accomplished by incubating increasing DNA on restriction enzyme activity has been investigated. amounts of the enzyme with supercoiled pUC19 DNA (2 mg/ Based on our results and the biochemical, structural and 3.37 pmol EcoP15I sites) for 1 h at 37°C in the reaction buffer sequence data of well characterised R-M systems, we propose and analysed by electrophoresis. The minimum amount of a model by which restriction enzymes might have evolved enzyme yielding complete DNA cleavage was considered to through domain duplication, deletion and shuf¯ing to acquire represent a 1:1 ratio of enzyme to EcoP15I site on DNA. the ability to cleave DNA within their recognition site. DNA preparations pUC19 DNA was prepared as described (22) and found to be 95% supercoiled. pUC19 DNA linearised by R.EcoP15I was MATERIALS AND METHODS prepared by incubating supercoiled pUC19 DNA with Chemicals used R.EcoP15I in the presence of 1 mM ATP for 1 h at 37°Cin AdoMet, S-adenosyl-L-homocysteine (AdoHcy), sinefungin, reaction buffer. Linearised DNA was puri®ed using the gel bovine serum albumin, adenosine-5¢-triphosphate (sodium elution kit. R.EcoP15I acts as methyltransferase in the absence salt), ampicillin, HEPES, polyethyleneimine, Coomassie of ATP and in the presence of AdoMet (23). DNA methylated Brilliant Blue R-250 and RNase A were from Sigma by R.EcoP15I was prepared by incubating the supercoiled Chemical Company, USA. Restriction enzymes and exo- pUC19 DNA with excess enzyme in the absence of ATP for 3 h nuclease III were from New England Biolabs, USA. The DNA at 37°C. DNA was checked for complete methylation by gel elution kit was from Amersham Pharmacia Biotech, Asia subjecting it to R.EcoP15I restriction digestion in the presence Paci®c Ltd, Hong Kong. All other reagents used were of the of ATP at 37°C for 1 h. The absence of any linearisation of highest grade. supercoiled DNA was considered as indicative of complete EcoP15I methylation. EcoP15I methylated DNA was further DNA cleavage assay linearised with PstI to yield a linear EcoP15I methylated DNA was incubated with puri®ed R.EcoP15I at 37°C for pUC19 DNA. Non-speci®c DNA was prepared by restriction 60 min in the HEPES reaction buffer [100 mM HEPES digestion of supercoiled pUC19 DNA with HinfI restriction 1890 Nucleic Acids Research, 2003, Vol. 31, No. 7 of cleavage could be limitation of one or other of the reaction component(s). Such a limitation might become more apparent at higher concentrations of the enzyme leading to the observed sub-stoichiometric cleavage seen in phase B. Sub- stoichiometric cleavage could be a consequence of oligomer- isation of enzyme at higher concentrations or shortage of ATP or AdoMet. The concentration of ATP and/or time of incubation could affect the amount of DNA cleaved. Increasing the concentra- tion of ATP in the restriction assay (1±5 mM) or increasing the incubation period up to 3 h, did not seem to have any signi®cant effect on DNA cleavage, at any enzyme concentration used in Figure 1 (data not shown). Recently, it has been shown that the cofactor AdoMet is required for cleavage by type III restriction enzymes and that Figure 1. DNA cleavage as a function of R.EcoP15I concentration in the R.EcoP15I copuri®es with bound AdoMet (16). The molecular absence and presence of exogenous AdoMet. The amount of supercoiled details of the role of AdoMet in cleavage are not yet clear, but pUC19 DNA (7.2 mg) in each assay corresponds to 12.14 pmol EcoP15I it has been suggested that AdoMet binding causes conforma- sites. The DNA cleavage assays were carried out in the HEPES reaction buffer at 37°C for 1 h as described. The amount of cleaved DNA was tional changes in the restriction enzyme which are essential for quantitated from intensities of the product and substrate bands after staining cleavage (16). AdoMet copuri®ed with R.EcoP15I might be the agarose gel with ethidium bromide. The linear phase, indicating suf®cient to support only one round of cleavage (Fig. 1, phase stoichiometric cleavage by R.EcoP15I, is labelled as A, the non-linear phase A). Therefore, the above restriction assays were repeated in of cleavage is labelled as B. The dashed line indicates the assay done with the presence of 20 mM exogenous AdoMet. The progress of supercoiled