Báo cáo khoa học: 2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase–substrate interaction docx

Gromova1 1 Chemistry Department, Moscow State University, Russia;2Engelghardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia EcoRII DNA methyltransferase M.E

2-Pyrimidinone as a probe for studying the Eco RII DNA

methyltransferase–substrate interaction

Oksana M Subach1, Anton V Khoroshaev1, Dmitrii N Gerasimov1, Vladimir B Baskunov1,

Anna K Shchyolkina2and Elizaveta S Gromova1

1

Chemistry Department, Moscow State University, Russia;2Engelghardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

EcoRII DNA methyltransferase (M.EcoRII) recognizes

the 5¢…CC*T/AGG…3¢ DNA sequence and catalyzes the

transfer of the methyl group from S-adenosyl-L-methionine

to the C5 position of the inner cytosine residue (C*) Here,

we study the mechanism of inhibition of M.EcoRII by DNA

containing 2-pyrimidinone, a cytosine analogue lacking an

NH2group at the C4 position of the pyrimidine ring Also,

DNA containing 2-pyrimidinone was used for probing

contacts of M.EcoRII with functional groups of pyrimidine

bases of the recognition sequence 2-Pyrimidinone was

incorporated into the 5¢…CCT/AGG…3¢ sequence

repla-cing the target and nontarget cytosine and central thymine

residues Study of the DNA stability using thermal

dena-turation of 2-pyrimidinone containing duplexes pointed

to the influence of the bases adjacent to 2-pyrimidinone

and to a greater destabilizing influence of 2-pyrimidinone

substitution for thymine than that for cytosine Binding of M.EcoRII to 2-pyrimidinone containing DNA and methy-lation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA–M.EcoRII interaction It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.Eco-RII In the absence of the cofactor S-adenosyl-L-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl-L-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs Keywords: 2-pyrimidinone; M.EcoRII; C5 cytosine DNA methyltransferase; inhibition; DNA recognition

DNA (cytosine-5)-methyltransferases (C5 MTases) catalyze

the transfer of a methyl group from S-adenosyl-L

-methio-nine (AdoMet) to cytosine C5 atom in specific DNA

sequences The methylation reaction of C5 MTases occurs

with the addition of a cysteine thiol group from the

conserved Pro-Cys motif to the C6 position of the target

cytosine, followed by methyl transfer from AdoMet to the

C5 position of the target base and the release of the

methylated substrate [1,2] (Fig 1A) It is important to note

that the target cytosine is flipped out of the DNA double

helix into the catalytic pocket of the enzyme and brought

into proximity of the cofactor [2]

Several cytosine analogues, 5-fluorocytosine (FC),

5-azacytosine (AzaC) and 2-pyrimidinone (2P), have been

reported as mechanism-based inhibitors of C5 MTases

[3–6] Introduction of a fluorine atom to the C5 position of

the target cytosine results in an irreversible covalent attack

of a cysteine residue and transfer of a methyl group to the C5 position of the target base [1] Replacement of C5 by a nitrogen atom in azacytosine (AzaC) facilitates nucleophilic attack of the cysteine residue at the C6 position which occurs in the presence or absence of AdoMet [5] Methyl or proton transfer to the N5 position occurs in the presence or absence of AdoMet, accordingly As a result, two structures

of the end product are possible: the enzyme linked to methylated AzaC or the enzyme linked to protonated AzaC [5] As there is no proton at C5 to take away when the N5 position becomes methylated an irreversible covalent com-plex is formed with the enzyme

2-Pyrimidinone (2P) is a cytosine analogue in which the exocyclic amino group is replaced by a hydrogen atom Removal of the exocyclic amino group from the cytosine results in an increase of reactivity at the C6 carbon atom in 2Pand in a reduction of the energy barrier for base flipping [7] 2P replacing the target cytosine in the recognition sequences for HhaI [6], MspI and HgaI-2 [7–9] C5 MTases evokes covalent bond formation with these MTases Zhou

et al [6] suggested the pathway of inhibition of C5 MTases

by 2P-containing DNA in the absence of AdoMet (Fig 1A) The reaction mechanism involves the addition

of a cysteine thiol group of the enzyme (from conserved Pro-Cys motif) to the C6 position of 2-pyrimidinone followed by proton transfer to the C5 position Due to the absence of the exocyclic amino group, b-elimination of the proton from the C5 position is retarded The mechanism of inhibition of C5 MTases by 2P-containing DNA in the

Correspondence to E S Gromova; Chemistry Department, Moscow

State University, Moscow, 119992, Russia Fax: + 7 095 939 31 81,

Tel.: + 7 095 939 31 44, E-mail: gromova@genebee.msu.ru

Abbreviations: C5 MTase, C5 cytosine DNA methyltransferase; FC,

5-fluorocytosine; AzaC, 5-azacytosine; M.EcoRII, EcoRII DNA

methyltransferase; AdoMet, S-adenosyl- L -methionine; AdoHcy,

S-adenosyl- L -homocysteine; 2P, 2-pyrimidinone.

Enzyme: EcoRII DNA methyltransferase (EC 2.1.1.37).

