Báo cáo Y học: The N-terminus of m5C-DNA methyltransferase Msp I is involved in its topoisomerase activity docx

The N-terminus of m5C-DNA methyltransferase Msp I is involved in its topoisomerase activity Sanjoy K. Bhattacharya* and Ashok K. Dubey† Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi, India DNA cytosine methyltransferase MspI(M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5C-MTases) to evoke the topoisomerase activity that it possesses in addition to DNA-methylation ability. This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5C-MTases. Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5C-MTase structures. Examination of the amino-acid sequence of M.MspI revealed similarity to human topoisomerase I at the N-terminus. Alignment of the amino-acid sequence of M.MspI also uncovered similarity (residues 245–287) to the active site of human DNA ligase I. To evaluate the role of the N-terminus of M.MspI, 2-hydroxy-5-nitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35. The purified HNBB-truncated protein has a mo- lecular mass of  45 kDa, retains DNA binding and meth- yltransferase activity, but does not possess topoisomerase activity. These findings were substantiated using a purified recombinant MspI protein with the N-terminal 34 amino acids deleted. Changing the N-terminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomerase I activity while the methyltransferase activity remains intact. Keywords: DNA binding; methyltransferase MspI; proteolysis; topoisomerase I. Methylation of DNA at C-5 of cytosine has been implicated in a number of biological processes in prokaryotes: control of immunity [1], gene expression [2], and replication [3]. In eukaryotes, it is also implicated in the control of develop- mental processes [4], transposition [5], recombination [6], X-chromosome inactivation [7] and genomic imprinting [8]. Cytosine methylation at C-5 is carried out by a class of methyltransferases (MTases) referred to as m5C-MTases. All m5C-MTases from bacteria to mammals share a common architectural plan [9,10]. They have six highly conserved and four not so well conserved motifs [10]. Most bacterial m5C-MTases, including those that have been well studied, have a very small N-terminal sequence before motif I: M.HhaI possesses a 13-amino acid N-terminal sequence, and M.HaeIII possesses only a 3-amino acid one [10]. However, m5C-MTases from higher eukaryotes possess a relatively large N-terminal sequence. In mouse m5C- MTase, the N-terminal domain is > 1000 amino acids [11,12]. Within the N-terminal domain, a DNA replication foci-targeting domain has been identified in mouse m5C- MTase. Multiple domains have been implicated in the targeting of mouse DNA MTase to the replication foci [13]. Independent DNA and multiple Zn-binding domains at the N-terminus of human DNA (cytosine-5) MTase have been characterized [14]. It has been shown that it is possible to modulate the properties of the DNA-binding domain by contiguous Zn-binding motifs [14]. Although all m5C-MTases share a high degree of sequence homology and are postulated to be similar in structure and function, there are important differences with respect to their kinetic, biological and chemical properties. M.Dcm and M.BsuRI, members of the m5C-MTase family, undergo self-methylation in the absence of substrate DNA [15,16]. M.MspI catalyzes the exchange of tritium at C-5 of cytosine from tritiated water in the absence of cofactor AdoMet, not common in many members of m5C-MTase family [17]. M.MspIandM.SssI MTases possess a topoisomerase activity not found in other m5C-MTases [18]. M.MspI induces a significant sequence-specific bend of 105 ° in DNA, which has not been reported for any other m5C-MTase [19]. Neither topoisomerase activity (S. K. Bhattacharya, unpublished observation) nor tritium exchange [17,20] in the absence of cofactor has been detected in M.HpaII, an isoschizomer of M.MspI. M.MspI is a 418-amino-acid protein with known DNA and amino- acid sequence [21]. It has one of the largest N-terminal sequences of any bacterial m5C-MTase. N-Terminal sequence here refers to the amino-acid sequence before motif I. The N-terminal sequence of M.MspI spans residues 1–107 [10]. Whether the presence of a large N-terminal sequence results in modulation of conformation/structure or activity in bacterial m5C-MTases such as M.MspIhas not been investigated. The phenomenon of sequence-specific Correspondence to S. K. Bhattacharya, Department of Ophthalmic Research/I31, Cole Eye Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Fax: + 1 216 297 9892, Tel.: + 1 216 445 0424, E-mail: bhattas@ccf.org Abbreviations: MTase, methyltransferase; M.MspI, methyltransferase MspI; m5C-MTase, DNA cytosine methyltransferase; HNBB, 2-hydroxy-5-nitrobenzyl bromide. *Present address: Department of Ophthalmic Research/I31, Cole Eye Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Present address:BiomolecularResearchInstitute,343RoyalParade, Parkville, Victoria 3052, Australia. (Received 8 February 2002, accepted 4 April 2002) Eur. J. Biochem. 