Using different fragments of pUC19 with varying numbers of GAGG sites fragments 3 to 6, or fragments not con-taining GAGG sites fragments 1 and 2, Table S1, as a substrates for the methy
Trang 1from Helicobacter pylori strain 26695 which methylates adjacent adenines on the same strand
Ritesh Kumar1, Asish K Mukhopadhyay2and Desirazu N Rao1
1 Department of Biochemistry, Indian Institute of Science, Bangalore, India
2 Division of Bacteriology, National Institute of Cholera and Enteric Disease, Kolkata, India
Introduction
DNA methylation is one of the most common forms
of DNA modification occurring in the prokaryotic
genome This modification does not affect the
Wat-son–Crick pairing, but creates a signature motif that
can be recognized by the proteins interacting with
DNA It has been shown that DNA methylation can
enhance or abrogate the affinity of transcription
fac-tors for DNA, thus affecting gene expression and
regu-lation These base modifications thus act as a second
line of genetic information [1]
Prokaryotic DNA methyltransferases (MTases) are
classified into two major groups – exocyclic amino
MTases and endocyclic MTases – based on the
position of the methyl group on the bases The exocy-clic amino MTases methylate adenine at the N6 position and cytosine at the N4 position, whereas endocyclic MTases methylate the cytosine at the C5 position [2,3] In prokaryotes most of the MTases are associated with a restriction enzyme and form a restriction-modification (R-M) system R-M systems are involved in the protection of bacteria from bacte-riophage invasion However, the identification of MTases without any associated restriction enzyme in many bacteria has compelled biologists to explore the functions of MTases beyond the distinction of self and nonself DNA Extensive work on solitary MTases,
Keywords
base flipping; DNA methyltransferase;
Helicobacter pylori; S-adenosyl- L -methionine;
site-directed mutagenesis
Correspondence
D N Rao, Department of Biochemistry,
Indian Institute of Science, Bangalore 560
012, India
Fax: +91 80 2360 814
Tel: +91 80 2293 2538
E-mail: dnrao@biochem.iisc.ernet.in
(Received 18 November 2009, revised 26
December 2009, accepted 25 January 2010)
doi:10.1111/j.1742-4658.2010.07593.x
Genomic sequences of Helicobacter pylori strains 26695, J99, HPAGI and G27 have revealed an abundance of restriction and modification genes hp0050, which encodes an N6adenine DNA methyltransferase, was cloned, overexpressed and purified to near homogeneity It recognizes the sequence 5¢-GRRG-3¢ (where R is A or G) and, most intriguingly, methylates both adenines when R is A (5¢-GAAG-3¢) Kinetic analysis suggests a nonpro-cessive (repeated-hit) mechanism of methylation in which HP0050 methyl-transferase methylates one adenine at a time in the sequence 5¢-GAAG-3¢ This is the first report of an N6 adenine DNA methyltransferase that methylates two adjacent residues on the same strand Interestingly, HP0050 homologs from two clinical strains of H pylori (PG227 and 128) methylate only 5¢-GAGG-3¢ compared with 5¢-GRRG-3¢ in strain 26695 HP0050 methyltransferase is highly conserved as it is present in more than 90% of
H pylori strains Inactivation of hp0050 in strain PG227 resulted in poor growth, suggesting its role in the biology of H pylori Collectively, these findings provide impetus for exploring the role(s) of this conserved DNA methyltransferase in the cellular processes of H pylori
Abbreviations
2AP, 2-aminopurine; AdoMet, S-adenosyl- L -methionine; Dam, DNA adenine methylase; DLS, dynamic light-scattering; IPTG, isopropyl thio-b- D -galactoside; KD, dissociation constant; LB, Luria–Bertani; MTase, methyltransferase; Rh, hydrodynamic radius; R-M, restriction-modification.
