FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2051225798> ?p ?o ?g. }

• W2051225798 endingPage "41755" @default.
• W2051225798 startingPage "41749" @default.
• W2051225798 abstract "We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5′-GATC/5′-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA « 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (k meth1 = 0.21 s–1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (k meth2 = 0.023 s–1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5′-GATC/5′-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (k meth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (k meth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced. We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5′-GATC/5′-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA « 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (k meth1 = 0.21 s–1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (k meth2 = 0.023 s–1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5′-GATC/5′-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (k meth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (k meth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced. Type II DNA-methyltransferases (MTases) 1The abbreviations used are: MTase, methyltransferase; AdoMet, S-adenosyl-l-methionine; AdoHcy, S-adenosyl-l-homocysteine; T4Dam, bacteriophage T4 encodes a DNA-(adenine-N 6)-MTase; ODN, oligodeoxynucleotide; M, N6 -methyladenine; N, 2-aminopurine; Z, 7-deazaguanine; MSC, model selection criterion. usually recognize short (4–6 bp) palindromic sequences and catalyze methyl group transfer from donor S-adenosyl-l-methionine (AdoMet) to position N6 of adenine (Ade) and either the C5 or N4 of cytosine (Cyt) (1Cheng X. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 293-318Crossref PubMed Scopus (287) Google Scholar); the reaction products are methylated DNA and S-adenosyl-l-homocysteine (AdoHcy). The elucidation of the mechanisms of action of these enzymes remains an important task in the field of biological DNA methylation. In addition, because of their relatively high specificity and simple structural organization, Type II MTases are attractive objects for detailed studies of specific protein-DNA interactions. The greatest advances in studying the mechanism of action were achieved with the (Cyt-C5)-MTases, for which not only the chemical mechanism of catalysis is known (2Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar), but also three-dimensional structures of enzyme-substrate complexes were solved (3Cheng X. Kumar S. Posfai J. Pflugrath J.W. Roberts R.J. Cell. 1993; 74: 299-307Abstract Full Text PDF PubMed Scopus (361) Google Scholar, 4Cheng X. Kumar S. Klimasauskas S. Roberts R.J. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 331-338Crossref PubMed Scopus (41) Google Scholar, 5Klimasauskas S. Kumar S. Roberts R.J. Cheng X. Cell. 1994; 76: 357-369Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 6Reinisch K.M. Chen L. Verdine G.L. Lipscomb W.N. Cell. 1995; 82: 143-153Abstract Full Text PDF PubMed Scopus (384) Google Scholar). A most surprising and important finding was that the target deoxycytidine “flips out” of the double helix (5Klimasauskas S. Kumar S. Roberts R.J. Cheng X. Cell. 1994; 76: 357-369Abstract Full Text PDF PubMed Scopus (924) Google Scholar), an event that is presumed to precede methyl transfer (7Schluckebier G. Labahn J. Granzin J. Saenger W. Biol. Chem. 1998; 379: 389-400PubMed Google Scholar). Among (Ade-N6)- and (Cyt-N4)-MTases, three-dimensional structures are published only for the TaqI, PvuII, DpnM, and RsrI enzymes (8Labahn J. Granzin J. Schluckebier G. Robinson D.P. Jack W.E. Schildkraut I. Saenger W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10957-10961Crossref PubMed Scopus (185) Google Scholar, 9Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (168) Google Scholar, 10Tran P.H. Korszun Z.R. Cerritelli S. Springhorn S.S. Lacks S.A. Structure. 