(Received 18 November 2003, revised 19 February 2004,

accepted 14 April 2004)

presence of AdoMet was not investigated It should be

noted that 1-(b-D-ribofuranosyl)-2-pyrimidinone, often

referred to as zebularine, was used in vivo as an antitumor

drug [10] By now it is clear that its antitumor properties are

likely attributed to inhibition of C5 MTase activity in tumor

cells 2-Pyrimidinone could also be considered as a mimic

of an intermediate in the minor deaminative pathway of

C5 MTases catalysis [6]

EcoRII DNA methyltransferase (M.EcoRII) catalyzes

the transfer of a methyl group from AdoMet to the C5

position of the inner deoxycytidine residue (C*) in the DNA

sequence 5¢…CC*T/AGG…3¢ It has been shown that

M.EcoRII forms an irreversible covalent complex with

DNA containing FC instead of the target cytosine [11,12]

Introduction of AzaC in this position also led to inhibition

of the enzyme [5] The mechanism of inhibition of

M.Eco-RII by 2P-containing DNA is still unknown Also, a study

of the contributions of different functional groups of the

5¢…CCT/AGG…3¢ sequence to specific interaction with

M.EcoRII is at the very beginning [13]

We aim to explore the effect of 2P substitution for C and

Twithin the recognition sequence of M.EcoRII on

recog-nition and catalysis performed by M.EcoRII In this work,

we have examined a DNA duplex containing

2-pyrimidi-none instead of the target cytosine as a potential inhibitor

of M.EcoRII, and also the role of AdoMet in the formation

of the covalent adduct between 2P-substituted DNA and

M.EcoRII Furthermore, we have studied the contribution

of the functional groups of pyrimidine residues in DNA–

M.EcoRII recognition by using duplexes containing

2-pyrimidinone in place of each pyrimidine base in the

5¢…CCT/AGG…3¢ sequence

Materials and methods

Chemicals and enzymes

AdoMet and AdoHcy were from Sigma (USA)

[CH3–3H]AdoMet (77 CiÆmmol)1) was from Amersham

(USA) [32P]ATP[cP] (1000 CiÆmmol)1) was from Izotop

(Obninsk, Russia) DNA-methyltransferase EcoRII (stock

solution, 52 lM) was overexpressed as an N-terminally

His6-tagged protein and chromatographically purified

on a nickel chelate column as described previously [14] T4 polynucleotide kinase was from MBI Fermentas (Vilnius, Lithuania)

Oligonucleotides Oligodeoxyribonucleotides containing 2-pyrimidinone were synthesized as described previously [15].32 P-phosphory-lation of the oligonucleotides was performed using T4-polynucleotide kinase and [32P]ATP[cP]

UV thermal denaturation and thermodynamic parameters

of duplex formation Heating of the samples containing 2.25 lMof duplexes in buffer A (40 mMTris/HCl, pH 7.9, 1 mMEDTA, 50 mM NaCl) at temperatures ranging from 15 to 85C was performed at a constant rate of 0.2CÆmin)1 Absorbance

of duplexes at 260 nm was measured using Cary 50 Bio spectrophotometer (Varian, Victoria, Australia) with tem-perature controller Thermodynamic analysis of helix-coil transition curves was performed using a two-state model The thermodynamic parameters were determined through a fitting procedure based on minimization of the integral mean square deviation between the theoretical transition curves and experimental absorbance data

Circular dichroism

CD measurements of duplexes (2.25 lM) in buffer A were performed with Mark V Jobin Yvon dichrograph (Paris, France) in 1 cm thermostated cells

Gel mobility-shift assay

To determine the active enzyme concentration, 20 nM M.EcoRII was incubated with 1–50 nM 32P-labeled DNA duplex I¢ in 20 lL of buffer B (40 mM Tris/HCl, pH 7.9,

5 mM dithiothreitol, 1 mM EDTA) containing 50 mM NaCl, 6% glycerol and 1 mMAdoHcy for 15 min at room temperature and 10 min at 0C The reaction mixtures

Fig 1 Proposed mechanism of inhibition of M.EcoRII by 2P-containing DNA duplexes in the absence [6] (A) or in the presence (B) of AdoMet In the case of M.EcoRII, the amino acid residue attacking the C6 position is Cys186 [11,12]; the general acid donating a proton to N3 is probably Glu233 [4,28].

were analysed by 8% native PAGE for 3 h at 120 V The gel

was prerun for 1 h at 100 V Autoradiographs of the gels

were prepared using Molecular Dynamics Phosphorimager

(Amersham Biosciences, USA) Radioactivities of

M.Eco-RIIỜDNA complex (cpmbound) and free DNA (cpmfree)