Trang 2such as DNA adenine methylase (Dam) and cell
cycle-regulated methylase (CcrM), have indeed shown the
role of DNA methylation in regulating cellular events
such as bacterial virulence, cell cycle regulation and
phase variation [4–6]
The Gram-negative bacterium Helicobacter pylori
persistently colonizes the human stomach and is
wide-spread throughout the world It is a major cause of
gastritis and peptic ulcer disease, and is an early risk
factor for gastric cancer H pylori is one of the most
genetically diverse species of bacteria, and
strain-spe-cific genetic diversity has been proposed to be involved
in the organism’s ability to cause different diseases
[7,8] Analysis of genome sequences of H pylori strains
26695 and J99 revealed the presence of 23 and 22 R-M
systems, respectively, far more than the number
detected in other bacterial genomes sequenced to date
[9–11] Two more H pylori strains – HPAG1 (isolated
from a patient with chronic atrophic gastritis) and
G27 – were sequenced and a similar number of
puta-tive R-M systems were identified [12,13] Comparison
of strains 26695 and J99 showed that the two genomes
are quite similar, with only 6–7% strain-specific genes
R-M systems are a major source of the strain
differ-ences [14] iceA-hpyIM, which encodes a cognate
restriction enzyme and an N6 adenine methylase has
been studied in various H pylori strains It was shown
that hpyIM expression is growth-phase regulated and
required for normal bacterial morphology Deletion of
hpyIM altered the expression of the stress-responsive
dnaK operon, suggesting that hpyIM may play a role
in H pylori physiology beyond its R-M function [15]
The Type II MTase, M.HpyAIV, which recognizes the
5¢-GANTC-3¢ site, has been shown to affect the
expression of the katA gene encoding the H pylori
catalase [16]
H pylori 26695 has three DNA MTases that lack
cognate restriction enzymes Vitkute et al [17] and Lin
et al [18] showed that HP0050, an orphan N6 adenine
MTase from H pylori 26695 recognizes 5¢-GAGG-3¢
and methylates adenine; these findings were based on
the results of a restriction endonuclease assay The
ORF hp0050 has been reported to be part of an R-M
system that contains two MTases and an inactive
restriction endonuclease This R-M system was later
assigned as HpyAVI, with hp0050 designated as
M1.HpyAVI, hp0051 as M2.HpyAVI and hp0052 as
HpyAVIP (putative) The hp0050 homolog of H pylori
strain HPAGI (HPAG1_0046) is a chronic atrophic
gastritis-associated gene [12]
Strain-specific DNA-modification genes are thought
to influence strain-specific phenotypic traits, host
speci-ficity, adaptability to changing micro-environmental
conditions or virulence [14] The identification and study of both species-specific and strain-specific MTases of H pylori could enhance our understanding
of the pathogenic mechanisms of this organism Our findings indicate that hp0050 from strain 26695 has evolved a relaxed specificity as a result of mutations, compared with other strains These observations fur-ther highlight the capability of this organism to undergo random mutations and evolve proteins with new functions
Results and Discussion HP0050 is an N6 adenine MTase from H pylori and belongs to the b subgroup of MTases, based on the linear arrangement of the S-adenosyl-l-methionine (AdoMet)-binding domain (FXGXG), the target rec-ognition domain and the catalytic domain (DPPY) HP0050 MTase is present in all the three sequenced strains of H pylori HP0050 MTase is present in all the three strains of H pylori (26695, J99 and HPAGI) for which genome has been sequenced The HP0050 proteins from H pylori J99 and HPAG1 have 91.7% and 90% identities respectively, to the HP0050 protein from H pylori 26695 [19] In H pylori 26695, hp0050 exists as an overlapping ORF with another MTase, hp0051 These MTases are remnant MTases of a defunct R-M system Both these ORFs have a high similarity with the MnlI DNA MTase that belongs to the Type IIS R-M system [20] However, in H pylori the functional MnlI restriction enzyme is absent [21]
Cloning, overexpression and purification of HP0050 protein
A 699 bp fragment (Fig S1A), representing the hp0050 gene from H pylori 26695, was PCR amplified using primers 1 and 2 (Table 1) and cloned between the BamHI and XhoI sites of the expression vector pET28a (data not shown) A polypeptide of the expected
Table 1 Primers used for cloning and mutagenesis The restriction enzyme site is indicated in bold letters SN, serial number.
SN Primer sequence (5¢- to 3¢)
Restriction site created(+) ⁄ lost(-)
5 TAGATCCTTCCATGGGGAGCGGCACCACCGGCT NcoI (+)
6 AACCGAAATGTTTAAAGGAGGGTCCGTGATGAT Psi I (-)
Trang 3molecular mass (32 kDa) was expressed at high levels
upon induction with 0.5 mm isopropyl
thio-b-d-galac-toside (IPTG) (Fig S1B) HP0050 was expressed as an
N-terminal His-tagged protein and was purified As
the purified HP0050 protein has an N-terminal
His-tag, western blot analysis was carried out with anti-His
IgG and a single band corresponding to HP0050
pro-tein was detected (Fig S1C) The His-tag was removed