1998; 6: 1563-1575Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 11Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar). Co-crystallization of these enzymes with DNA and cofactor has not been successful, with the exception of the TaqI MTase. The crystal structure of an AdoMet analog-DNA-TaqI MTase ternary complex (12Goedecke K. Pignot M. Goody R.S. Scheidig A.J. Weinhold E. Nat. Struct. Biol. 2001; 8: 121-125Crossref PubMed Scopus (206) Google Scholar) revealed flipping of the target deoxyadenosine. Flipping of the target deoxynucleoside has not been shown directly with other (Ade-N6)-MTases. However, alternative methodologies, in which the target base is replaced with the fluorescent analog 2-aminopurine (N), and modeling of spatial structures confirm such a DNA deformation upon interaction with (Ade-N6)-MTases (13Allan B.W. Beechem J.M. Lindstrom W.M. Reich N.O. J. Biol. Chem. 1998; 273: 2368-2373Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 14Holz B. Klimasauskas S. Serva S. Weinhold E. Nucleic Acids Res. 1998; 26: 1076-1083Crossref PubMed Scopus (198) Google Scholar, 15Allan B.W. Reich N.O. Biochemistry. 1996; 35: 14757-14762Crossref PubMed Scopus (133) Google Scholar, 16Pues H. Bleimling N. Holz B. Wolcke J. Weinhold E. Biochemistry. 1999; 38: 1426-1434Crossref PubMed Scopus (53) Google Scholar, 17Jeltsch A. Roth M. Friedrich T. Mol. Biol. 1999; 285: 1121-1130Crossref Scopus (34) Google Scholar, 18Schluckebier G. Labahn J. Granzin J. Schildkraut I. Saenger W. Gene. 1995; 157: 131-134Crossref PubMed Scopus (36) Google Scholar). Another important line of study is on kinetic mechanisms of DNA methylation, because the detailed understanding of recognition specificity and catalysis requires knowledge of the rate constants for individual reaction stages. DNA MTases utilize polymeric DNA as their natural substrates in vivo. However, analysis of in vitro methylation of polymeric DNA, even if it contains a single target sequence, is complicated by the need for linear diffusion of the DNA-bound enzyme to find its specific recognition site. The use of relatively short oligodeoxynucleotide (ODN) duplexes (usually 12–30 bp) containing a single specific site allows for simplification of the experimental conditions and acquisition of more precise data for calculating reaction parameters. In addition, substitutions of chemical groups or other modifications can be introduced, and the effect of these defined alterations on the reaction course can be assessed (19Reich N.O. Danzitz M.J. Nucleic Acids Res. 1991; 19: 6587-6594Crossref PubMed Scopus (19) Google Scholar, 20Buryanov Y.I. Zinoviev V.V. Gorbunov Yu.A. Tuzikov F.V. Rechkunova N.I. Malygin E.G. Bayev A.A. Gene. 1988; 74: 67-69Crossref PubMed Scopus (16) Google Scholar, 21Gromova E.S. Oretzkaya T.S. Eritja R. Guschlbauer W. Biochem. Mol. Biol. Int. 1995; 36: 247-255PubMed Google Scholar, 22Brevnov M.G. Kubareva E.A. Volkov E.M. Romanova E.A. Oretskaia T.S. Gromova E.S. Shabarova Z.A. Gene. 1995; 157: 149-152Crossref PubMed Scopus (6) Google Scholar). Because a natural DNA may contain some structural defects such as nicks, gaps, deletions, or chemical modification of nucleotide residues, it is interesting to know how these alterations affect the methylation process. Earlier we studied the influence of various defects in synthetic ODN duplexes upon binding of the bacteriophage T4Dam (DNA-(Ade-N6)-MTase) and on the steady-state parameters of methylation (23Malygin E.G. Petrov N.A. Gorbunov Yu.A. Kossykh V.G. Hattman S. Nucleic Acids Res. 1997; 25: 4393-4399Crossref PubMed Scopus (34) Google Scholar, 24Malygin E.G. Zinoviev V.V. Petrov N.A. Evdokimov A.A. Jen-Jacobson L. Kossykh V.G. Hattman S. Nucleic Acids Res. 1999; 27: 1135-1144Crossref PubMed Scopus (30) Google Scholar, 25Zinoviev V.V. Evdokimov A.A. Gorbunov Yu.A. Malygin E.G. Kossykh V.G. Hattman S. Biol. Chem. 1998; 379: 