were determined The ratio of bound to free DNA was

calculated as (cpmbound)/(cpmfree); the concentration of

bound DNA was calculated as [S0][(cpmbound)/(cpmbound+

cpmfree)] Data were analyzed by linear regression using the

MicrocalORIGIN6.0 software For all further experiments

the concentration of active form of M.EcoRII was used

To determine the apparent dissociation constants (Kdapp),

1.5Ờ150 nM M.EcoRII were incubated with 15 nM 32

P-labeled DNA duplexes IỜIV in 15 lL of buffer B (40 mM

Tris/HCl, pH 7.9, 5 mMdithiothreitol, 1 mMEDTA,

con-taining 8% glycerol and 1 mMAdoHcy for 15 min at room

temperature and 15 min at 0C The reaction mixtures were

processed as described above Radioactivities of M.EcoRIIỜ

DNA complex (cpmbound) and free DNA (cpmfree) were

determined and the fraction of bound DNA was calculated

as (cpmbound)/(cpmbound+ cpmfree) Kdappwas calculated by

fitting the data to the following equation derived from a

standard bimolecular binding equilibrium as described [16]:

cpmbound

đcpm bound ợcpm free ỡỬ

ơES

ơS 0 

Ử A

2ơS 0  đơS0ợơE0ợK

app

d ỡ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi đơS 0 ợơE 0 ợKdappỡ24ơE 0 ơS 0  q

where [S0] is total DNA concentration; [E0] is total

M.EcoRII concentration; A is the factor accounting for

nonideal equilibrium conditions during electrophoresis

(cage effect, thermal dissociation) Nonlinear regression

was performed using MicrocalORIGIN6.0 software

Detection of DNA-enzyme adducts

All reactions of32P-labeled duplexes IỜIII (100 nM) with

M.EcoRII (200 nM) were performed in 15 lL of buffer B

containing 0.5 mMAdoMet or 0.5 mMAdoHcy Reaction

mixtures were incubated for 30 min at room temperature

and for 15 min on ice and processed by one of the following

ways: (a) incubated with 1% SDS at room temperature for

10 min and analyzed by 8% native PAGE; (b) incubated

with 1% SDS at 65C for 5 min and analyzed by 8% native

PAGE; (c) incubated with 0.8% SDS at room temperature

for 10 min and analyzed by 12% SDS/PAGE (Laemmli

gel)

For autoradiography of the electrophoretic pattern,

Kodak-XOMAT-S film was exposed with an intensifier

screen at)20 C overnight

Methylation assay

For determination of initial methylation rates (V0),

methy-lation reactions were performed in 26 lL of buffer B,

containing DNA duplexes IỜIV or ImỜIVm (1 lM),

M.Eco-RII (30 nM), and [CH3Ờ3H]-AdoMet (1.3 lM) Reactions

were started by adding the enzyme After a 1, 2, 3, 4 and

5 min incubation at 15C, 5 lL of reaction mixture were

pipetted onto DE81 (Whatman) paper disks The disks were

washed 5ở for 5 min with 50 mM KHPO, 3 min with

water and 3 min with ethanol, air dried and placed into

5 mL of ZHS-106 scintillation fluid The filter-bound radioactivity was measured on Tracor Analytic Delta 300 scintillation counter (ThermoQuest/CE Instruments, Piscataway, USA) and the amount of methylated DNA was determined as described [17] Data were analyzed by linear regression using the MicrocalORIGIN6.0 software For quantification of the transfer of methyl groups to a 2-pyrimidinone residue in DNA duplexes by M.EcoRII methylation reactions were performed in 10 lL of buffer B, containing duplexes I, Im, III, IIIm or V (500Ờ1000 nM), M.EcoRII (12.5Ờ1000 nM) and [CH3Ờ3H]-AdoMet (1.3 lM) In the case of M.HhaI methylation reactions were performed in 10 lL of buffer C (50 mMTris/HCl, pH 7.5,

5 mM 2-mercaptoethanol, 10 mM EDTA, 0.2 mg mL)1 bovine serum albumin), containing duplexes I, Vm or VIm (670 nM), M.HhaI (20Ờ16 000 nM) and [CH3Ờ3H]-AdoMet (0.42 lM) Reactions were started by adding the enzyme After a 30 min incubation at 15C (reactions with M.Eco-RII) or at 20C (reactions with M.HhaI), 8 lL of reaction mixture were pipetted onto DE81 (Whatman) paper disks and processed as described for determination of V0 The time-dependent methyltransferase assays were per-formed by incubating M.EcoRII (500 nM) with duplexes Im

or IIIm (500 nM) and [CH3Ờ3H]-AdoMet (1.3 lM) in buffer

B at 15C for the indicated time periods Reaction mixtures (8 lL) were pipetted onto DE81 (Whatman) paper disks and processed as described for determination of V0

Results and discussion

To elucidate the mechanism of inhibition of M.EcoRII

by 2-pyrimidinone modified DNA, and to understand the role of functional groups of pyrimidine bases of the recognition sequence in specific DNAỜM.EcoRII inter-action, a series of 2P-containing substrate analogues was synthesized (duplexes IIỜIV, Table 1 and duplexes IimỜIVm, Table 2)

Thermodynamics of formation of the2P-containing DNA Insertion of 2P in place of C or Tresulted in a marked destabilization of DNA duplexes [18Ờ21] To ascertain whether the 2P-containing DNA duplexes IIỜIV had a double helix structure under conditions of methylation by M.EcoRII, the thermodynamic stability of these duplexes was evaluated (Table 1) The values of the free energy of transition, as well as of the transition temperature (melting temperature, Tm), point to the following duplex stabilities:

I > II > III > IV In order to elucidate the contribution

of a single nucleotide substitution, the value of the transition free energy of the nonmodified duplex I was subtracted from that of the modified duplexes IIỜIV (Table 1, DDG) The greatest energy penalty, DDG, for substitution of 2P for Tin the TA base pair was 4.4 kcalẳmol)1 T he substitution of 2P for C appeared to be much more energetically disadvantageous in the base context C2PT (III) than in the context A2PC (II) The presence of only two H-bonds in the 2PG base pair vs the three H-bonds in the CG base pair [19] as well as electrostatic interactions of 2P in the duplexes [21] may largely contribute to the destabilization of the duplexes

Conformation of the2P-containing DNA

To answer the question of whether 2P substitution for

pyrimidines led to a marked distortion of DNA

conforma-tion, we compared CD spectra of duplexes I–IV (Fig 2)

There are minor changes in the CD spectra, predominantly

an intensity drop in the longwave CD band This may be attributed to a strongly reduced absorption of the 2P base at

260 nm [18,19], which eliminates a potential contribution of the nearest-neighbor stacking contacts involving the 2PG

or 2PA base pair to the CD signals in the corresponding spectral region The difference in the CD spectra (Fig 2, open symbols) reflects the decreased contribution of 2P to the conservative CD spectrum in comparison with the contributions of cytosine and thymine Thus, the observed minor dissimilarity in the CD spectra of duplexes II–IV from CD spectrum of duplex I can be attributed to the distinctive optical features of the 2P analog, rather than to a marked distortion of the DNA conformation

Determination of concentration of active form

of M.EcoRII

It is known that the concentration of the active form of DNA methyltransferases does not correspond accurately to the total protein concentration [5,22] The concentration of active form of M.EcoRII was estimated by titration of the enzyme (20 nM) with M.EcoRII substrate in the presence of AdoHcy (Fig 3, inset) The ratio of bound to free DNA was plotted vs concentration of bound DNA (a Scatchard plot, Fig 3) [23] An 18-mer DNA duplex 5¢-GAG CCAACCTGGCTCTGA-3¢/3¢-CTCGGTTGGACCGAG ACT-5¢ (I¢) was used as M.EcoRII substrate The horizontal axis intercept gives the total concentration of DNA binding sites (n[E0]) equal to 2.35 ± 0.12 nM (Fig 3) or 2.04 ± 0.25 (data not shown) As the number of DNA binding sites per molecule of enzyme (n) is 1 for M.EcoRII, the average concentration of M.EcoRII active form is 2.2 ± 0.2 nM So, the amount of enzyme bound to DNA was assumed to be only 11 ± 1% of the total enzyme molecules For all further experiments the concentration of active form of M.EcoRII was used

Binding and methylation of2P-containing DNA

by M.EcoRII Different heterocyclic base analogues have proven to be useful for studies of DNA–enzyme interactions [24] In order to investigate the influence of introducing 2P into the different positions of the substrate DNA on the sequence-specific interaction of M.EcoRII with DNA, we studied

Table 2 Relative initial methylation rates of hemimethylated

2P-con-taining DNA duplexes by M.EcoRII Relative initial methylation rates

(V rel

0 ) were calculated as ratio of V 0 of duplexes Iim–IVm to V 0 of

duplex Im M, 5-methylcytosine Recognition sequence is in bold; 2P is

underlined.

No DNA duplex V 0 (n M Æmin)1) V rel

0 (%)

Im 5 ¢ -GCCAA CCTGG CTCT-3 ¢/ 172.0 ± 34.4 100 ± 20

3 ¢ -CGGTT GGAMC GAGA-5 ¢

IIm 5 ¢ -GCCAA 2PCTGG CTCT-3 ¢/ 197.8 ± 53.3 115 ± 31

3 ¢ -CGGTT- GGAMC GAGA-5 ¢

IIIm 5 ¢ -GCCAA C2PTGG CTCT-3 ¢/ 0 0

3 ¢ -CGGTT G-GAMC GAGA-5 ¢

IVm 5 ¢ -GCCAA CC2PGG CTCT-3 ¢/ 34.4 ± 3.4 20 ± 2

3 ¢ -CGGTT GG-AMC GAGA-5 ¢

Fig 2 CD spectra of DNA duplexes I–IV ––, I; –d–, II; –m–, III;

–j–, IV Difference of CD spectra of I and duplexes: –s–, II; –n–, III;

–h–, IV Temperature was 20 C.

Table 1 Thermodynamic parameters of formation of 2P-containing DNA duplexes determined from thermal denaturation curves Thermodynamic parameters and their standard deviations were determined from fitting the theoretical melting curves to experimental curves (see Materials and methods) Standard deviation of DS was less than 0.1 calÆmol)1ÆK)1 Experimental conditions see Materials and methods 2P, 2-pyrimidinone;

T m , melting temperature Recognition sequence is in bold; 2P is underlined.