using the Thrombin cleancleaveTM kit, according to
the manufacturer’s instructions (see the Experimental
procedures) The protein was purified to > 95%
homogeneity, as judged by SDS⁄ PAGE followed by
silver staining (Fig S1D)
Peptide finger mapping of HP0050
A peptide finger map of the HP0050 protein was
obtained by digesting purified HP0050 protein with
trypsin and subjecting it to MALDI analysis The
finger map thus obtained was then matched with
the expected finger map It was found that eight
pep-tide ions matched with the expected ions, as shown by
the asterisk in Fig S2A, suggesting the authenticity of
the purified protein
Oligomeric status of HP0050 protein
HP0050 protein elutes as a monomer, and the molecular
mass was determined to be 28 kDa by analytical
gel-fil-tration chromatography (Fig S2B) Dynamic
light-scat-tering (DLS) measurements on HP0050 MTase were
performed on a DynaPro DLS instrument using 20 lL
of 1.5 mgÆmL)1 of protein with a data-acquisition time
of 10 s Scattering intensities at various time intervals
(ls) with the initial (t = 0 s) intensity were compared
and a combined correlation function was constructed
(inset, Fig S2C) As seen in Fig S2C, DLS data, when
fitted to the Stokes–Einstein equation, gave a
hydrody-namic radius (Rh) of 2.2 nm The frictional ratio was
calculated as 0.89, suggesting that HP0050 is more or
less spherical in structure An ideal spherical protein
would give a value of 1.0 Higher values indicate an
anisotropic structure
Kinetics of methylation reaction
To establish the relationship between the initial
veloc-ity of the reaction and the enzyme concentration, the
rate of DNA methylation catalysed by HP0050 was
determined pUC19 DNA was used as a substrate
Different concentrations of HP0050 protein (10–
100 nm) were added to the reaction mixture containing
DNA (80 nm) and AdoMet (2.0 lm) and incubated at
37C When the initial velocities were plotted against increasing enzyme concentrations, a linear relationship was obtained (Fig 1A) This indicated that the initial velocity of the reaction was directly proportional to the enzyme concentration Next, the initial velocities were determined at various concentrations of the sub-strates, [3H]AdoMet and pUC19 DNA For the deter-mination of Km (DNA), a series of similar reactions containing HP0050 MTase (100 nm), [3H]AdoMet (2.0 lm) and increasing concentration of pUC19 DNA (10–80 nm) were performed and a conventional hyper-bolic dependence was obtained Nonlinear regression analysis of initial velocity versus DNA concentration established the Km (DNA)as 19.9 ± 3 nm (Fig 1B)
To determine Km (AdoMet), a series of reactions containing HP0050 MTase (100 nm), DNA (50 nm) and increasing concentration of [3H]AdoMet (0.3–
12 lm) were performed Increasing the concentration
of AdoMet led to a progressive stimulation in the reaction rate Whereas the initial portion of the concentration-dependence curve corresponded approx-imately to a conventional hyperbolic dependence, saturation was not achieved (Fig 1C) Similar obser-vations have been reported for T4 Dam and EcoDam [22,23]
Determination of site of methylation The recognition sequence of HP0050 MTase was previ-ously reported by Vitkute et al [17], based on restric-tion enzyme digesrestric-tion, to be 5¢-GAGG-3¢, where A is methylated by HP0050 MTase in the target site Using different fragments of pUC19 with varying numbers of GAGG sites (fragments 3 to 6), or fragments not con-taining GAGG sites (fragments 1 and 2, Table S1), as
a substrates for the methylation reaction by HP0050 MTase, it was observed that besides fragments with GAGG sites, fragment 2 (without GAGG site) was also methylated It should be noted that fragment 2 has one GAAG site, which could be a recognition site for HP0050 MTase
There are 20 GAAG and 13 GAGG sites per mole-cule of pUC19 To further confirm this observation we used 26 mer duplex substrates (Table 2) with GAGG (duplex 1), GGAG (duplex 2), GAAG (duplex 3), GTGG (duplex 4), GAGA (duplex 5) or GmAmAG (duplex 8) site to determine the specificity of HP0050 MTase It was found that HP0050 MTase recognized and methylated GAGG, GGAG and GAAG, but did not methylate GTGG, GmAmAG or GAGA (Fig 2)
As HP0050 was able to recognize and methylate both GAGG and GGAG, it was of interest to determine which A was the target for the MTase in the
Trang 4oligonu-cleotide with the GAAG sequence To address this, two 26-mer duplex substrates – one with 5¢- GAmAG-3¢ (duplex 6) and the other with 5¢-GmAAG-GAmAG-3¢ (duplex 7) (where mA is the methyl-adenine) site – were used individually as a substrate in the methylation assay It was found that HP0050 MTase was able to methylate duplex 6 and duplex 7, suggesting that both adenine residues were targets for HP0050 MTase (Fig 2), and
Table 2 Duplex DNA used in this study The underlined region of the oligonucleotide represents the HP0050 MTase recognition sequence, and restriction enzyme sites are shown in bold 2, 2-amino purine; Bt, biotin; mA, methyl adenine.