481-488Crossref PubMed Scopus (35) Google Scholar, 26Evdokimov A.A. Zinoviev V.V. Malygin E.G. Schlagman S.L. Hattman S. J. Biol. Chem. 2002; 277: 279-286Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We showed that the addition of T4Dam to the reaction mixture led to an initial reaction “burst,” which was then followed by a slower steady-state phase of methylation. This result indicated that the rate-limiting stage of the reaction is the release of product(s) from the complex with the enzyme. Certain defects in duplex structure reduced the k cat value but had only small effects on the Km or the ability of T4Dam to bind the duplex. Other alterations reduced T4Dam binding ability but retained good substrate characteristics, viz. relatively high k cat and low Km values. Thus, diverse effects on the interaction with T4Dam were exerted by different changes of the specific site. In these steady-state studies, methylation was carried out under conditions where substrate DNA was in excess (enzyme/DNA « 1) and T4Dam has a monomeric structure; in contrast, when enzyme/DNA > 1, a dimeric enzyme structure is favored (27Malygin E.G. Evdokimov A.A. Zinoviev V.V. Ovechkina L.G. Lindstrom Jr., W.M. Reich N.O. Schlagman S.L. Hattman S. Nucleic Acids Res. 2001; 29: 2361-2369Crossref PubMed Google Scholar). In this regard, the x-ray crystal structure of a T4Dam-DNA-AdoHcy ternary complex (28Yang Z. Horton J.R. Zhou L. Zhang X.J. Dong A. Zhang X. Schlagman S.L. Kossykh V. Hattman S. Cheng X. Nat. Struct. Biol. 2003; 10 (in press)Google Scholar) showed that the enzyme:DNA (a 12-mer ODN duplex) ratio was 2:1. Hence, it is important to examine the catalytic characteristics of dimeric T4Dam and compare them with the properties of the monomeric form. Therefore, we have extended our studies on T4Dam to analyses of pre-steady-state kinetics under single turnover conditions. Enzymes and Chemicals—[3H]CH3-AdoMet was purchased from Amersham Biosciences. Unlabeled AdoMet (Sigma) was purified further by chromatography on a C18 reversed-phase column, as described previously (29Kossykh V.G. Schlagman S.L. Hattman S. FEBS Lett. 1995; 370: 75-77Crossref PubMed Scopus (15) Google Scholar). ODNs were synthesized and purchased from Midland Certified Reagent Co. (Midland, TX). They were purified further as described (13Allan B.W. Beechem J.M. Lindstrom W.M. Reich N.O. J. Biol. Chem. 1998; 273: 2368-2373Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar); concentrations were determined spectrophotometrically from the molar extinction coefficients of individual nucleotides and the known sequence of each ODN. T4Dam MTase was purified to homogeneity, as described previously (30Kossykh V.G. Schlagman S.L. Hattman S. J. Biol. Chem. 1995; 270: 14389-14393Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Protein concentrations were determined by the Bradford method (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Defined synthetic duplexes were obtained by heating and annealing individual ODN chains (Table I) from 90 to 20 °C over a 7–12-h period. For example, annealing complementary ODN I (designated as the upper strand) and ODN II produced duplex 1 with a centrally located T4Dam recognition site GATC/GATC (Table II). The hemimethylated variant duplex 1m was produced by annealing ODN Im (contains an M residue in the recognition site) with ODN II. Other target site variants contained a modified base substitution in the “bottom” strand II; e.g. G was replaced by 7-deazaG (G → Z, ODN IIz), or Ade was replaced by 2-aminopurine (A → N, ODN IIn). Structurally defective duplexes lacking some element of the recognition site (either a nucleotide or phosphate) were prepared by annealing equimolar mixtures of upper strand ODN I with one or more of ODN III-IX (Table I).Table ISynthetic ODNsODNSequenceaUnderlined bases denote the recognition site GATC and its variants or portion thereof. M = N 6-methyladenine; Z = 7-deazaguanine; N = 2-aminopurine.I5′-CAGTTTAGGATCCATTTCACIM5′-CAGTTTAGGMTCCATTTCACII5′-GTGAAATGGATCCTAAACTGIIZ5′-GTGAAATGZATCCTAAACTGIIN5′-GTGAAATGGNTCCTAAACTGIII5′-TCCTAAACTGIV5′-CCTAAACTGV5′-ATCCTAAACTGVI5′-GTGAAATGGVII5′-GTGAAATGGATVIII5′-GTGAAATGGAIX5′-GTGAAATGGTACCTAAACTGa Underlined bases denote the recognition site GATC and its variants or portion thereof. M = N 6-methyladenine; Z = 7-deazaguanine; N = 2-aminopurine. Open table in a new tab Table IIKinetic parameters for T4Dam methylation of 20-mer duplexes with canonical GATC/GATC or modified sites Single Turnover Assays—Methyl transfer assays were carried out at 25 °C, as described previously (25Zinoviev V.V. Evdokimov A.A. Gorbunov Yu.A. Malygin E.G. Kossykh V.G. Hattman S. Biol. Chem. 1998; 379: 481-488Crossref PubMed Scopus (35) Google Scholar). Reaction mixtures contained 100 mm Tris-HCl, pH 8.0, 10 mm EDTA, 10 mm dithiothreitol, and 0.2 mg/ml bovine serum albumin. The microvolume rapid quench instrument RQF-3 (KinTek Corp.) was used for pre-steady-state assays. Syringes, mixers, and age-loops were equilibrated to 25 °C. The feeding syringe containing the enzyme preparation was kept at 4 °C to avoid inactivation of the T4Dam MTase during the experiment. SDS (0.05%, w/v) in 25 mm Tris-HCl (pH 8.3) was used as the quench solution. The quenched samples were collected in Eppendorf tubes and evaporated to a volume of 100 μl using an Eppendorf vacuum concentrator. Duplicate 50-μl aliquots were spotted onto DE81 anion-exchange filter paper (Whatman, 2.0 cm) for counting 3H counts/min. The molar concentration of [3H]CH3 groups incorporated into DNA was quantitated as described earlier (32Thielking V. Du Bois S. Eritja R. Guschlbauer W. Biol. Chem. 1997; 378: 407-415PubMed Google Scholar). Each experiment was performed at least twice, and the mean values were used for analysis. Kinetic parameters were obtained by using the computer program “Scientist” (MicroMath Inc.) for a regression analysis to fit the experimental data. Single Turnover Kinetics of Methylation of Duplexes Containing a Canonical T4Dam Recognition Site: a Two-stage Reaction—T4Dam methylation of defined synthetic duplexes (Table II) was assayed under single turnover conditions; i.e. AdoMet (8 μm) was saturating relative to T4Dam (2.7 μm), and both were saturating relative to the ODN duplex (0.2 μm). Under these conditions, two T4Dam (presumably bound with AdoMet) are bound per duplex (32Thielking V. Du Bois S. Eritja R. Guschlbauer W. Biol. Chem. 1997; 378: 407-415PubMed Google Scholar). In the classic case of single turnover, a substrate molecule has a single reactive group that is converted to product. The course of the reaction can be described by a simple exponential function [[ 3H]CH3])[duplex]=Pmax⋅(1-e-kmeth⋅t)(Eq. 1) where P max is the maximum level of substrate conversion, and k meth is the rate constant of the chemical stage of the reaction. However, the curves did not obey a simple exponential dependence (representative results are presented in Fig. 1, and statistical evidence is presented under “Appendix”). All data sets were best described by using Equation 2 below for two-step methylation of duplexes; however, it is not ruled out that some other more complex model might fit the data even better. Duplex 1 (Table II) contains GATC on both strands of the target site. During a single turnover reaction, T4Dam should catalyze a methyl transfer to every duplex, and it might even methylate both Ade residues. Because the overall DNA-MTase reaction is irreversible (2Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar, 26Evdokimov A.A. Zinoviev V.V. Malygin E.G. Schlagman S.L. Hattman S. J. Biol. Chem. 2002; 277: 279-286Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), it can be represented as a two-stage conversion of the initial oligonucleotide duplex containing two target Ade residues. where E is T4Dam bound with AdoMet and/or AdoHcy, D is the initial duplex, mD is the half-methylated duplex; mDm is the fully methylated reaction product, k meth1 is the rate constant of the first stage of the methylation reaction, and k meth2 is the rate constant of the second stage. Because the technique used only allows registration of the sum of the products of reaction p = mD + mDm, the kinetic scheme (Scheme 1) is described by Equation 2 (33Fersht A. Enzyme Structure and Mechanism. 