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
Trang 1The 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.MspI and M.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-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.MspI has 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: Biomolecular Research Institute, 343 Royal Parade,
Parkville, Victoria 3052, Australia.
(Received 8 February 2002, accepted 4 April 2002)
Trang 2DNA 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
E X P E R I M E N T A L P R O C E D U R E S
Bacterial strain and plasmid
Escherichia coli K-12 strain ER 1727 [(mcr BC–) hsd
RMS– (mrr)2::Tn10, mcrA1272::Tn10, F¢¢lacproAB lacIq
-(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 37C 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
1 mM, 2-mercaptoethanol 14 mM, glycerol 10%)
con-taining unlabeled 1459-bp BstXI DNA fragment of
/X174 as substrate (50 nM DNA) and 200 nM 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 37C 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 0.2M NH4HCO3, twice with
80% ethanol in 50 mM phosphate buffer (pH 8.0), and
once with 90% ethanol in 50 mM 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 mM sodium phosphate buffer (pH 10) at 25C The enzyme was quickly brought to pH 7.4 with buffer A [26] HNBB (1–5 mM) was added to 5 lMMspI 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) A410was measured An absorption coefficient
of 18 000M )1Æcm)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 mM HNBB in dimethyl sulfoxide is added to reaction mixture containing 5 lMMspI
in a reaction volume of 40 mL in 20 mMsodium phosphate buffer (pH 8.5) at 25C 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 mMNaCl
Optimization of trypsin concentration and reaction time for proteolysis Trypsin, dissolved in 20 mMHCl (20%, w/v), was used to cleave the M.MspI The tryptic digestion was previously optimized: 5% (w/v) trypsin, with 5 lM M.MspI in a reaction volume of 30 lL containing 50 mM Tris/HCl (pH 8.0), 2.0 mM EDTA, 1 mM 2-mercaptoethanol and
100 mM NaCl The cleavage reaction was terminated by adding 0.5 mM phenylmethanesulfonyl fluoride after
90 min of incubation at 37C 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 mM Tris/HCl,
pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 20% (v/v) glycerol] The assay was performed by incubating DNA (1 lg) and enzyme at 37C 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 37C 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
Trang 3the 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
AACAGGCGGAATCAGGTAAA-3¢ (W34A) and 5¢-AT
ATTCTAGAAAGCTAACCAGAATCAA-3¢ (Y74A)
The mutants were identified by DNA sequencing of the
plasmid DNA
Expression and purification of truncatedMsp 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
a site for the NdeI 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 coliBL21 DE3 plysS and induced as described above to
produce the recombinant truncated protein The truncated protein was purified as described above
R E S U L T S
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.MspI amino-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
Trang 4Proteolysis 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 molecumolecu-lar
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.MspI and the marker proteins were 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.
Trang 5proteolytic 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 truncatedMspI (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
D I S C U S S I O N
Alignment of the protein sequence of M.MspI with that of
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.
Trang 6interesting 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 aBLASTsearch, 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,
MspI and SssI methylase, recognize 5¢-CGCCGGC-3¢,
5¢-CCGG-3¢ and 5¢-CG-3¢, respectively The common
element in their recognition sequences is 5¢-CG-3¢ It is also
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 bsequenc-est are Plasmodium carinii and P falciparum;
both are human parasites Nocardia aerocolonigenes (the
host of NaeI) and Moraxella (host of M.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.MspI also
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.MspI and
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]
Promiscuous domains 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 methyltopoisomer-ase/endonucletopoisomer-ase 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.MspI to evolve in Moraxella, 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
A C K N O W L E D G E M E N T S
This work was supported by internal grants of the Department of Biochemical Engineering and Biotechnology-IIT, Delhi We thank Dr Richard J Roberts for research gifts of plasmid and host strain We thank Dr Jack Benner and Dr H Liu for sequencing the peptide fragments.
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