No DNA duplex

T m (C)

DH (kcalÆmol)1)

DS (kcalÆmol)1ÆK)1)

DG (T ¼ 20 C) (kcalÆmol)1)

DDG (T ¼ 20 C) (kcalÆmol)1)

I 5 ¢ -GCCAA CCTGG CTCT-3 ¢/ 52.6 ± 0.1 )65.0 ± 0.6 )173 )14.3 ± 0.6 –

3 ¢ -CGGTT GGACC GAGA-5 ¢

II 5 ¢ -GCCAA 2PCTGG CTCT-3 ¢/ 46.0 ± 0.1 )43.9 ± 0.7 )111 )11.5 ± 0.7 2.8

3 ¢ -CGGTT- GGACC GAGA-5 ¢

III 5 ¢ -GCCAA C2PTGG CTCT-3 ¢/ 41.4 ± 0.1 )36.3 ± 0.4 )88 )10.3 ± 0.4 4

3 ¢ -CGGTT G-GACC GAGA-5 ¢

IV 5 ¢ -GCCAA CC2PGG CTCT-3 ¢/ 35.4 ± 0.1 )41.7 ± 0.7 )106 )9.9 ± 0.7 4.4

3 ¢ -CGGTT GG-ACCG AGA-5 ¢

binding and methylation of canonical (I) and the

2P-containing DNA duplexes (II-IV) with M.EcoRII (Table 3)

The formation of complexes was monitored by gel

mobility-shift assays in the presence of AdoHcy because of the

known increase in the affinity of M.EcoRII for DNA in the

presence of the cofactor [12] DNA duplexes were incubated

with increasing M.EcoRII concentrations at saturating

AdoHcy concentrations A binding isotherm and

corres-ponding autoradiograph of a typical experiment are shown

in Fig 4 The calculated apparent dissociation constants

(Kdapp) are summarized in Table 3

The methylation reactions were performed under

steady-state conditions (Tables 2 and 3) Reactions of M.EcoRII

with hemimethylated duplexes Im–IVm were performed in

order to determine the influence of the 2P on methylation of

2P-containing strands of the DNA duplexes (Table 2)

M.EcoRII binds to the substrate analogue II with the

same affinity as to the parent duplex I (Table 3)

Replace-ment of the outer C (duplexes II and IIm) by 2P does not

affect methylation of either strand (II) or the 2P-containing

strand (IIm) (Tables 2 and 3) According to the

thermo-dynamic and conformational analysis of the 2P-containing

duplexes, the substitution of 2P for C could be a good probe

for DNA–protein interactions in the major groove of DNA Substitution of 2P for C appears to cause a small destabilizing effect and duplex II is conformationally similar

to duplex I The explanation of the observed equal binding affinities and methylation rates can be attributed to the difference in the chemical structure between 2P and C Therefore, the 4-NH2group of the outer cytosine residue in the recognition sequence is not likely to be essential for sequence-specific DNA binding by M.EcoRII In contrast, 2Psubstitution for both nontarget cytosine residues in the recognition sequence of MspI and HpaII C5 MTases prevents these MTases from binding, and the C4 amino functional groups of the nontarget cytosine residues are essential for DNA binding by these MTases [25]

M.EcoRII binds to duplex III containing 2P in place of the target cytosine with a Kdapp similar to that of duplex I (Table 3) It could be that the 4-NH2 group of the target cytosine is not essential for recognition of DNA by M.EcoRII as was suggested for several other MTases, as base flipping probably occurs with any base at the target position [26] However, in the case of the M.EcoRII complex with duplex III such a simple conclusion is ambiguous because in addition to the noncovalent complex a stable

Fig 3 Scatchard plot of the ratio of bound to

free DNA substrate vs concentration of bound

DNA substrate Inset: autoradiograph of gel

shift assay of M.EcoRII with DNA substrate

I¢ Lanes 1–4: 20 n M M.EcoRII with 1 m M

AdoHcy and increasing concentrations of

duplex I¢ (1, 2, 3.5 and 10 n M ).

Table 3 Binding and substrate properties of 2P-containing DNA duplexes Apparent dissociation constants (Kdapp) of complex M.EcoRII–DNA– AdoHcy were calculated as described in Materials and methods Relative Kdapp[Kdapp(rel.)] were calculated as ratio of Kdappof duplexes II–IV to

Kdappof duplex I Relative initial methylation rates (V rel

0 ) were calculated as ratio of V 0 of duplexes II–IV to V 0 of duplex I Recognition sequence is

in bold; 2P is underlined.

0 (%)

I 5 ¢ -GCCAA CCTGG CTCT-3 ¢/ 4.9 ± 1.8 100 ± 37 195.6 ± 39.1 100 ± 20

3 ¢ -CGGTT GGACC GAGA-5 ¢

II 5 ¢ -GCCAA 2PCTGG CTCT-3 ¢/ 3.9 ± 1.4 80 ± 28 170.2 ± 56.7 87 ± 29

3 ¢ -CGGTT- GGACC GAGA-5 ¢

III 5 ¢ -GCCAA C2PTGG CTCT-3 ¢/ 5.3 ± 2.4 108 ± 49 3.5 ± 0.3 1.8 ± 0.1

3 ¢ -CGGTT G-GACC GAGA-5 ¢

IV 5 ¢ -GCCAA CC2PGG CTCT-3 ¢/ 96.0 ± 35.6 1959 ± 726 41.1 ± 11.7 21 ± 6

3 ¢ -CGGTT GG-ACC GAGA-5 ¢

covalent M.EcoRII complex with duplex III can be formed

(see below) Thus, the Kdappobtained does not represent true

binding affinity of M.EcoRII to duplex III In the case of

duplexes III and IIIm, methylation was essentially not

detected under steady-state conditions The transfer of a

methyl group is blocked or occurs at very low levels

In the case of duplex IV containing 2P in place of the

central Tin the recognition sequence, we observed a

substantial increase in Kdapp (Table 3) Initial methylation

rates of both strands (IV) or 2P-containing strand (IVm)