269, 2491–2497 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02913.x DNA bending and topoisomerase activity shown by M.MspI, in addition to cytosine methylation, suggests that the interaction of M.MspI with DNA may be different from that of other m5C-MTases. The additional DNA-modifying activity (topoisomerase activity) possessed by M.MspI would require a different type of interaction of protein with DNA. To achieve this, M.MspI may require a different structural organization in the solution phase from normal. We used limited proteolysis and specific chemical modifi- cation to investigate this. Attempts were also made to discover whether the N-terminus has a role in any of the biological activities shown by M.MspI. EXPERIMENTAL PROCEDURES Bacterial strain and plasmid Escherichia coli K-12 strain ER 1727 [(mcr BC – ) hsd RMS – (mrr)2::Tn10, mcrA1272::Tn10, F¢¢lacproAB lacI q - (lacZ) – M15] used for overexpression of the target gene was placed at the downstream region of the T7 promoter regulated by the lac operator in the expression vector pMSP [22]. E. coli strain ER 1727 harboring recombin- ant plasmid pMSP was cultivated in Luria broth containing 150 lgÆmL )1 ampicillin at 37 °C. M.MspI was purified using column chromatography as described previously [23]. The purified M.MspI was tested for homogeneity by SDS/PAGE (10% gel) using Coomassie Blue R250 staining. Assay of MTase activity Reactions of MspI MTase were performed in 30 mL buffer A (potassium phosphate, pH 7.4, sodium EDTA 1m M , 2-mercaptoethanol 14 m M , glycerol 10%) con- taining unlabeled 1459-bp BstXI DNA fragment of /X174 as substrate (50 n M DNA) and 200 n M AdoMet (80 mCiÆmmol )1 ). The reactions were initiated by adding M.MspI solution (3 mL); the reaction mixtures were then incubated for 30 min at 37 °C. Then 20-mL samples from a 30-mL reaction mixture were spotted on DE81 filter discs placed in a filter manifold. The filter discs were washed twice with 0.2 M NH 4 HCO 3 ,twicewith 80% ethanol in 50 m M phosphate buffer (pH 8.0), and once with 90% ethanol in 50 m M phosphate buffer (pH 8.0). The filters were dried under vacuum on the filter manifold and subsequently under a lamp and measured for radioactivity in 4 mL scintillant (Supertron; Kontron). The activity of M.MspI was measured as radioactive methyl groups transferred (d.p.m.). Filter discs treated in an identical manner using a reaction mixture that lacked the enzyme served as background or blank counts. Typically the background counts were less than 50 d.p.m. Protein analysis Protein concentration was determined on the basis of Bradford’s principle [24] using the Bio-Rad Coomassie plus kit. Standard curves were established using BSA. Protein samples were subjected SDS/PAGE (10% gels) using Laemmli buffer [25]. Proteins were visualized by staining with Coomassie Brilliant Blue R250. Modification of M. Msp I with 2-hydroxy-5-nitrobenzyl bromide (HNBB) Modification reactions were carried out in 20 m M sodium phosphate buffer (pH 10) at 25 °C. The enzyme was quickly brought to pH 7.4 with buffer A [26]. HNBB (1–5 m M )was addedto5l M MspI in a reaction volume of 40 mL. The reaction mixture was analyzed for MTase activity after HNBB treatment (HNBB is readily inactivated under these conditions). A 410 was measured. An absorption coefficient of 18 000 M )1 Æcm )1 [27] was used to calculate the mol of tryptophan residue modified by the reagent. MTase activity of HNBB-modified protein was determined [17]. A slightly modified protocol of the chemical modification with HNBB leads to truncation of the N-terminal residue between residues 34 and 35. Briefly,  10 m M HNBB in dimethyl sulfoxide is added to reaction mixture containing 5 l M MspI in a reaction volume of 40 mL in 20 m M sodium phosphate buffer (pH 8.5) at 25 °C. The HNBB-truncated protein can be readily separated from the intact protein using either Q-Sepharose or SP-Sepharose chromatography. The puri- fied HNBB-digested protein is eluted