Duplex Sequence (5¢- to 3¢)
ATGTTACATGGCTCCTAGATAACTAG
ATGTTACATGGCCTCTAGATAACTAG
ATGTTACATGGCTTCTAGATAACTAG
ATGTTACATGGCACCTAGATAACTAG
ATGTTACATGGCTCTTAGATAACTAG
ATGTTACATGGCTTCTAGATAACTAG
ATGTTACATGGCTTCTAGATAACTAG
ATGTTACATGGCTTCTAGATAACTAG
ATGTTACATGGCTCCTAGATAACTAG
ATGTTACATGAGCTTCGATAGATAACTAG
ATGTTACATAGTACTTCATGAGATAACTAG
ATGTTACATAGCGCTTCGCGAGATAACTAG
GAAGCTGATCGAGTC ATGTTACATGAGCTCGATCTATAGATAAACCTT CGACTAGCTCAG
ATGATGTTGT TATGACATGGCTCCGACGCTAGATCCAGACGAC TCCTACTACAACA
ATGTTACATGGCTTCTAGATAACTAG
GAGGATGATGTTGT TATGACATGGCTCCGACGCTAGATCCAGACGAC TCCTACTACAACA
ACGCTCCTACCAGACAGCTTCGACTACAA
ATGTTACATGGCTTCGAGATAACTAG
A
B
C
600
400
200
0
500
400
300
200
100
0
2500
2000
1500
1000
500
0
HP0050 (n M )
pUC19 DNA (n M )
AdoMET (µ M )
Fig 1 Kinetics of methylation (A) Initial ,velocity versus the
con-centration of HP0050 MTase Increasing concon-centrations of HP0050
MTase (10–100 n M ) were incubated with 80 n M pUC19 and 2.0 l M
AdoMet in standard reaction buffer at 37 C for 15 min, then the
reaction was stopped and analyzed as described in the
Experimen-tal procedures (B) Determination of Km (DNA) Methylation assays
were carried out in reactions containing 2.0 l M [3H]AdoMet and
increasing concentrations of pUC19 DNA (10–80 n M ) in standard
reaction buffer at 37 C for 15 min HP0050 MTase (100 n M ) was
added to start the reaction The data points were analysed using
nonlinear regression analysis (C) Initial velocity versus the
concen-tration of AdoMet Methylation assays were carried out in
reactions containing 50 n M pUC19 DNA and increasing
concentra-tions of [ 3 H] AdoMet (0.3–12 l M ) in standard reaction buffer at
37 C for 15 min HP0050 MTase (100 n M ) was added to start the
reaction.
Trang 5this was carried out at different protein concentrations
(data not shown)
Experiments were then performed to estimate the
kinetic constants for these DNA substrates by
nonlin-ear regression analysis The duplex with GAGG was
the substrate most preferred, with a Kmof 5.2 lm, and
DNA with a GAAG site was a preferred substrate over
DNA with GGAG, GAmAG or GmAAG sites, with a
Km of 13 lm compared with Km values of 17 lm,
27 lm or 29 lm, respectively (Table 3) In addition, the
kcat⁄ Km(specificity constant) was calculated for
differ-ent DNA substrates and it was found that the
specific-ity constant for duplex 1 was 10 times higher than the
specificity constant for duplex 2, suggesting that duplex
1 was a better substrate than duplex 2 (Table 3) The
specificity constant for duplex 3 was 2.1-fold higher
than the specificity constant for duplex 2, and the Km
values were very similar, which again suggests that both
the adenines are methylated by HP0050 MTase
Furthermore, to confirm the observation that both
adenines in GAAG are methylated by HP0050 MTase,
duplex 10 (Table 2) was used as a substrate Duplex 10
contains an HP0050 MTase recognition sequence (GAAG) with overlapping AluI (AGCT) and TaqI (TCGA) restriction sites Upon methylation with HP0050 MTase if both adenine bases were modified, the DNA would be resistant to both AluI and TaqI digestion It is clear from Fig 3A that the methylated duplex is resistant to restriction with AluI and TaqI, confirming that HP0050 MTase indeed methylates both the adenines in 5¢-GAAG-3¢ Furthermore, duplex 11 (Table 2) was used, which contains an HP0050 MTase site (GAAG) with an overlapping ScaI site (AG-TACT), as a substrate in the methylation assays Upon methylation, if the second adenine was methylated in GAAG, the DNA would be resistant to ScaI digestion
It was found that upon methylation with HP0050 MTase the duplex DNA was resistant to ScaI digestion (Fig S3A) It is possible that HP0050 MTase binds strongly to the duplex and thus inhibits the cleavage
To rule out this possibility we used duplex 12, which contains an HP0050 MTase site (GAAG) with an over-lapping AfeI site (AGCGCT), as a substrate in the methylation assays AfeI is not sensitive to the methyla-tion status of adenine in its cognate sequence It was found that, upon methylation, the duplex DNA was sensitive to digestion with AfeI (Fig 3B) In addition, duplex 13 was used, which contains two AluI sites – one overlapping with the HP0050 MTase site (GAAG) and other 15 bp away from it When duplex 13 was methylated by HP0050 MTase and then digested with AluI, two fragments were obtained It was observed that, upon methylation, the AluI site overlapping with the HP0050 MTase cognate sequence became resistant
to AluI digestion However, three fragments of same size were obtained when unmethylated duplex 13 was digested with AluI (Fig S3B) To eliminate the possibil-ity that AluI is blocked by modification immediately outside its recognition site, duplex 18 was used Duplex
18 has a GAAG site with an overlapping AluI site and
in which the first A was methylated (GmAAG) Duplex
18 was completely digested with AluI, suggesting that the modification immediately outside the recognition site of AluI has no effect on its activity (Fig S3C)
1600
1200
800
400
0
Oligonucleotide (µ M )
GGAG GmAAG GAmAG GTGG GAGA GmAmAG
Fig 2 Specificity of HP0050 MTase (A) Methylation activity of
HP0050 MTase as a function of increasing concentrations of
differ-ent 26-mer duplex DNA species Methylation assays were carried
out in reactions containing 2.0 l M [ 3 H]AdoMet and increasing
con-centrations of 26-mer duplex DNA (2.5–15 l M ), with one GAGG site
or with a modified GAGG site, in standard reaction buffer at 37 C.