were decreased (Tables 2 and 3) It is probable that the

decrease in V0is attributed to the weak binding affinity of

these substrate analogues to the enzyme Recently, it was

suggested that ATvs GC discrimination is achieved by

interactions between the large domain of M.EcoRII and the

minor groove of DNA [13] M.EcoRII did not bind to a

substrate analogue with 2-aminopurine having been

substi-tuted for adenine (M G Brevnov, O A Rechkoblit and

E S Gromova, unpublished results) Hence, the

appear-ance of the amino group in the minor groove in the case of

2-aminopurine for adenine substitution, disturbs the

recog-nition of the specific DNA sequence by M.EcoRII This is in

agreement with the role of the minor groove in substrate

recognition by M.EcoRII [13] In the case of a substitution

of Tby 2P (duplex IV), the pattern of functional groups

exposed into the minor groove remains the same, with the

groups of the central thymine residue exposed into the

major groove being disturbed Therefore, it is likely that

weak binding of duplex IV to M.EcoRII may be attributed

to the elimination of some DNA–protein contacts in the

major groove of the double helix Alternatively, this effect

may be caused by a greater destabilization of duplex IV in

comparison with duplexes II and III (Table 1) However,

the conformations of duplexes I–IV are similar It has also

been shown that substitution of ATby CI in the 5¢…GGT/

ACC…3¢ sequence for SinI C5 MTase led to a considerable

increase in Km[13] This observation corresponds to our suggestion that specific contacts of C5 MTases with the central base pair could be mediated by contacts not only in the minor but also in the major groove

Comparison of methylation of unmethylated and hemi-methylated DNA duplexes (Tables 2 and 3) permits us to speculate about influence of 2P on methylation of unmodi-fied DNA strand in duplexes II–IV Equal methylation rates

of duplexes II and IIm allow us to suggest that rates of methylation of unmodified and 2P-containing strands in duplex II are virtually the same Analogously, we suppose equal methylation rates of unmodified and 2P-containing strands in duplex IV The unmodified strand in duplex III was not methylated under steady-state conditions – prob-ably due to formation of the stable covalent adduct of M.EcoRII with 2P-containing strand

Mechanism-based inhibition of M.EcoRII

by2P-containing DNA

To examine the possibility of covalent adduct formation between M.EcoRII and DNA containing 2P in place of the target C in the presence of AdoMet or AdoHcy, duplex III was incubated with the enzyme The resulting samples were analyzed under different conditions First, the enzyme was denaturated by adding SDS to a final concentration of 1% and subjected (or not) to heating with a subsequent analysis

by 8% native PAGE (Fig 5A) Without heating in the presence of AdoHcy, a small part of the noncovalent complex remains in the case of canonical duplex I and duplex II in which the outer cytosine residue of the recognition sequence was replaced by 2P, however, this complex is absent after heating We did not observe the formation of the covalent adduct in the case of duplex II in the presence of AdoMet Figure 5A (lanes 4, 5 and 9, 10) demonstrates that duplex III forms in the presence or in the absence of AdoMet a covalent intermediate with M.EcoRII stable to heating at 65C for 5 min Thus, 2-pyrimidinone for the target C substitution results in the inhibition of M.EcoRII

The adducts of 2P-containing duplex III with M.EcoRII obtained in the presence of AdoHcy or AdoMet are not resistant to heating in SDS solution at 90C for 5 min (data not shown) or to the addition of SDS and analysis by SDS/ PAGE (Fig 5B, lanes 6 and 10) However, we observed products moving faster than the protein and slower than the oligonucleotides These products seem to be oligonucleo-tides generated from the duplex III–M.EcoRII adducts The SDS gel (Laemmli) exhibits two components at different pH: an upper part at pH 6.8 (stacking gel) and a lower part

at pH 8.8 (separating gel) (Fig 5B) Due to a pH change from 6.8 to 8.8, b-elimination of the proton from the C5 position of 2P and dissociation of the covalent intermediates

of M.EcoRII and duplex III take place The appearance of the slowly moving oligonucleotides is attributed to retarda-tion of the duplex III–M.EcoRII covalent intermediates in the upper part of the gel before dissociation

It is interesting to compare the stabilities of the adducts of C5 MTases with DNA duplexes containing AzaC, FC or 2Pin place of the target C in the presence of AdoMet The adducts of M.EcoRII with AzaC DNA [5] and M.HhaI with FC DNA [27] are resistant to heating in SDS solution