pUC19 DNA containing 24.28 pmol EcoP15I sites as described in the text. DNA cleavage as a function of R.EcoP15I concentration in the presence of 20 mM exogenous AdoMet is shown in Figure 1 (open circles). As there was no detectable increase in DNA enzyme followed by agarose gel electrophoresis and cleavage in phase A, it would imply that exogenously added puri®cation of the 1419 bp pUC19 DNA HinfI fragment. AdoMet did not increase the catalytic activity of the enzyme. DNA was quanti®ed by absorbance at 260 nm assuming an These results clearly demonstrate that R.EcoP15I does not OD of 1.0 corresponds to 50 mg/ml of double stranded DNA. perform multiple rounds of catalysis in vitro. However, a 10% DNA cleavage in the presence of exonuclease III increase in the amount of DNA cleaved was observed in the non-linear phase B in the presence of 20 mM exogenous These reactions were carried out in Lac buffer (10 mM AdoMet (open circles) relative to assays without exogenous Tris±HCl, pH 8.0, 10 mM KCl, 10 mM MgCl , 0.1 mM AdoMet (closed circles). This 10% increase in cleavage at all EDTA, 0.1 mM dithiothreitol) instead of HEPES reaction enzyme concentrations in phase B indicates that 10% of the buffer for 3 or 6 h as indicated in the text. Fifty units of R.EcoP15I had lost AdoMet and became re-activated upon the exonuclease III (NEB) was added to each reaction at time addition of exogenous AdoMet. zero. We next studied the effect of an increased DNA substrate concentration. Incubation of double the amount of supercoiled pUC19 DNA (24.28 pmol sites) with 11.0 pmol of R.EcoP15I RESULTS AND DISCUSSION for 1 h at 37°C resulted in stoichiometric cleavage of half of DNA cleavage versus R.EcoP15I concentration the pUC19 DNA (11.8 pmol sites) (closed inverted triangle on dashed line in Fig. 1). This indicates that although excess The amount of DNA cleaved by increasing amounts of substrate DNA alleviated the decrease in enzyme activity seen R.EcoP15I was studied to establish the relationship between DNA cleavage activity and enzyme concentration. Super- in phase B of Figure 1, there was still only a single round of coiled pUC19 DNA containing a pair of recognition sites in cleavage by the enzyme. This rules out the possibility of inverse orientation (®¬) was the substrate. Increasing oligomerisation of enzyme at higher concentrations, as the amounts of R.EcoP15I enzyme (1.82±21.84 pmol) were added activity of oligomers would not have been affected by to supercoiled pUC19 DNA (7.2 mg/12.14 pmol EcoP15I increasing substrate DNA concentration. sites) in a standard restriction assay and incubated for1hat To study the effect of DNA concentration upon cleavage by 37°C. The result is shown in Figure 1 (®lled circles). Initially, R.EcoP15I enzyme in phase A of Figure 1, 4.2 pmol of the amount of DNA cleaved is directly proportional to the R.EcoP15I was incubated with supercoiled pUC19 DNA enzyme concentrations (Fig. 1, phase A), indicating that the (4.6±13.9 pmol EcoP15I sites) for 1 h at 37°C. When the R.EcoP15I catalysed reaction is stoichiometric with respect to amount of substrate DNA (4.6 pmol EcoP15I sites) was enzyme concentration and that it performs a single round of slightly more than that of enzyme (4.2 pmol of R.EcoP15I) catalysis in vitro. However, there is a non-linear relationship (Fig. 2A, lane 1), a detectable amount of supercoiled DNA between the amount of DNA cleaved and higher concentra- (~8%) was left uncleaved. Even when the total amount of tions of R.EcoP15I (Fig. 1, phase B). Phase B shows that substrate DNA was in great excess over the enzyme, DNA at concentrations >6.3 pmol of enzyme, there is sub- corresponding to only 4.2 pmol EcoP15I sites was cleaved stoichiometric cleavage. One possibility for the stoichiometric (Fig. 2A, lanes 2 and 3). When an assay containing 4.2 pmol of cleavage observed in phase A as opposed to multiple rounds R.EcoP15I and supercoiled pUC19 DNA containing 4.6 pmol Nucleic Acids Research, 2003, Vol. 31, No. 7 1891 by ATP hydrolysis. ATP hydrolysis by R.EcoPI was shown to continue even after DNA cleavage (S.Saha and D.N.Rao, unpublished results). It has been reported that R.EcoP15I binds both methylated and unmethylated recognition sites and hydrolyses comparable amounts of ATP in a manner