HP0050 MTase (100 n M ) was added to start the reaction (d,
GAGG; , GAAG; , GGAG; , GmAAG; r, GAmAG; s, GTGG; h,
GAGA; D, GmAmAG) mA, methyl adenine.
Table 3 Kinetic parameters for HP0050 N 6 adenine methyltransferase.
Trang 6To further confirm the methylation of adjacent
ade-nines in GAAG, we used duplex 17, which contains a
FokI site (GGATG) and GAAG, which is 7 bp away
from the FokI site Duplex 17 was used as a substrate
and the methylation reaction was carried out in the
presence of [3H] AdoMet The methylated duplex was
purified and then digested by FokI, which resulted in
two fragments, each containing one adenine from
GAAG These fragments were separated by
electro-phoresis on a 20% polyacrylamide gel and then
checked for the incorporation of radiolabel It was
found that both fragments were labelled, confirming
that HP0050 MTase indeed methylates both the
ade-nines in 5¢-GAAG -3¢ (Fig 4) It is worth mentioning
here that the Type IIS MnlI R-M system, comprising
N6 adenine and C5 cytosine MTase and a restriction
endonuclease, recognizes the nonpalindromic
nucleo-tide sequence 5¢-CCTC(N)7 ⁄ 6-3¢ While the C5 MTase modifies the first cytosine base within the 5¢-CCTC-3¢ sequence, the N6 adenine MTase methylates the bottom strand of the MnlI target, resulting in 5¢-G mAGG Interestingly, these two MTases share the greatest degree of similarity with HP0050 MTase and HP0051 MTase from H pylori 26695 [20] In the case of the FokI MTase, two domains are responsible for methylating two adenine residues – one in the upper strand and one in the lower strand [24].Yet another variation is seen in the case of MmeI, where it has been reported that MmeI modifies the adenine in the top strand of the recognition sequence 5¢-TCC RAC-3¢ and uses modification only on one of the two DNA strands for host protection [25] Interestingly, M.Alw261, M.Eco31l and M.Esp3l methylate both strands of their recognition sites, yielding C5 methyl
AluI TaqI
*
29 mer
12 mer/13 mer
Unmethylated duplex 12
Methylated duplex 12 M
50 bp
30 bp
A
B
Fig 3 Comparison of restriction digestion patterns of methylated and unmethylated duplex DNA (A) Restriction digestion of 29-mer duplex
10 M, molecular mass marker, AluI and TaqI denote digestion of duplex 10 with these respective enzymes Schematic representation of the 29-mer duplex 10 is shown with HP0050 MTase and overlapping AluI and TaqI sites (B) Restriction digestion of the 30-mer duplex 12 AfeI denotes digestion of duplex 12 Schematic representation of the 30-mer duplex 12 is shown with HP0050 MTase and an overlapping AfeI site The underlined region of the oligonucleotide represents the HP0050 MTase recognition sequence UD, undigested duplex.
* Corresponds to a 50-bp band in the marker.