at 350 m M NaCl. Optimization of trypsin concentration and reaction time for proteolysis Trypsin, dissolved in 20 m M HCl(20%,w/v),wasusedto cleave the M.MspI. The tryptic digestion was previously optimized: 5% (w/v) trypsin, with 5 l M M.MspIina reaction volume of 30 lL containing 50 m M Tris/HCl (pH 8.0), 2.0 m M EDTA, 1 m M 2-mercaptoethanol and 100 m M NaCl. The cleavage reaction was terminated by adding 0.5 m M phenylmethanesulfonyl fluoride after 90 min of incubation at 37 °C. The fragments were purified by FPLC column chromatography using Mono Q and Mono S columns and a modification of a protocol for intact M.MspI [23]. Peptide sequencing After SDS/PAGE separation, peptides were transferred to poly(vinylidene difluoride) membranes using a Novablot multiphor semidried Western blotting apparatus. The first 20-amino-acid p26 and p18 band was sequenced using an Applied Biosystems automated sequencer. Assay of topoisomerase I activity Topoisomerase I was assayed using CsCl-purified plasmid pBR322 in topoisomerase assay buffer [50 m M Tris/HCl, pH 7.5, 1 m M EDTA, 1 m M dithiothreitol, 20% (v/v) glycerol]. The assay was performed by incubating DNA (1 lg) and enzyme at 37 °C for 30 min in a reaction volume of 30 lL. The DNA was then analyzed by electrophoresis on a 0.8% agarose gel. One unit of topoisomerase activity is defined as the amount of enzyme required to convert 0.25 lg supercoiled pBR322 into a relaxed form in 30 min at 37 °C. The topoisomerase I was used as the positive control. Mutagenesis Plasmid pMSP [22] with the M.MspI coding region placed at the downstream region of the T7 promoter regulated by 2492 S. K. Bhattacharya and A. K. Dubey (Eur. J. Biochem. 269) Ó FEBS 2002 the lac operator was used as the template for mutagenesis. W34A and Y74A mutants were constructed with the QuikChange/Chameleon site-directed mutagenesis kit from Stratagene. The sequence of the oligonucleotides for the mutations at the underlined positions were: 5¢-ACATGGC AACAG GCGGAATCAGGTAAA-3¢ (W34A) and 5¢-AT ATTCTAGAAA GCTAACCAGAATCAA-3¢ (Y74A). The mutants were identified by DNA sequencing of the plasmid DNA. Expression and purification of truncated Msp I (del 34aa) Using the intact M.MspI gene in pMSP plasmid as template, a PCR amplification of truncated MspI (deletion of 34 N-terminal amino acids; del34aa) was made using the following set of primers: 5¢-CATATGgaatcaggtaaaaca-3¢ and 5¢-tgttttacctgattccCATATG-3¢ (forward and reverse primers, respectively. These primers ensured introduction of asitefortheNdeI enzyme, which has no recognition site in the MspI gene sequence. The amplification was carried out using Taq polymerase (Gibco/BRL) following the protocol recommended by the manufacturer. The PCR product was ligated in PGEMT vector using a PGEMT kit (Promega). The amplified fragment was separated from the PGEMT vector using NdeI and ligated with NdeI-digested pET3a vector. The plasmid with the correct orientation of the fragment was selected using restriction digestion; DNA sequencing was subsequently performed to confirm that the orientation was correct. The plasmid was introduced into E. coli BL21 DE3 plysS and induced as described above to produce the recombinant truncated protein. The truncated protein was purified as described above. RESULTS Alignment of M. Msp I sequence with topoisomerase I and DNA ligase In a computer search with a basic local alignment search tool [28] (BLAST network service at the National Center for Biotechnology Information), no similarities were detected between the MspI amino-acid sequence and sequences of members of the topoisomerase, recombinase, transpose or ligase families. Subsequently, the protein sequences were obtained from GenBank and aligned using MEGALIGN AND EDITSEQUENCE programs (DNASTAR Inc.) 1 . Comparison of amino-acid sequences of M.MspI (P11408) with those of topoisomerases (Fig. 1A) and DNA ligases (Fig. 1B) revealed a region of similarity; better matches were observed with eukaryotic than prokaryotic protein sequences. The amino-acid sequence 32–98 of M.MspI was found to have similarity to a region of the topoisomerases, and the sequence 245–287 had similarity to members of the DNA ligase family. The N-terminal portion of M.MspI (1–107) has similarity to topoisomerase I (region 399–458 of human topoisom- erase I; P11387 [29]; Fig. 1A), whereas the C-terminal portion of M.MspI (245–418) has sequence similarity to the active site of DNA ligases (region 543–584 of human DNA ligase I; P18858; [30]; Fig. 1B). Fig. 1. Alignment