inde- pendent of DNA length and restriction (15). One interpretation of these ®ndings could be that continued ATP hydrolysis after cleavage leads to a low dissociation or `off' rate of the enzyme from the cleaved DNA. The lack of increase in DNA cleavage in our assays carried out over 3 h supports this interpretation. In addition, binding of any free restriction enzyme to cleaved DNA, thus decreasing the concentration of free enzyme in the assay, could elicit site-speci®c ATPase activity and a low off rate of enzyme from DNA. To test if cleaved DNA was responsible for the decrease in R.EcoP15I concentration and thus its restriction activity, supercoiled pUC19 substrate DNA (4.6 pmol EcoP15I sites) was incubated with R.EcoP15I enzyme (4.2 pmol) in the presence of either increasing amounts of R.EcoP15I cleaved pUC19 DNA or R.EcoP15I methylated pUC19 DNA or non- speci®c DNA (1419 bp HinfI fragment of pUC19). DNA cleavage by R.EcoP15I decreased (as evidenced by the increase in uncleaved supercoiled DNA) in a dose-dependent manner in the presence of R.EcoP15I cleaved DNA (Fig. 3A) or R.EcoP15I methylated DNA (Fig. 3B) but not in the presence of non-speci®c DNA (Fig. 3C). Hence, the seques- tration of enzyme by DNA is dependent upon there being a Figure 2. DNA cleavage by R.EcoP15I as a function of supercoiled pUC19 recognition site on the DNA. These results demonstrate that DNA concentration. (A) In the absence of exogenous AdoMet. Increasing binding of enzyme molecules to cleaved DNA or methylated amounts of supercoiled pUC19 DNA were incubated with R.EcoP15I DNA decreases the restriction activity of the enzyme. As (4.2 pmol) and DNA cleavage assays were carried out as described. Lane 1, demonstrated previously with methylated and unmethylated 4.63 pmol EcoP15I sites; lane 2, 9.27 pmol EcoP15I sites and lane 3, 13.9 pmol EcoP15I sites. (B) Time course of cleavage. Supercoiled pUC19 DNA (15), DNA cleaved by EcoP15I restriction enzyme and DNA (4.63 pmol EcoP15I sites) was incubated with R.EcoP15I (4.2 pmol) methylated DNA (Fig. 3A and B) seem to elicit ATPase in the absence of exogenous AdoMet for different periods of time and the activity of enzyme and result in a low off rate of enzyme from assay performed as described. Lane 1, 20 min; lane 2, 40 min; lane 3, DNA. Sequestration of enzyme by cleaved DNA thus leads to 60 min and lane 4, 90 min. (C) In the presence of exogenous AdoMet a decrease in the effective number of enzyme molecules. (20 mM). Increasing amounts of supercoiled pUC19 DNA were incubated with R.EcoP15I (4.2 pmol) and DNA cleavage assays performed as Non-linearity in the amount of DNA cleaved at high described. Lane 1, 4.63 pmol EcoP15I sites; lane 2, 9.27 pmol EcoP15I concentrations of R.EcoP15I (Fig. 1, phase B) could, there- sites and lane 3, 13.9 pmol EcoP15I sites. Lanes U and M, uncut super- fore, be a consequence of the accumulation of cleaved DNA coiled pUC19 DNA and marker DNA fragments (sizes given to the right in sequestering free enzyme molecules. This would require that base pairs). I and II represent supercoiled and linearised pUC19 DNA, respectively. the recognition site be exposed on the accumulating cleaved DNA to allow site-speci®c sequestration of free enzyme molecules. In the absence of multiple rounds of catalysis, as of EcoP15I sites was carried out, the kinetics of cleavage evident in Figure 1 (phase A), it can be postutated that the showed that most of the DNA was cleaved within 20 min enzyme molecules once they cleave stay bound to DNA but (Fig. 2B, lane 1). The effect of exogenous AdoMet was tested not necessarily at the recognition site. Absence of turnover by by repeating the above assays in the presence of 20 mM R.EcoP15I enzyme, demonstrated here, combined with an exogenous AdoMet. As can be seen in Figure 2C, the extent of earlier observation of a weak footprint in the presence of both cleavage was not signi®cantly different compared with that in ATP and AdoMet (28) support this interpretation. Doubling Figure 2A, indicating that addition of exogenous AdoMet had the amount of supercoiled pUC19 DNA in an assay would no appreciable effect on the extent of DNA cleavage. decrease the proportion of accumulating cleaved DNA and also enhance the binding of enzyme molecules to supercoiled