Trang 7cytosine and N6 methyl adenine on opposite strands
[26] To the best of our knowledge, M.CviPII is the
only other DNA MTase that modifies adjacent
resi-dues in the cognate sequence In addition to modifyng
the first cytosine in CCD (D = A, G or T) sequences,
M.CviPII also modifies both the cytosines in CCAA
and CCCG sites [27]
Processivity of DNA methylation
HP0050 MTase methylates adjacent adenines in
GAAG The methylation can take place either in a
sin-gle binding event or in two separate binding events
To address this, 100 nm HP0050 MTase was
pre-incu-bated with 5 lm AdoMet for 10 min at 37C to
pro-mote the formation of the protein–AdoMet complex
This complex was then made catalytically competent
by adding 2 lm duplex 15 and incubated for an
addi-tional 5 min on ice to allow the formation of a ternary
complex Following the second incubation, the
reac-tion mixture was split in two and 40 lm duplex 3 was
added in one set as a trap and the other set was
allowed to proceed without the DNA trap Both the
reaction mixes were incubated at 25C, and reaction
aliquots withdrawn at 2 min intervals were checked for
methylation The reaction mixes were incubated at
25C in order to decrease the turnover rate so that
the first turnover could be monitored
If HP0050 MTase methylates in a nonprocessive
manner, it would dissociate from the substrate
mole-cule after each round of methylation and would
re-associate in the next round of catalysis If, however,
the MTase works in a processive manner, then it would
dissociate from the substrate molecule after methylating both the adenines in GAAG (duplex 15) A biotin– avidin microplate assay was used to separate biotiny-lated substrate from nonbiotinybiotiny-lated duplex DNA and
to monitor the methylation of biotinylated substrate The addition of a molar excess of duplex 3 to duplex
15 at different time-points of the modification reaction
of the GAAG substrate resulted in a decrease in the rate of methylation of duplex 15 (Fig 5A) This result clearly suggests that HP0050 methylates adjacent adenines in a nonprocessive manner
To determine if HP0050 MTase methylates the duplex with two recognition sites in a nonprocessive manner, we used duplex 16 containing two GAGG sites (duplex 14 with a 5¢ biotin tag) as a substrate and duplex 14 as competitor DNA The biotin–avidin mi-croplate assay was used to separate biotinylated sub-strate from nonbiotinylated duplex DNA and to monitor the methylation of biotinylated substrate It was observed that, in the presence of a 20-fold excess
of nonbiotinylated duplex DNA, the extent of the methylation reaction did not increase, but in the absence of nonbiotinylated competitor, methylation was observed (Fig 5B) This suggests a distributive mechanism of methylation In this assay, EcoDam was used as a positive control for the processive mecha-nism of methylation (data not shown)
Yet another approach was used to show the proces-sivity of HP0050 A 294 bp dsDNA containing a GAAG site with overlapping AluI and TaqI sites (simi-lar to duplex 10) was used for the methylation assay The master mix (400 lL) containing 1 lm HP0050 MTase was incubated with 5 lm [3H] AdoMet and
Duplex 17
FokI
29 bp
1
19 10
2200 ± 150
Fragment c.p.m.
Duplex 17
Fragment 1 (19 bp) 950 ± 100
Fragment 2 (10 bp) 850 ± 100
20% PAGE
Fig 4 Analysis of the methylation pattern
of HP0050 MTase Duplex 17 was
methylat-ed by HP0050 MTase using [ 3 H]AdoMet After cleavage with FokI, the DNA was electrophoresed through a 20% polyacryl-amide gel and specific restriction fragments (Fragments 1 and 2) were isolated The labelled methyl group contents of the fragments are shown in counts per minute (c.p.m.).
Trang 83 lm of 294 bp dsDNA at 25C (to decrease the
turn-over) Two aliquots (of 25 lL each) were withdrawn at
3-min intervals up to 15 min and the reactions were
stopped by snap-freezing in liquid nitrogen One
ali-quot from each time-point was analyzed for protection
from AluI digestion and the other for protection from
TaqI digestion (Fig 6) If HP0050 methylates adjacent
adenines in a processive manner there should not be
any difference in the resistance to AluI digestion and
to TaqI digestion However, if HP0050 methylates
adjacent adenines in a nonprocessive manner then the
substrate DNA should show early resistance to TaqI
digestion compared with AluI digestion
It is evident from Fig 6 that after 3 min the
sub-strate starts showing resistance to TaqI digestion but
shows AluI resistance only at the 6-min time-point
These results suggest that HP0050 methylates adjacent
residues in a nonprocessive manner In general, DNA
MTases accompanied with a restriction enzyme, such
as M.EcoRI exhibit a nonprocessive mechanism of
action, whereas solitary MTases, such as T4 Dam and
EcoDam, methylate DNA in a processive manner [3]
Purification and characterization of
AdoMet-binding motif (F195S) and catalytic
motif (Y32L) HP0050 mutant proteins
All N6adenine MTases have conserved characteristic
motifs such as the AdoMet-binding motif (FXGXG)
and the catalytic motif (DPPY) Several research groups have performed mutational studies on amino acids in these motifs, which, in turn, have revealed the significance of these motifs in catalysis [1,3] For instance, Pues et al [28] have shown in the case of M.TaqI that replacement of Y108 with alanine or glycine resulted in mutant MTases with reduced enzy-matic activities, which highlights the importance of tyrosine in the methylation activity It was shown that the replacement of F39 with alanine in the AdoMet-binding motif of M.EcoRV abrogated AdoMet AdoMet-binding [29] Site-directed mutagenesis was performed to replace F195 and Y32 of HP0050 MTase by serine and lysine, respectively Both the AdoMet-binding motif (F195S) and the catalytic motif (Y32L) HP0050 mutant proteins were purified to near homogeneity and analyzed on an SDS-polyacrylamide gel for altera-tions in their electrophoretic mobilities Both mutant proteins fractionated like the wild-type HP0050 protein and no apparent changes were detected To determine the size and subunit structure of the HP0050 mutant proteins in solution, gel-filtration chromatography was performed and it was found that the mutant proteins eluted as monomers with a molecular mass of 28 kDa (data not shown) Analysis of the wild-type, F195S and Y32L mutants did not reveal significant differ-ences in the CD spectra (data not shown), indicating that the amino acid exchanges did not affect the over-all structure of the mutant proteins
HP0050+AdoMet
HP0050-AdoMet Binary complex
at 37°C
Ternary complex at 4°C
+ competitor – competitor
0 2 4 6 8
Time (min)
Without competitor
Without competitor Competitor added at 0 min
Competitor added at 0 min Competitor added at 4 min
Competitor added at 4 min Competitor added at 8 min
Competitor added at 8 min
10 12 14 16 0 2 4 6 8
Time (min)
10 12 14 16
100
200 300
400
200 0
Fig 5 Nonprocessive methylation catalyzed by HP0050 MTase HP0050 MTase (100 n M ) was incubated with 5 l M [3H]AdoMet at 37 C for
10 min to facilitate formation of the HP0050–AdoMet binary complex and then the 2 l M duplex 15 or duplex 16 was added The mixture was incubated on ice for 5 min to allow the formation of a ternary complex Then, the mixture was divided into two sets and 40 l M duplex
3 or duplex 14 was added to one set at different time-points ( , 0 min; , 4 min; and , 8 min) and the other set was allowed to proceed without a DNA trap (d), as described in the Experimental procedures The reaction was monitored at 2-min time intervals by processing
25 lL of the reaction mixture in duplicate (A) Duplex 15; (B) duplex 16.