of sequences. Alignments were performed using the Megalign program initially in a pairwise fashion followed by multiple alignment. The protein sequences were aligned using the Lipman–Pearson protein alignment method with a gap penalty of 4, Ktuple of 2, and gap length penalty of 12. Black and gray outlines indicate identical and similar amino acid residues, respectively. (A) Alignment of the M.MspIamino- acid sequence with sequences from members of the DNA topoisomerase I family. Comparisons were made with human (P11387), Xenopus laevis (P451512), Mus musculus (Q04750), Caenorhabditis elegans (CAA65537), Plasmodium carinii (AF061533), Schizosaccharomyces pombe (P07799), Drosophila melanogaster (P30189), Plasmodium falciparum (S54174), Saccharomyces cerevisiae (K03077), and Candida albicans (U41342). (B) Amino-acid sequence of M.MspI was aligned with members of DNA ligase family, namely human (P18858), X. laevis (P51892), mouse (P37913), M. thermoformicum (P54875), Variola virus (CAB54763), A. thaliana (Q42572), S. pombe (CAB08176), S. cerevisiae (CAA27005), Thermostable Pfu DNA ligase (P56709), M. musculus DNA ligase III (P97386), Myxoma virus (AF170726) and rabbit fibrosarcoma virus (AF170722). 3 Ó FEBS 2002 N-Terminal of MspI and topoisomerization (Eur. J. Biochem. 269) 2493 Proteolysis of M. Msp I Digestion of M.MspI with trypsin resulted in the generation of two bands of molecular mass 26 kDa and 18 kDa, designated p26 and p18, respectively (Figs 2A,B). The first 15 amino acids of fragment p26 are LKLIRSKLDLTQK QA. The sequence obtained for the first 20 amino acids of p18 is GIPQKRKRFYLVAFLNQNIH. This is in agree- ment with the C-terminal portion of M.MspI that would contain 166 amino acids. The molecular mass for this fragment was calculated to be  17 kDa, which is in good agreement with the experimental value of 18 kDa. Trypsin digestion at two sites (between six and 251 amino acids) as observed with N-terminal sequencing of the p26 and p18 fragments, the p26 fragment should contain 246 amino acids which should correspond to an approximate molecu- lar mass of 28 kDa consistent with the measured molecular mass of this fragment. 2 Topoisomerase activity of HNBB-modified M. Msp I Treatment with HNBB resulted in modification or trunca- tion at tryptophan residues. There are only two tryptophan residues in M.MspI, at positions 31 and 34, respectively. Thus, modification of a tryptophan residue in M.MspI should result in modification/truncation of the N-terminal portion only. Indeed, on HNBB modification (50-fold excess HNBB at pH 8.5), the N-terminal portion of the protein is truncated, which was confirmed by SDS/PAGE (20% gel) followed by amino-acid sequencing (Fig. 3A). HNBB-modified M.MspI does not show any topoisomerase activity in either the absence or presence of ATP (Fig. 3B). Amino-acid sequencing of the first 15 N-terminal residues of the HNBB-truncated protein yielded ESGKTEMHPA YYSFL, which is consistent with the sequence of M.MspI truncated between residues 34 and 35. The HNBB-modified protein retains both DNA binding [26] and AdoMet- dependent MTase activity (Fig. 3C). The loss of topoisom- erase activity is in agreement with the alignment data, which suggest that an intact N-terminal domain is necessary for topoisomerase I activity. Determination of MTase and topoisomerase activity of tryptic fragments The MTase and topoisomerase activities of fragments p26 and p18 were determined using standard assays as des- cribed in Experimental procedures. None of the purified Fig. 2. Proteolysis of M.MspI. (A) Proteolysis and molecular mass determination of frag- ments. Purified fragments p26 and p18 and intact M.MspIandthemarkerproteinswere subjected to 10% polyacrylamide gel electro- phoresis in the presence of 0.1% SDS. (B) The mobilities of the marker proteins were used to calibrate the curve, and the molecular masses of the denatured fragments were calculated by interpolation. Fig. 3. Modification of M. MspI. (A) M.MspI treated with 50-fold excess ethanolic HNBB and purified chromatographically. Lane 1, M.MspI control column; lane 2, M.MspI treated with 50-fold excess of HNBB at pH 8.5; lane 3, HNBB-treated M.MspI puri- fied on a Q-Sepharose column; lane 4, HNBB- treated M.MspI purified on an SP-Sepharose column. (B) Topoisomerase activity of HNBB-modified protein. Lane 1, M.MspI control; lane 2, HNBB-treated protein; lane 3, pBR 322 control; lane 4, EcoRI digest. (C) MTase activity of M.MspI and HNBB-treated protein. The amino acid residues 1–34 of M.MspI are given below panel (A); the arrows below W (tryptophan) indicate the site of modification by HNBB. 2494 S. K. Bhattacharya and A. K. Dubey (Eur. J. Biochem. 