2nd ed. W. H. Freeman & Co., New York1985: 133Google Scholar): [[ 3H]CH3]/[duplex]=P2-(P1⋅kmeth1-P2⋅kmeth2)⋅e-kmeth1⋅/(kmeth1-kmeth2)-(P2-P1)⋅e-kmeth2⋅t⋅kmeth1/(kmeth1-kmeth1)(Eq. 2) where P 1 is the number of Ade residues methylated per duplex during the first stage of the reaction, and P 2 is the number of Ade residues methylated per duplex in the final product. For duplex 1, containing the canonical palindromic GATC/GATC site, the first stage of the single turnover reaction involves methylation of one of the two Ade residues (P 1 = 1.03 [3H] methyl groups transferred per duplex) with rate constant k meth1 = 0.21 s–1 (Fig. 1A and Table II). This value is 14-fold higher than the k cat under steady-state conditions (24Malygin E.G. Zinoviev V.V. Petrov N.A. Evdokimov A.A. Jen-Jacobson L. Kossykh V.G. Hattman S. Nucleic Acids Res. 1999; 27: 1135-1144Crossref PubMed Scopus (30) Google Scholar, 25Zinoviev V.V. Evdokimov A.A. Gorbunov Yu.A. Malygin E.G. Kossykh V.G. Hattman S. Biol. Chem. 1998; 379: 481-488Crossref PubMed Scopus (35) Google Scholar). This finding confirms that, as in the case of the EcoRI MTase (34Reich N.O. Mashhoon N. J. Biol. Chem. 1993; 268: 9191-9193Abstract Full Text PDF PubMed Google Scholar), the chemical step of the reaction (methyl group transfer) is not rate-limiting. In the first stage after binding, T4Dam methylates one strand only. The second methylation stage proceeded at a rate that was an order of magnitude slower than in the first stage, and the P 2 was 1.90 methyl groups per duplex. Thus, both Ade residues in the canonical GATC/GATC site were methylated under single turnover conditions, where two Dam-AdoMet complexes can be bound to the duplex (27Malygin E.G. Evdokimov A.A. Zinoviev V.V. Ovechkina L.G. Lindstrom Jr., W.M. Reich N.O. Schlagman S.L. Hattman S. Nucleic Acids Res. 2001; 29: 2361-2369Crossref PubMed Google Scholar). This result suggests that the two distinct k meth values reflect differing methylation conditions; e.g. the second methylation is rate-limited by the dissociation of T4Dam-AdoHcy or its clearance from the site on the opposite strand. Single Turnover Kinetics of Methylation with Duplexes Containing a Nucleotide Substitution in the T4Dam Recognition Site—Duplexes 1m, 3, 4, and 5 (GMTC/GATC, GMTC/ZATC, GATC/GNTC, and GATC/GTAC, respectively) contain only a single Ade residue as a potential methyl acceptor. Thus, for these duplexes, random T4Dam binding should result in one of two mutually exclusive orientations (productive and non-productive) to the substrate, resulting in modification of only 50% of the duplexes. In all cases we observed a rapid exponential phase of methyl transfer followed by a slower increase in product accumulation; the results for duplex 1m are shown in Fig. 1B. Nevertheless, the two-exponential equation (Equation 2) adequately described the methylation of these duplexes, indicating that their methylation also proceeds in two stages. Fig. 1B shows the fitting of the experimental points for duplex 1m, and it worked satisfactorily in all the other cases, allowing determination of all four reaction parameters, k meth1, k meth2, P 1, and P 2 (Table II). However, it must be pointed out that here k meth2 applies to a different process. For example, consider methylation of the hemimethylated duplex 1m, where the rate of methyl group transfer was almost 2-fold higher (k meth1 = 0.37 s–1) than for duplex 1 (Fig. 1B, Table II). The extent of methyl transfer during the first-stage P 1 was 0.45 [3H]methyl groups per