Trang 9The methylation activity of both the mutant
proteins was analysed as a function of increasing
enzyme concentration It was found that both the
mutant proteins were catalytically inactive compared
with wild-type HP0050 MTase (Fig 7A) The loss of
activity could be a result of the inability of these
mutant proteins to bind to one or both substrates To
investigate the AdoMet binding of the F195S mutant,
fluorescence emission spectra and fluorescence
intensi-ties were measured in the presence of different
concen-trations of AdoMet The F195S mutant protein
showed significantly less quenching in the presence of
AdoMet (up to 80 lm) compared with wild-type
HP0050 MTase The Ka value for AdoMet was
calcu-lated (using a modified Stern-Volmer plot) as 7 lm for
the wild-type protein and (using a Stern–Volmer plot)
as 64 lm for the F195S mutant (Fig S4), which is
nine times higher than that obtained for the wild-type
MTase This result showed that the F195S mutant was
not able to bind to the AdoMet as effectively as the
wild-type protein, therefore resulting in the loss of
activity When the Y32L mutant protein was analysed for its AdoMet-binding property, it was found to binds to AdoMet as efficiently as wild-type HP0050 MTase (Fig S4) but was catalytically inactive (Fig 7A)
DNA distortion induced by wild-type HP0050 MTase upon binding to 2-aminopurine-containing duplexes
Most DNA MTases flip the target base within the cog-nate sequence [30] The fluorescence of 2-aminopurine (2AP) is often used as a signal for base flipping because it shows enhanced fluorescence when its envi-ronment is perturbed However, it is now well estab-lished that the enhancement of 2AP fluorescence is a more general measure of DNA distortion [31] To study the change in DNA conformation in the enzyme–DNA complex, we used the 2AP fluorescence-based assay Irradiation of oligonucleotide (upper strand, duplex 9) containing 2AP at a target base instead of at an adenine base, at 320 nm produced a strong fluorescence emission spectrum with a kmax at
375 nm (Fig 7B) Annealing of this oligonucleotide with the complementary strand resulted in a decrease
of approximately threefold in fluorescence intensity at
375 nm When HP0050 MTase (100 nm) was incubated with 200 nm double-stranded 2AP DNA (duplex 9, Table 2), a fivefold increase in 2AP fluorescence was observed The increased fluorescence observed upon enzyme binding was more substantial than the fluores-cence of the single-stranded 2AP oligonucleotide This suggests that the increased fluorescence was not just caused by an enzyme-induced local unwinding of the helix resulting in a region of single-stranded DNA sur-rounding the 2AP, but possibly a result of DNA dis-tortion caused by binding of the protein The addition
of 1 lm sinefungin (an AdoMet analog) resulted in further enhancement of fluorescence Interestingly, the addition of sinefungin shifted the fluorescence emission spectrum 10 nm towards a longer wavelength This could be because of a change in the environment of the adenine base upon the addition of sinefungin By contrast, the Y32L mutant of the HP0050 MTase failed to show any increase in fluorescence, suggesting that, unlike the wild-type MTase, the mutant protein was not able to interact with DNA, and this could be the reason for being catalytically inactive When the F195S mutant was incubated with double-stranded 2AP DNA (duplex 9, Table 2), an increase in 2AP fluorescence was observed, but the addition of 1 lm sinefungin did not lead to further enhancement of fluorescence This is in agreement with the observation
TaqI
294 bp
150/146 bp
1.6% Agarose gel
AluI
0
10
20
30
40
50
Taql
Alul
Time (min)
Fig 6 Nonprocessive methylation of adjacent adenines in
5¢-GAAG-3¢ by HP0050 MTase HP0050 MTase (1 l M ) was incubated
with 5 l M [3H]AdoMet and 3 l M of 294-bp dsDNA at 25 Two
aliquots (each 25 lL) were withdrawn at 3-min intervals up to
15 min and the reactions were stopped by snap-freezing in liquid
nitrogen One of these aliquots was analyzed for protection from
digestion with AluI ( ) and the other was analyzed for protection
from digestion with TaqI (d).