269) Ó FEBS 2002 proteolytic fragments possessed either of these activities (Fig. 4A,B). Effect of mutation on enzyme activities The purified mutant enzymes W34A and Y74A were compared for topoisomerase and MTase activity. Both possessed more than 80% MTase activity (Fig. 4C), but both were completely inactive with respect to topoisom- erase I/relaxation activity, even when 200 lg of the mutant enzyme was used, a more than 20-fold excess over the amount needed to obtain relaxation by the wild-type enzyme(Fig. 4D). Characterization of truncated Msp I (del34aa) The truncated M.MspI protein (del 34aa) was purified to apparent homogeneity (Fig. 5A). It possessed MTase activ- ity at levels similar to that of the wild-type protein (Fig. 5B), but lacked topoisomerase activity even when excess amounts compared with wild-type controls were used. DISCUSSION Alignment of the protein sequence of M.MspIwiththatof topoisomerases using the MEGALIGN program revealed similarity. As topoisomerase I activity involves a ligation step, we also searched for sequence similarity to DNA ligase. The sequence alignment showed that the amino acid region 32–90 at the N-terminal portion of M.MspI has weak similarity to topoisomerases, and region 245–287 has similarity to members of the DNA ligase family. It is Fig. 5. SDS/PAGE analysis and activity determination of purified truncated (del34aa) MspI. (A) The purified intact and 34 N-terminal amino-acid-deleted protein was expressed and purified as described in Experimental procedures. The purified preparations were electro- phoresed for 2 h at 100 V on a 4–20% gradient gel (Bio-Rad), stained with Coomassie Blue and visualized. (B) The MTase activity of trun- cated protein was determined in triplicate using the methylation assay described in Experimental procedures. Fig. 4. MTase (A) and topoisomerase (B) activity of M.MspI and trypsin-digested fragments p26 and p18, and MTase (C) and topoisomerase (D) activity of M.MspI mutants. (A) The methylation assay was carried out as described in Experimental Procedures. Background counts were less than 50 d.p.m. The counts obtained using M.MspI were taken as 100%. (B) A plasmid DNA was treated with M.MspI in topoisomerase I buffer. Lane 1, DNA incubated with topoisomerase I (control); lane 2, DNA incubated with M.MspI; lane 3, DNA incubated with p26; lane 4, DNA incubated with p18; lane 5, DNA control. (C) Methylation assay was carried out as described in Experimental procedures. M.MspI activity was taken as 100% and served as control; the tryptophan mutant is represented as W34A and the tyrosine mutant as Y74A. (D) Lane 1, DNA incubated with topoisomerase I (control); lane 2, DNA incubated with M.MspI; lane 3, DNA incubated with tryptophan mutant W34A; lane 4, DNA incubated with tyrosine mutant Y74A; lane 5, DNA control. Ó FEBS 2002 N-Terminal of MspI and topoisomerization (Eur. J. Biochem. 269) 2495 interesting to note that the better matches were obtained with eukaryotic than prokaryotic sequences. The best match was with human DNA topoisomerase I and human DNA ligase I. This better match with eukaryotic sequences, human proteins in particular, has also been observed for NaeI restriction endonuclease [31]. The amino-acid sequence 245–287 of M.MspI shows similarity to the active- site sequence of DNA ligases, which has also been found for NaeI restriction. In NaeI [31], as well as in M.MspI, the sequence with similarity around the active site of the DNA ligase sequence differs from human ligase active site in one important respect: the lysine that forms the adenylated intermediate essential for catalysis by the DNA ligase active site in NaeI has been replaced with a leucine (L43) at this position, whereas in M.MspI it has been replaced with a histidine (H271). Using a BLAST search, sequence similarity was not observed between NaeI MTase and topoisomer- ases. Changing L43 to K43, however, enables NaeI restriction to possess topoisomerase activity [31]. The three members of the restriction-modification family that show topoisomerase activity (or with the potential to do so on mutation of a single residue), NaeI restriction endonuclease, MspIandSssI methylase, recognize 5¢-CGCCGGC-3¢, 5¢-CCGG-3¢ and 5¢-CG-3¢, respectively. The common element in their recognition sequences is 5¢-CG-3¢.Itisalso noteworthy that the regions in NaeI restriction