Sequestration of the enzyme DNA. As expected, stoichiometric cleavage was restored in R.EcoP15I has been shown to hydrolyse ATP in the presence the assay when double the amount of supercoiled DNA was of a DNA recognition site (15). Similar observations were used (Fig. 1, closed inverted triangle on dashed line). obtained with the closely related type III enzyme R.EcoPI Sequestration of the enzyme by cleaved DNA resulting in a (24), whose Res subunit is almost identical to that of single round of cleavage (Fig. 3A and Fig. 1, phase A) with R.EcoP15I (25). Mutations in the helicase motifs of R.EcoPI R.EcoP15I may also explain the single round of cleavage affecting ATP hydrolysis abolished DNA cleaving ability observed with the functionally related type I restriction (26,27). DNA translocation during the cleavage reaction enzymes (29). It is possible that other types of restriction catalysed by these enzymes has been proposed to be assisted enzymes that cleave DNA suf®ciently far away from their 1892 Nucleic Acids Research, 2003, Vol. 31, No. 7 resistant to cleavage by cognate restriction enzymes. Sequestration of restriction enzyme by cleaved DNA per se provides the phage with a restriction alleviation mechanism. A somewhat similar kind of sequestration phenomenon has been reported when bacteriophage T7 infects Escherichia coli and bacteriophages PBS-1 and -2 infect Bacillus subtilis. The gene 0.3 protein of phage T7, also known as ocr (overcome classical restriction) (31) competitively inhibits type I DNA restriction enzymes by preventing them from binding to their DNA target (32). Biochemical observations that ocr is a competitive inhibitor of DNA binding (33) and that it contains a large excess of negatively charged amino acids, prompted the suggestion that ocr was not only a polyanionic inhibitor but that it could also be a mimic of an extended DNA structure (34). The atomic structure of ocr reveals remarkable molecular mimicry of B-form DNA (35). Ugi (UDG inhibitor) is an early gene product of B.subtilis bacteriophage PBS-1 and -2 and a protein mimic of DNA. These phages have genomes contain- ing uracil and UGI protects the genome by forming an extremely speci®c and physiologically irreversible complex with the host uracil DNA glycosylase (UDG) (36). Clearly, bacteriophages have a number of ways of regulating the activity of cellular nucleases. Exonuclease activity is essential for multiple rounds of DNA cleavage by EcoP15I restriction enzyme Type I restriction enzymes have been shown to perform only one round of cleavage reaction in vitro, i.e. each enzyme molecule cuts the DNA once only. No turnover of the enzyme Figure 3. Sequestration of EcoP15I restriction enzyme. (A) Supercoiled can be measured with respect to its cleavage function or with pUC19 DNA (4.63 pmol EcoP15I sites) was incubated with R.EcoP15I respect to its capacity to bind unmodi®ed DNA to nitrocellu- (4.2 pmol) in the presence of increasing amounts of R.EcoP15I cleaved lose membrane (29). Therefore, it has been an interesting pUC19 DNA. Lane 1, 1.375 mg; lane 2, 2.75 mg; lane 3, 5.5 mg and lane 4, 11 mg. (B) Supercoiled pUC19 DNA (4.63 pmol EcoP15I sites) was incu- question, whether one is justi®ed calling these proteins bated with R.EcoP15I (4.2 pmol) in the presence of increasing amounts of `enzymes' (37). The results presented in Figures 1 and 2 R.EcoP15I methylated pUC19 DNA. Lane 1, 2.75 mg and lane 2, 5.5 mg. indicate that type III restriction enzymes also show single (C) Supercoiled pUC19 DNA (4.63 pmol EcoP15I sites) was incubated with turnover kinetics in vitro. The ability of R.EcoP15I to perform R.EcoP15I (4.2 pmol) in the presence of increasing quantities of non- multiple rounds of catalysis was tested as follows. When the speci®c DNA. Lane 1, 2.75 mg; lane 2, 5.5 mg; lane 3, 11 mg and lane 4, 22 mg. I, II and Ns represent supercoiled pUC19 DNA, linearised pUC19 restriction assay is performed in the presence of exonuclease DNA and 1419 bp pUC19 DNA HinfI fragment, respectively. Lane C in all III, DNA cleaved by R.EcoP15I would be further digested by three panels: supercoiled pUC 19 DNA (4.63 pmol EcoP15I sites) incubated the exonuclease. The removal of cleaved DNA should release with R.EcoP15I (4.2 pmol). Lanes U and M, uncut