duplex. This value is close to the theoretical value of 0.50, assuming that Dam-AdoMet molecules bound to the productive and nonproductive strands of the hemimethylated target have an equal probability of flipping a target A or N6 -methyladenine deoxynucleoside and that interacting with the methylated Ade precludes methylation during the first stage. During the second methylation stage (which was also faster than with duplex 1), the P 2 value attained the theoretical maximum of 1.01 [3H]methyl groups per duplex. Therefore, we propose that under single turnover reaction conditions, methylation of the hemimethylated duplex 1m (and other duplexes having one Ade residue capable of methylation) also occurs in two stages. Here, k meth1 reflects the rate of the methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the Ade residue capable of methylation, whereas the slower k meth2 reflects methylation by enzyme molecules that were oriented first to the non-productive GMTC chain and have to reorient (27Malygin E.G. Evdokimov A.A. Zinoviev V.V. Ovechkina L.G. Lindstrom Jr., W.M. Reich N.O. Schlagman S.L. Hattman S. Nucleic Acids Res. 2001; 29: 2361-2369Crossref PubMed Google Scholar) to the opposite productive chain. The substitution of Z for G (in duplexes 2 and 3) represents only a minor structural change, which does not alter normal hydrogen bonding. Duplex 2 contains two Ade residues accessible for methylation, whereas duplex 3 has one. With both duplexes 2 and 3, there was a 1.5- to 2-fold lower k meth1 than for canonical duplex 1 (Table II). Earlier it was shown that the G → Z substitution disrupts an important contact between T4Dam and guanine atom N7 , which is exposed in the major groove (24Malygin E.G. Zinoviev V.V. Petrov N.A. Evdokimov A.A. Jen-Jacobson L. Kossykh V.G. Hattman S. Nucleic Acids Res. 1999; 27: 1135-1144Crossref PubMed Scopus (30) Google Scholar). In particular, the Kd value (according to gel retardation data) increased from 18 nm for the canonical duplex to 950 nm for the Z-substituted duplex. Nevertheless, the theoretical extent of methylation (P 2) of each duplex (2 and 3) was attained (Table II). An → N substitution (duplex 4) shifts the location of the exocyclic amino group from C6 to C2 (as well as shifting hydrogen bonding with thymine to this position). Although duplex 4 exhibits a 4-fold decrease in the k cat value, the chemical stage rates k meth1 and k meth2 increased 2-fold (Table II). The extent of methylation at both stages (P 1 = 0.58, P 2 = 1.12) corresponded to the expected values. Introduction of a double-base mismatch opposite the central upper strand AT (duplex 5) decreased the k cat value 30-fold (Table II). However, k meth1 was not affected, and k meth2 showed only a 2-fold decrease. Because the target Ade residue in the upper strand cannot form a hydrogen bond, it might be expected that its flipping out of the helix would be facilitated. However, because k meth1 was not increased, we conclude that either the stacking effect continues to stabilize the structure, or that flipping of the target base is not rate-limiting. The latter would be in agreement with earlier findings (35Allan B.W. Reich N.O. Beechem J.M. Biochemistry. 1999; 38: 5308-5314Crossref PubMed Scopus (80) Google Scholar). The expected extents of methylation P 1 and P 2 were observed with duplex 5. Single Turnover Kinetics of Methylation with Duplexes Containing a Deletion in the T4Dam Recognition Site—Absence of a single internucleotide phosphate in the target site influences methylation in a position-dependent fashion. For example, deletion of a phosphate between T and C (duplex 6) had no effect on k cat, k meth1, and k meth2 (Fig. 1C, Table II), and the extent of methylation in the first stage was as expected, although slightly lower after the second stage. In contrast, absence of the phosphate between A and T (duplex 7) lowered k meth2 by 1.5-fold and k cat by 2.5-fold (Fig. 