Trang 10that the F195S protein does not bind AdoMet Using
the 2AP fluorescence-based assay, enhancements in
flu-orescence upon enzyme binding to canonical sequences
have been reported with other MTases, such as
EcoDam [32] and T4Dam [33]
Distribution of hp0050 in clinical H pylori isolates
The strains of H pylori (26695, J99 and HPAG1) for
which the genome sequence is available were isolated
from patients with superficial gastritis, duodenal ulcer
and chronic atrophic gastritis, respectively In the
pres-ent study a number of clinical isolates of H pylori
were screened for the presence of hp0050 hp0050 was
found to be present in 97.14% of strains obtained
from patients [n = 73 (Kolkata strains)] compared
with 90.63% of strains from healthy volunteers
[n = 32 (Santhali strains)] (data not shown) Primers 3
and 4 (Table 1) were used to amplify hp0050
homo-logs The functionality of HP0050 MTase in the strains
was checked by digestion with MnlI If a strain has a
functional MTase then the genomic DNA will be
resis-tant to digestion with MnlI It was found that all
strains which were positive for the presence of hp0050
by PCR were resistant to digestion with MnlI (data
not shown) Kolkata strains are H.pylori isolates from
patients suffering from ulcer, gastritis or cancer,
whereas Santhali strains are isolates from healthy
vol-unteers [34] The hp0050 gene from two clinical isolates
(strain PG227 isolated from a patient suffering from
duodenal ulcers and strain 128 isolated from a patient
with antral gastritis) was cloned into the BamHI and
XhoI sites of pET28a, overexpressed and the proteins purified as mentioned in the Experimental procedures Both were found to be as active as wild-type HP0050 MTase (from H pylori 26695), and, in the presence of
1 lm sinefungin, which is a competitive inhibitor of all MTases, methylation activity was inhibited by 70%, similarly to the wild-type MTase (data not shown) The hp0050 gene from H pylori strains PG227 and
128 was sequenced and found to be 89% similar to its homolog from strain 26695 (Fig 8A)
Interestingly, when HP0050 MTase homologs were checked for their specificity, it was found that HP0050 MTase from strains PG227 and 128 methylate GAGG but do not methylate GAAG or GGAG (Fig 8B)
A dot-blot assay was performed to further confirm this observation using duplexes 1, 2 and 3 (Table 2) (Fig 8C–D) These observations suggest that because
of mutations, hp0050 from strain 26695 has evolved relaxed specificity HP0050 MTase from strain 26695 is able to methylate GAAG and GGAG, whereas its homologs from strains PG227 and 128 lack this speci-ficity as they methylate only GAGG Because HP0050
is an orphan MTase and lacks a cognate restriction enzyme, it can afford to undergo mutations that result
in changed specificity
Isolation and characterization of the Dhp0050 derivative of H pylori
Transcriptional regulation by methylation patterns has been described for a number of prokaryotes, where promoter methylation alters the interaction of
Duplex 9 + wild type HP0050 MTase + Sf
a
Enzyme (n M )
Wild type
a
c Duplex 9 + F195S + Sf
d
F195S Y32L
Duplex 9 + wild type HP0050 MTase
d
Duplex 9 + Y32L
e
ss 2AP DNA
f g
Duplex 9
B A
Y32L F195S
550 440
330 220 110 0
Wavelength (nm)
600
400
200
0
Fig 7 Characterization of HP0050 MTase Y32L and F195S mutants (A) Initial velocity versus enzyme concentration Increasing concentra-tions of wild-type or mutant HP0050 MTase (10–80 n M ) were incubated with 80 n M pUC19 and 2.0 l M AdoMet in the presence of 10 m M
Tris ⁄ HCl, pH 8.0, containing 5 m M b-mercaptoethanol, at 37 C for 15 min The reactions were stopped and analyzed as described in the Experimental procedures ( ) wild type, (•) Y32L, ( ) F195S (B) Steady-state fluorescence emission spectra of 2AP-substituted DNA with HP0050 MTase Spectra were recorded after incubating 100 n M enzyme and 200 n M duplex 9 for 15 min on ice in 10 m M Tris ⁄ HCl, pH 8.0, containing 5 m M b-mercaptoethanol The total volume of the reaction mixture was 400 lL Curve a, HP0050 MTase with duplex 9 in the presence of 1 l M sinefungin; curve b, F195S mutant with duplex 9; curve c, F195S mutant with duplex 9 in the presence of 1 l M sinefungin; curve d, HP0050 MTase with duplex 9; curve e, Y32L mutant with duplex 9; curve f, 2AP ssDNA; curve g, duplex 9; curve h, HP0050 MTase with sinefungin.