Fig 4 Binding of M.EcoRII to DNA duplex I in the presence of

AdoHcy Relative amount of M.EcoRII–DNA–AdoHcy complex

obtained from the gel-shift autoradiograph vs protein concentration is

plotted M.EcoRII (1.5–94 n M ) was incubated with duplex I (15 n M ) in

the presence of AdoHcy (1 m M ) Inset, autoradiograph of gel-shift

assay of M.EcoRII with duplex I Lanes: 1–8, duplex I with 1 m M

AdoHcy and increasing concentrations of M.EcoRII (5, 6, 7, 10, 40,

50, 62.5 and 78 n M ); 9, duplex I.

and analysis by SDS/PAGE In contrast, the covalent

intermediates of M.EcoRII with 2-pyrimidinone (duplex

III) dissociate upon heating in SDS solution or during

analysis by SDS/PAGE, probably because of b-elimination

of the proton from the C5 position of 2-pyrimidinone

Analysis of possibility of 2-pyrimidinone methylation

To clarify the role of AdoMet in the formation of the

covalent adduct between 2P-containing DNA and

M.Eco-RII it is important to examine the possibility of a methyl

group transfer to the 2P residue 2P-modified DNA was

not methylated by MspI and HhaI C5 MTases [6] We

also did not observe the methylation of duplex III under

steady-state conditions (Table 3) However, the proposed

mechanism of inhibition of C5 MTases by 2-pyrimidinone

[6] does not contradict the transfer of a methyl group to

the 2P residue

The possibility of the methylation of DNA duplexes containing 2P in place of the target cytosine (III and IIIm)

by M.EcoRII was tested at different enzyme concentrations

in the presence of AdoMet at 15C for 30 min (Fig 6) Hemimethylated duplex IIIm was used to exclude methy-lation of the unmodified strand Under the same conditions, methylation of canonical duplexes I and Im was performed The increase of enzyme concentration favoured methylation

of 2P-containing DNA duplexes III and IIIm (Fig 6) No methyl transfer was detected at all enzyme concentrations in the case of duplex 5¢-GAGCCAAGCGCACTCTGA-3¢/ 3¢-CTCGGTTCGCGTGAGACT-5¢(V) lacking the

EcoR-II recognition sequence (Fig 6) There was also no methy-lation in a control sample containing the same amount of enzyme and AdoMet but no DNA Methylation of duplex III may be due to methyl transfer to the target unmethylated cytosine residue However, this is impossible in the case of duplex IIIm Hence, one can suggest that a methyl group transfer occurs to the 2-pyrimidinone base

The methylation of duplex IIIm may be stopped at the stage of formation of the covalent intermediate (Fig 1B, step1) or may proceed with dissociation of the covalent intermediate and release of the methylated 2P-containing DNA (Fig 1B, step2) In the first case, the quantity of methyl groups incorporated into duplex IIIm should correspond to the quantity of methyl groups incorporated into canonical DNA after the first turnover of the methylation reaction In the second case, we should observe more than one turnover

of the methylation reaction for duplex IIIm To clarify the nature of this new effect, we compared the dependence of methylation of duplexes Im and IIIm on an enzyme concentration (Fig 6) Complete methylation of duplex Im was observed even at low enzyme concentration M.EcoRII transfers the methyl group to unmodified DNA strand, turns

Fig 6 Dependence of methylation of unmethylated (III), hemimethyl-ated (Im and IIIm) and nonspecific (V) DNA duplexes on concentration of M.EcoRII M.EcoRII was incubated with indicated duplexes (500 n M )

in buffer B in the presence of [CH 3 – 3 H]AdoMet (1.3 l M ) at 15 C for

30 min Relative methylation was calculated as the ratio of radio-activities of duplexes Im, III, IIIm and V to the radioactivity of duplex I Methylation of duplex I (not shown) was accepted as 100% s, Canonical duplex Im; j, duplex III; d, duplex IIIm and m, duplex V.

Fig 5 Covalent adduct formation of M.EcoRII with DNA duplexes

I–III in the presence of AdoMet or AdoHcy M.EcoRII (200 n M ) was

incubated with indicated duplexes (100 n M ) in buffer B in the presence

of AdoHcy or AdoMet (0.5 m M ) (A) Autoradiograph of 8% native

PAGE Reactions were incubated with 1% SDS and heated for 5 min

at 65 C prior to electrophoresis if indicated Lanes: 1, without

M.EcoRII; 2–11, with M.EcoRII (B) Autoradiograph of 12% SDS/

PAGE (Laemmli) Reactions were incubated with 0.8% SDS prior to

electrophoresis After autoradiography gel was stained with

Coomas-sie Blue G-250, protein band is indicated by arrow; Enz is for

M.EcoRII Composition of the reaction mixtures is indicated on the

top of the gels; III T (lane 1) is the upper strand of the duplex III.