1E, Table II). Absence of a phosphate between G and A (duplex 8) had the largest effect on kinetic parameters (Table II). Thus, the k cat value was reduced almost 10-fold, whereas the k meth1 increased more than 2.5-fold, but P 1 decreased. The latter change indicates that the proportion of catalytically competent complexes was lower than expected. Finally, k meth2 was lowered 3-fold, but the P 2 = 1.2 suggests there was complete methylation of the native strand. Duplex 9, which lacks C and additional 3′-nucleotides in the bottom strand, had single turnover parameters quite close to those for duplex 6 (Fig. 1D, Table II), which lacks a phosphodiester bond between the T and C residues of the bottom strand. In contrast, duplex 10, which lacks G and additional 5′-nucleotides in the bottom strand, gave entirely different single turnover parameters (Table II). For example, the P 1 value was less than 0.1; nevertheless, the k meth1 of 1.17 s–1 is 6-fold higher than that for the native duplex. On the other hand, the second stage of methylation showed an 8-fold lower k meth2 compared with the native duplex. Still, the P 2 value of 1.10 suggests complete methylation of the native strand. The k meth2 and k cat values were identical and at least five times lower than the respective values for native duplex 1. Duplex 11, which lacks T in the bottom strand, had a 4-fold higher k meth1 value compared with native duplex 1 (Fig. 1F, Table II), yet it had a low P 1 value of 0.16. The sharp reduction in the proportion of stage 1 productive complexes for duplexes 7, 8, 10, and 11, each devoid of some structural element, might be attributed to some distortion in the normal bending of DNA in the recognition site, which was shown to accompany interaction with DNA-MTases (36Garcia R.A. Bustamante C.J. Reich N.O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7618-7622Crossref PubMed Scopus (55) Google Scholar). Under Single Turnover Conditions T4Dam Acts as a Dimer and Methylates Both Strands of Canonical GATC/GATC Duplex 1—We showed earlier (by gel filtration and sucrose gradient ultra-centrifugation) that two T4Dam molecules are present in complexes formed with 20- and 32-mer ODN duplexes under conditions of single turnover (37Zinoviev V.V. Ovechkina L.G. Malygin E.G. Mol. Biol. (Mosc.). 1996; 30: 1203-1208PubMed Google Scholar). To study this further, we took advantage of the ability of glutaraldehyde to cross-link protein subunits, because of the formation of covalent bonds between closely located lysine residues (27Malygin E.G. Evdokimov A.A. Zinoviev V.V. Ovechkina L.G. Lindstrom Jr., W.M. Reich N.O. Schlagman S.L. Hattman S. Nucleic Acids Res. 2001; 29: 2361-2369Crossref" @default.
• W2051225798 created "2016-06-24" @default.
• W2051225798 creator A5010171665 @default.
• W2051225798 creator A5028009074 @default.
• W2051225798 creator A5030607627 @default.
• W2051225798 creator A5031601960 @default.
• W2051225798 creator A5059725617 @default.
• W2051225798 creator A5064531954 @default.
• W2051225798 creator A5089653624 @default.
• W2051225798 date "2003-10-01" @default.
• W2051225798 modified "2023-09-27" @default.
• W2051225798 title "Bacteriophage T4Dam (DNA-(Adenine-N)-methyltransferase)" @default.
• W2051225798 cites W1517414895 @default.
• W2051225798 cites W1535452281 @default.
• W2051225798 cites W1548141954 @default.
• W2051225798 cites W1585338920 @default.
• W2051225798 cites W1867958153 @default.
• W2051225798 cites W1973011601 @default.
• W2051225798 cites W1973496342 @default.
• W2051225798 cites W1974152305 @default.
• W2051225798 cites W1986844235 @default.
• W2051225798 cites W1991776265 @default.
• W2051225798 cites W1993855539 @default.
• W2051225798 cites W1994162276 @default.
• W2051225798 cites W1997974036 @default.
• W2051225798 cites W2010880750 @default.
• W2051225798 cites W2019367624 @default.
• W2051225798 cites W2019492821 @default.
• W2051225798 cites W2033735656 @default.