supercoiled pUC19 R.EcoP15I to perform a further round of catalysis. DNA and marker DNA fragments, respectively. Supercoiled pUC19 DNA (4.5 mg/7.6 pmol EcoP15I sites) was incubated with R.EcoP15I (3.8 pmol) and 50 U of recognition site are also sequestered by cleaved DNA and may exonuclease III (where indicated) for 3 h at 37°C. Under exhibit slower cleavage kinetics. stoichiometric conditions, R.EcoP15I should cleave only Phage restriction is not generally absolute and phage 2.25 mg (3.8 pmol EcoP15I sites) of supercoiled pUC19 genomes that do escape cleavage normally become modi®ed. DNA. However, the cleavage of 4.5 mg of DNA (7.6 pmol Progeny of such modi®ed phages are protected against EcoP15I sites) would indicate a second round of catalysis by restriction by bacteria with R-M system of the same speci®city the enzyme. In this assay, linearisation of 2.25 mg DNA was (30). The molecular basis of this phenomenon has so far observed both in the absence and presence of 20 mM AdoMet remained obscure. The sequestration phenomenon described and in the absence of exonuclease III (Fig. 4A, lanes 1 and 2). here provides a clue to understanding how non-resistant In the assays performed in the presence of exonuclease III, phages acquire resistance to a given R-M system. Infection of DNA linearised by R.EcoP15I will be subjected to complete a bacterium with an `overwhelming number' of non-resistant degradation by exonuclease III, hence the amount of super- phages might result in a scenario where all the intracellular coiled pUC19 DNA left at the end of the assay would indicate restriction enzyme is involved in stoichiometric cleavage of the amount of DNA cleaved by R.EcoP15I. Complete infecting phage and gets sequestered by the cleaved phage disappearance of DNA would indicate cleavage of 4.5 mg DNA. Such a situation provides the remaining phages with an supercoiled pUC19 DNA by 3.8 pmol of R.EcoP15I. As can oppurtunity to be completely methylated by the cognate be seen from Figure 4A (lane 3) the enzyme showed complete methyltransferase (MTase) before the endonuclease acts on cleavage of 4.5 mg supercoiled pUC19 DNA only in the them. The phages with methylated recognition sites are presence of exonuclease III. As a control, we show that the Nucleic Acids Research, 2003, Vol. 31, No. 7 1893 that cleave DNA suf®ciently far outside their recognition site. An earlier report demonstrating reduction in restriction by EcoKI in the absence of the RecBCD exonuclease supports this suggestion (20). Assistance of multiple rounds of catalysis by the restriction enzyme in the presence of exonuclease is characterised by a time lag between DNA cleavage and dissociation of the restriction enzyme from the cleaved DNA. The time lag provides an opportunity for the infecting phage to acquire methylation by the cognate methyltransferase before the restriction enzyme acts on them under situations discussed in the previous section. Implications for the evolution of R-M enzymes Sequence comparisons and biochemical experiments in con- junction with site-directed mutagenesis studies have estab- lished the fact that the Mod subunit of type III R-M systems contains the target recognition domain (TRD) (9,13,23). The Res subunit contains the characteristic endonuclease motif (PD¼D/E-X-K) involved in phosphodiester bond cleavage (27), and the helicase motifs responsible for DNA trans- Figure 4. Multiple rounds of DNA cleavage by EcoP15I restriction enzyme location (driven by ATP hydrolysis) (26). Consequently, the in the presence of exonuclease III. (A) Supercoiled pUC19 DNA (7.6 pmol amino acid residues involved in target recognition and the EcoP15I sites) was incubated with EcoP15I restriction enzyme (3.8 pmol) active site residues involved in DNA cleavage are likely to be in the absence or presence of 20 mM exogenous AdoMet or exonuclease III spatially distant in the holoenzyme and thus lead to cleavage or both as indicated on top of each lane. (B) Supercoiled pUC19 DNA was incubated with R.EcoP15I (3.8 pmol) for 3 or 6 h at 37°C in the presence of some distance from the recognition site. This is probably true exonuclease III. Lane 1, 11.4 pmol EcoP15I sites; lane 2, 11.4 pmol for other restriction enzymes cleaving DNA outside their EcoP15I sites; lane 