Stacking (upper) and separating (lower) components of the gel are

shown schematically.

over several times and, as a result, methylates all target

cytosine residues for 30 min The observed level of

methy-lation of duplex IIIm was low There was a linear increase of

methylation with the increase of the enzyme concentration

This effect may be due to the arrest of the reaction after one

turnover One can suggest that the stable covalent adduct

between M.EcoRII and 2P residue in DNA was formed Its

amount grew with the increase of the enzyme concentration

Therefore, for duplex IIIm, inhibition of M.EcoRII by

2P-containing DNA (i.e the covalent intermediate is formed)

occurs with methyl group transfer to the C5 position of 2P,

and all active enzyme molecules become covalently bound to

2P-containing DNA (Fig 1B, step 1) In duplex III, only one

strand is modified However, the level of methylation of

duplex III was unexpectedly low (Fig 6) We suppose that

formation of the stable covalent adduct with strand

containing 2P prevents effective methylation of the duplex

III unmodified strand

The time dependence of methyl transfer to duplexes Im

and IIIm was studied (data not shown) Most of the methyl

groups were transferred to duplexes Im and IIIm by

M.EcoRII within 1–2.5 min During the remainder of the

time there was very little or no further methyl transfer to

DNA We suggest that duplex IIIm forms a covalent adduct

with M.EcoRII within the first few minutes of the reaction

Taken together, the results obtained suggest that the

mechanism of C5 MTases inhibition by 2P in the presence

of AdoMet involves methyl group transfer to the C5

position of 2P The methylation of duplex IIIm is attributed

to the formation of the stable covalent intermediate

(Fig 1B, step 1)

To ascertain whether other C5 MTases can methylate 2P,

methyl transfer reactions by M.HhaI to hemimethylated

duplexes

G2PGCACTCTGA-3¢(VIm)/3¢-CTCGGTTCGMGT

GAGACT-5¢ were performed at different enzyme

concen-trations (Fig 7) Duplex VIm contained 2P in place of the

target cytosine in the HhaI recognition sequence (GCGC)

We did not observe methylation of 2P-containing duplex VIm at low enzyme concentrations as was mentioned earlier [6] However, methylation of duplex VIm took place at increased M.HhaI concentrations No methyl transfer was detected in the case of duplex I lacking the HhaI recognition sequence (data not shown) Therefore, as

in the case of M.EcoRII inhibition of M.HhaI by 2P was accompanied by a methyl group transfer to the 2-pyrimid-inone base

Our study allows us to assume that there are two ways of formation of covalent adducts between C5 MTases and 2P-containing DNA In the absence of AdoMet, proton transfer to the C5 position of 2-pyrimidinone occurs (Fig 1A) [6] In the presence of AdoMet, methyl transfer

to the C5 position of 2-pyrimidinone occurs (Fig 1B) Similar complexes of M.EcoRII with AzaC containing DNA were reported [5] The formation of the stable covalent intermediate between M.EcoRII and 2P-contain-ing DNA in the presence of AdoMet causes the inhibition

of methylation One can suggest that the potency of 2-pyrimidinone as an inhibitor arises from the retardation

of proton elimination from the covalent intermediate in the course of catalysis as a consequence of the absence of the N4 amino group in the pyrimidinone ring

In summary, our data suggest that the conformation of DNA is not markedly affected by substitution of 2P for

C or Tin the sequences studied 2-Pyrimidinone signifi-cantly destabilizes the DNA double helix in the order of sequence contexts: ACCTG > A2PCTG > AC2PT G > ACC2PG The amino group of the outer cytosine residue

in the recognition sequence does not take part in the recognition of DNA by M.EcoRII Functional groups of the central thymine exposed in the major groove are probably involved in the recognition by the enzyme EcoRII C5 MTase is inhibited by DNA containing 2-pyrimidinone instead of the target cytosine, two types

of covalent intermediates are possible depending on the presence of AdoMet or AdoHcy Both types of adducts undergo decomposition under heating in the presence of SDS or under analysis by SDS/PAGE The revised mechanism of inhibition of C5 MTases by 2-pyrimidinone containing DNA may be useful in the application of 2-pyrimidinone containing DNA as a MTase inhibitor 2-pyrmidinone incorporation in DNA sequences may also serve as a specific probe for studying discrimination contacts formed by proteins and functional groups of pyrimidine bases exposed in the major groove of DNA

Acknowledgements

The research was supported by a US Public Health Service grant from the Fogarty International Center (No TW05689) grants from the Russian Foundation for Basic Research (48637,

01-04-48561, 02-04-48790 and 02-04-06804) We thank S N Mikhailov for preparation of 2-pyrimidinone phosphoramidite, S Mu¨ller for oligo-nucleotide synthesis, S Klimasˇauskas for M.HhaI, A S Bhagwat for plasmid pT71 used for construction of a hybrid plasmid carrying the gene for M.EcoRII and O V Kirsanova for help on purification of M.EcoRII We are grateful to N E Geacintov, C Crean and

A Kolbanovskiy for critically reading the manuscript and to V L Florentiev for helpful discussion.

Fig 7 Dependence of methylation of hemimethylated DNA duplex VIm

on concentration of M.HhaI M.HhaI was incubated with indicated

duplex (670 n M ) in buffer B in the presence of [CH 3 – 3 H]AdoMet

(420 n M ) at 20 C for 30 min Relative methylation was calculated as

the ratio of radioactivity of duplex VIm to the radioactivity of duplex

Vm Methylation of duplex Vm (not shown) was accepted as 100%.

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