• W2051225798 cites W2040797344 @default.
• W2051225798 cites W2046079483 @default.
• W2051225798 cites W2048978731 @default.
• W2051225798 cites W2050728905 @default.
• W2051225798 cites W2055479492 @default.
• W2051225798 cites W2056377405 @default.
• W2051225798 cites W2059475921 @default.
• W2051225798 cites W2060248442 @default.
• W2051225798 cites W2076129724 @default.
• W2051225798 cites W2077890483 @default.
• W2051225798 cites W2095084342 @default.
• W2051225798 cites W2097404504 @default.
• W2051225798 cites W2100394396 @default.
• W2051225798 cites W2104587229 @default.
• W2051225798 cites W2106359847 @default.
• W2051225798 cites W2126916732 @default.
• W2051225798 cites W2134326554 @default.
• W2051225798 cites W2142316966 @default.
• W2051225798 cites W4293247451 @default.
• W2051225798 doi "https://doi.org/10.1074/jbc.m306397200" @default.
• W2051225798 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12893823" @default.
• W2051225798 hasPublicationYear "2003" @default.
• W2051225798 type Work @default.
• W2051225798 sameAs 2051225798 @default.
• W2051225798 citedByCount "8" @default.
• W2051225798 countsByYear W20512257982012 @default.
• W2051225798 crossrefType "journal-article" @default.
• W2051225798 hasAuthorship W2051225798A5010171665 @default.
• W2051225798 hasAuthorship W2051225798A5028009074 @default.
• W2051225798 hasAuthorship W2051225798A5030607627 @default.
• W2051225798 hasAuthorship W2051225798A5031601960 @default.
• W2051225798 hasAuthorship W2051225798A5059725617 @default.
• W2051225798 hasAuthorship W2051225798A5064531954 @default.
• W2051225798 hasAuthorship W2051225798A5089653624 @default.
• W2051225798 hasBestOaLocation W20512257981 @default.
• W2051225798 hasConcept C104317684 @default.
• W2051225798 hasConcept C153911025 @default.
• W2051225798 hasConcept C185592680 @default.
• W2051225798 hasConcept C2776441376 @default.
• W2051225798 hasConcept C33288867 @default.
• W2051225798 hasConcept C54355233 @default.
• W2051225798 hasConcept C547475151 @default.
• W2051225798 hasConcept C552990157 @default.
• W2051225798 hasConcept C55493867 @default.
• W2051225798 hasConcept C86803240 @default.
• W2051225798 hasConcept C91965660 @default.
• W2051225798 hasConceptScore W2051225798C104317684 @default.
• W2051225798 hasConceptScore W2051225798C153911025 @default.
• W2051225798 hasConceptScore W2051225798C185592680 @default.
• W2051225798 hasConceptScore W2051225798C2776441376 @default.
• W2051225798 hasConceptScore W2051225798C33288867 @default.
• W2051225798 hasConceptScore W2051225798C54355233 @default.
• W2051225798 hasConceptScore W2051225798C547475151 @default.
• W2051225798 hasConceptScore W2051225798C552990157 @default.
• W2051225798 hasConceptScore W2051225798C55493867 @default.
• W2051225798 hasConceptScore W2051225798C86803240 @default.
• W2051225798 hasConceptScore W2051225798C91965660 @default.
• W2051225798 hasIssue "43" @default.
• W2051225798 hasLocation W20512257981 @default.
• W2051225798 hasOpenAccess W2051225798 @default.
• W2051225798 hasPrimaryLocation W20512257981 @default.
• W2051225798 hasRelatedWork W1828691184 @default.
• W2051225798 hasRelatedWork W1903732681 @default.
• W2051225798 hasRelatedWork W1991523530 @default.
• W2051225798 hasRelatedWork W2000709210 @default.
• W2051225798 hasRelatedWork W2002128513 @default.
• W2051225798 hasRelatedWork W2020824267 @default.
• W2051225798 hasRelatedWork W2074481477 @default.
• W2051225798 hasRelatedWork W2075611159 @default.

• next