endonuc- lease and MspI methylase that are similar to topoisomerase or ligase are better matched to eukaryotic sequences, human ones in particular. The prokaryotic topoisomerase sequenc- es that match best are Plasmodium carinii and P. falciparum; both are human parasites. Nocardia aerocolonigenes (the host of NaeI) and Moraxella (hostofM.MspI) are also capable of being human parasites. Two prototype bacterial m5C-MTases, M.HhaI [32] and M.HaeIII [33], have been crystalized and their structures resolved. A bilobal or two-domain structure was found. Limited proteolysis of MspI with trypsin agrees with the two-domain structure for this m5C-MTase. M.MspIalso yielded two bands of  26 kDa and 18 kDa on digestion with SV8 protease (data not shown). Previous reports on limited proteolysis of adenine MTase suggested a two- domain structure [34,35], which is in agreement with the findings presented here. Only two tryptophan residues are present in M.MspIand both are in the N-terminal region; one is residue 34, the region that shows similarity in sequence alignment. Conse- quently, we attempted to modify the tryptophan residues to investigate the role of the N-terminus in M.MspI. With HNBB modification, we indeed observed a protein that had retained the specificity and kinetic properties of a MTase [26] but had lost its topoisomerase activity, indicating the role of the N-terminal portion in M.MspI. To substantiate this finding, a deletion mutant of MspI with the N-terminal 34 amino acids removed (MspI del 34aa) was generated using PCR. The purified MspI del 34aa retains MTase activity but lacks topoisomerase activity (Fig. 5). Deletion of 85 residues of the N-terminal region does not affect either the MTase activity or specific DNA binding in M.EcoRII, which possesses one of the largest N-terminal sequences (98 residues) [36]. This is commensurate with our observation that a HNBB-treated protein does retain MTase activity. However, deletion of 97 amino acids in M.EcoRII resulted in a decrease in enzyme activity. Further deletions caused complete loss of activity. The N-terminus is a variable region present in many prokaryotic DNA (cytosine-5) methylases which plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA; it has been investigated in detail for the EcoRII methylase [36]. ÔPromiscuous domainsÕ are widespread components of many proteins; the fusions found may simply represent permutations and combinations of a set of common components and may not imply interactions [37]. Even though the organisms that harbor these members of restriction-modification (r-m) systems (R.NaeI, M.MspI and solitary M.SssI) are capable of being human parasites, it is unlikely that there could have been a fusion of two genes conferring MTase and topoisomerase activity in which the topoisomerase/ligase part was derived from a eukaryotic counterpart. Equally intriguing is the fact that topoisomer- ase activity is associated with the methylase/endonuclease with a 5¢-CG-3¢ in the recognition sequence. In the light of the similarities at the amino-acid level observed between MspI and ligase and topoisomerase, and the experimental observations presented here, it is debatable whether it was a single progenitor protein with different regions that diverged into different discrete activities (namely MTase and topo- isomerase) or it was duplication of genes [38] and their subsequent mixing up that caused a restriction-modification protein such as M.MspItoevolveinMoraxella,which possesses both methylation and topoisomerase activities. It is quite likely that a progenitor protein acquired muta- tions and possessed different recognition sequences, but with 5¢-CG-3¢ common to them. It is also likely that some of these mutations were common and led to incorporation of a topoisomerase/ligase-like region resulting in these activities, but, in some of them, the process was not complete as with NaeI, which still requires a leucine to be changed into a lysine. 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Biotechnol. 18,27. 38. Lauster, R. (1989) Evolution of type II DNA methyltransferases. A gene duplication model. J. Mol. Biol. 206, 313–321. Ó FEBS 2002 N-Terminal of MspI and topoisomerization (Eur. J. Biochem. 269) 2497 . topoisomerase I activity while the methyltransferase activity remains intact. Keywords: DNA binding; methyltransferase MspI; proteolysis; topoisomerase I. Methylation. Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi, India DNA cytosine methyltransferase MspI(M.MspI) must require