3, 15.2 pmol EcoP15I sites; lanes U and M, uncut recognition site (39). In contrast, these sequence recognition supercoiled pUC19 DNA and marker DNA fragments, respectively. and cleavage residues are in close proximity in the tertiary structures of orthodox type II enzymes, allowing cleavage to occur within the recognition site (40). supercoiled DNA was not affected by exonuclease III in the Since the amino acid sequences of R-M enzymes do not absence of EcoP15I restriction enzyme (Fig. 4A, lane 4). This share signi®cant similarity and are not easily amendable to demonstrated that removal of DNA, once cleaved by standard alignment procedures, it has been dif®cult to ®nd an R.EcoP15I, allowed a second round of catalysis by the evolutionary link among R-M systems through sequence restriction enzyme. analysis alone. Despite limited sequence similarities, from the To check for multiple rounds of catalysis, supercoiled crystal structures of restriction enzymes and methyltrans- pUC19 DNA (11.4 pmol EcoP15I sites) was incubated with ferases and predicted secondary structures of other R-M R.EcoP15I (3.8 pmol) and 50 U of exonuclease III for 3 h at enzymes, the presence of common folds responsible for 37°C. As evident from Figure 4B (lane 1) 2.25 mg supercoiled enzyme activity has been shown (41±47). It has been DNA was left uncleaved, indicating only two rounds of suggested that spatial conservation of side-chain locations cleavage by the enzyme. The above assay was repeated by plays a dominant role and that the primary sequence of increasing the incubation period from 3 to 6 h at 37°C. conserved active site residues is less important when searching Disappearance of the entire supercoiled DNA in Figure 4B for similarities among restriction enzymes (5). This would (lane 2) clearly shows that a third round of DNA cleavage is suggest that an evolutionary link among these enzymes could possible. A fourth round of catalysis was also demonstrated by be deduced from their catalytic ef®ciency (as they share the incubating supercoiled pUC19 DNA (15.2 pmol EcoP15I same folds) in conjunction with sequence analysis. Based on sites) for 6 h at 37°C with 3.8 pmol of R.EcoP15I in the the results described here, which indicate that the ef®ciency of presence of exonuclease III (Fig. 4B, lane 3). restriction enzymes increases with decreasing af®nity for These results indicate that the type III restriction enzyme cleaved DNA, we propose a functional evolutionary hierarchy R.EcoP15I, in the presence of an exonuclease, can perform for R-M systems illustrated schematically in Figure 5. Our multiple rounds of DNA cleavage. This is the ®rst report scheme suggests that restriction enzymes are under pressure to demonstrating functional cooperation between a restriction evolve the ability to cut within their recognition site rather endonuclease and an exonuclease and this may re¯ect the than cutting away from the site and, as a consequence, having situation in vivo. Such functional cooperation between DNA to rely upon other enzymes to release them from the DNA for transaction proteins has been reported recently (38). The further catalytic cycles. unexpected functional cooperation between an exonuclease Restriction enzymes have structural homology with and an endonuclease provides a highly ef®cient mechanism enzymes involved in DNA repair and recombination which for an increase in the ef®ciency of the R.EcoP15I restriction also perform phosphodiester bond cleavage (5,46,48±51). enzyme. Our observations make it attractive to suggest that Type I R-M systems (e.g. R.EcoKI) have high structural exonucleases might play a similar role in the in vivo activity of complexities and can be considered as the most suitable forms other restriction enzymes, such as type I restriction enzymes, for the evolution of new types of R-M systems through domain 1894 Nucleic Acids Research, 2003, Vol. 31, No. 7 enzymes. One such form would be a type IIB R-M enzyme. Type IIB enzymes (e.g. BcgI) have two polypeptides, one containing only TRDs, the other containing both MTase and ENase domains (56). The polypeptide with TRDs associates with the polypeptide having both ENase and MTase domains to form a functional type IIB restriction enzyme that can also methylate DNA. Alternatively, duplication of a domain such as TRD in a type IIG enzyme is probable. A polypeptide with a duplicated TRD allows simultaneous segregation of the two TRDs. Segregation of one TRD with MTase domains would produce a protein resembling the MTase of type IIS R-M enzymes, while segregation of other TRD with the ENase domain would produce a protein resembling a type IIS Figure 5. Schematic representation of a functional evolutionary hierarchy restriction enzyme. Based on the biochemical properties of the of R-M systems. Different colours indicate different domains. All domains prototype type IIG enzyme, Eco57I, it was suggested in single polypeptide are represented as fused domains. (Purple, target previously that the enzyme might be regarded as an inter- recognition domain; green, endonuclease domain; yellow, AdoMet binding mediate type re¯ecting evolutionary link between the enzymes domain; red, helicase domain; black, methyltransferase catalytic domain.) of types III and IIS (55). Later it was argued that type IIG R-M systems might have evolved from a progenitor formed by the fusion of a single strand-speci®c MTase of type IIS with a shuf¯ing, deletions, insertions and duplication (2,52). The DNA endonuclease (57). However, acquisition of MTase HsdS subunit determines DNA speci®city and it is composed domains, which are not related to restriction activity, by an of two separate DNA binding domains, each recognising one endonuclease to form the predecessor of type IIG seems rather speci®c part of a non-palindromic sequence. The HsdM unlikely. Type IIS enzymes (FokI) have DNA binding domain subunit contains the AdoMet binding site and the catalytic site in both the modi®cation and the restriction subunits. A type for DNA methylation (53). The HsdR subunit is essential for IIS system has two MTases, one for each strand, which could restriction and contains a set of seven amino acid sequence also be a consequence of these genetic rearrangements. An motifs typical of the superfamily of helicases, the so-called endonuclease capable of recognising DNA without requiring DEAD box proteins, involved in ATP binding and ATP- association with an MTase allows DNA cleavage independent dependent DNA translocation (54). Fusion of one DNA of AdoMet. However, both types IIB and IIS cleave outside binding domain of a type I HsdS subunit, recognising one their recognition sequence. FokI endonuclease supplemented speci®c part of a non-palindromic sequence, with a type I with a DNA binding-de®cient mutant of FokI endonuclease HsdM subunit, would, in conjunction with rearrangements of the tertiary structure, result in a subunit similar to the Mod containing an active catalytic domain was shown to increase the rate of DNA cleavage 10±20-fold relative to the wild-type subunits of type III R-M enzymes. This would be a step FokI alone (58). This increased cleavage is possibly a towards increasing the ef®ciency of the enzyme with now only consequence of an increased off rate from cleaved DNA for one subunit performing the functions of two subunits, HsdS the heterodimer (wild-type + mutant) compared with the and HsdM. However, both types I and III restriction enzymes homodimer (wild-type). Earlier quantitative evaluation of perform ATP and AdoMet-dependent DNA cleavage and cleave outside the recognition sequence (Table 1). In type III sequence and structure compatibility has shown that cleavage R-M enzymes, ATP hydrolysis and DNA translocation are domain of homologues of FokI (type IIS) have a higher degree of similarity to type I and type III R-M systems than to essential for oligomerisation of enzyme molecules involved in orthodox type II enzymes such as EcoRV and PvuII. DNA cleavage. Although type I restriction enzymes possess Furthermore, the cleavage domain of FokI is apparently ATPase activity and have been shown to translocate DNA, evolutionarily older than type II restriction enzymes (51). there has been evidence from atomic force microscopy Amino acid residues involved in site recognition and indicating that type I R-M complexes interact and oligomerise even in the absence of ATP-hydrolysis (54). phosphodiester bond cleavage are spatially separated in type Fusion of the Mod and Res subunits of type III enzymes, IIS enzymes. Any change(s) bringing these residues into close accompanied by deletion of the helicase domains, would proximity (in tertiary structure) might have resulted in probably result in type IIG enzymes (e.g. Eco57I) (53) that orthodox type II enzymes. Orthodox type II restriction enzymes have amino acid residues involved in recognition cleave DNA independent of ATP. Type IIG enzymes would and cleavage in close proximity in tertiary structure and cleave thus have four domains, namely TRD, AdoMet-binding DNA within the recognition site (4). These are the most domain, MTase catalytic domain and an endonuclease (ENase) domain in a single polypeptide chain. Type IIG ef®cient restriction enzymes with respect to DNA cleavage. enzymes also cleave DNA in an AdoMet-dependent manner Dissociation of these enzymes from cleaved DNA can be (55). Interestingly, association of endonuclease domains with expected to be faster than that of restriction enzymes, which MTase catalytic domains (type I, type III, type IIG, type IIB) cleave DNA away from the recognition site. The evolutionary hierarchy described here re¯ects the fact that although the leads to requirement for AdoMet in DNA cleavage (3,16,56). enzymes need to maintain high af®nity for their substrates, A type IIG restriction enzyme could give rise to a number of variants. Deletion of one of the four domains with the fusion of they should have low af®nity for their products to be remaining three domains can result in four different forms of functionally ef®cient. Nucleic Acids Research, 2003, Vol. 31, No. 7 1895 12. Meisel,A., Kru È ger,D.H. and Bickle,T.A. (1991) M.EcoP15 methylates It must be mentioned that the above model does not take the second adenine in its recognition sequence. Nucleic Acids Res., 19, into consideration a lot of variants that might have appeared between and within each step of the functional evolutionary 13. Ahmad,I. and Rao,D.N. (1994) Interaction of EcoP15I DNA hierarchy. Identi®cation and characterisation of new restric- methyltransferase with oligonucleotides containing the asymmetric sequence 5¢-CAGCAG-3¢. J. Mol. Biol., 242, 378±388. tion enzymes such as HaeIV (59) and AloI (60) provide 14. Meisel,A., Bickle,T.A., Kruger,D.H. and Schroeder,C. (1992) Type III enzymes that share properties with more than one existing restriction enzymes need two inversely oriented recognition sites for type. As new R-M systems are characterised, it is likely that DNA cleavage. Nature, 355, 467±469. the biochemical properties of the various types of enzymes 15. Meisel,A., Mackeldanz,P., Bickle,T.A., Kru È ger,D.H. and Schroeder,C. will eventually represent a continuum. (1995) Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-speci®c ATP hydrolysis. EMBO J., 14, Collectively, our data demonstrate that the type III EcoP15I 2958±2966. restriction enzyme is sequestered by the intact recognition site 16. Bist,P., Srivani,S., Krishnamurthy,V., Asha,A., Chandrakala,B. and remaining after DNA cleavage. The presence of cleaved DNA Rao,D.N. (2001) S-adenosyl-L-methionine is required for DNA cleavage with an intact recognition site decreases the effective by type III restriction enzymes. J. Mol. 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(1997) hierarchy proposed here suggests that product release after Modulation of EcoKI restriction in vivo: role of the lambda Gam protein DNA cleavage, independent of exonuclease, has been the and plasmid metabolism. J. Bacteriol., 179, 1852±1856. driving force for the evolution of ef®cient restriction enzymes. 21. Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680±685. 22. Sambrook,J. and Russell,D.W. (2001) Molecular Cloning. A Laboratory Manual, 3rd Edn. Cold Spring Harbor Laboratory Press, New York, NY. ACKNOWLEDGEMENTS 23. Hadi,S.M., Ba Èchi,B., Iida,S. and Bickle,T.A. (1983) DNA restriction- modi®cation enzymes of phage P1 and plasmid p15B. Subunit functions N.K.R. acknowledges Shashi Bhusan Pandit, S. Varadarajan and structural homologies. J. Mol. Biol., 165, 19±34. and Surbhi Gupta for stimulating discussions, and Pradeep 24. Saha,S. and Rao,D.N. (1995) ATP hydrolysis is required for DNA Bist and S. 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Published: Apr 1, 2003

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