Mutational Analysis of Target Base Flipping by the EcoRV Adenine-N6 DNA Methyltransferase

Self Cite

The M.EcoRV DNA methyltransferase recognizes GATATC sites. It is related to EcoDam, which methylates GATC sites. The DNA binding domain of M.EcoRV is similar to that of EcoDam suggesting a similar mechanism of DNA recognition. We show that amino acid residue Lys 11 of M.EcoRV is involved in recognition of Gua 1 and Arg 128 contacts the Gua in base pair 6. These residues correspond to Lys 9 and Arg 124 in EcoDam, which recognize the Gua residues in both strands of the Dam recognition sequence, indicating that M.EcoRV and EcoDam make similar contacts to outermost base pairs of their recognition sequences and M.EcoRV recognizes its target site as an expanded GATC site. In contrast to EcoDam, M.EcoRV considerably bends the DNA (59 ؎ 4°) suggesting indirect readout of the AT-rich inner sequence. Recognition of an expanded target site by DNA bending is a new principle for changing DNA recognition specificity of proteins during molecular evolution. R128A is inefficient in DNA bending and binding, whereas K11A bends DNA with relaxed sequence specificity. These results suggest a temporal order of the formation of protein-DNA contacts in which the Gua 6 -Arg 128 contact forms early followed by DNA bending and, finally, the formation of the Lys 11 -Gua 1 contact.Sequence specific recognition of DNA by proteins is essential for life, because the biological effect of each gene is regulated by transcription factors, which bind to the promoter elements of target genes and stimulate or repress the expression of the corresponding gene (1). Also many nucleic acid interacting enzymes, like DNA methyltransferases (2-4), aminoacyl-tRNA synthetases (5), DNA repair enzymes (6), and restriction endonucleases (7) interact with nucleic acids in a highly sequencespecific manner. In general, DNA recognition follows two paradigms: direct and indirect readout (1). For direct readout, proteins form contacts (including hydrogen bonds and van der Waals contacts) in the major (and to a lesser degree also the minor) groove of the DNA to the edges of the base pairs to probe the DNA sequence (8). For indirect readout, proteins form contacts to the DNA backbone. Because the structural preferences and dynamics of the DNA are sequence dependent, the interaction energy associated with a particular DNA conformation can be used to read the DNA sequence (9).DNA methyltransferases are an important example of enzymes that recognize specific DNA sequences (2-4). DNA-(adenine N 6 )-MTases, as studied here, transfer a methyl group to the N 6 -position of adenine residues embedded in a specific recognition sequence. In Escherichia coli, the E. coli DNA adenine methyltransferase (EcoDam) 3 enzyme that methylates DNA at GATC sites is involved in the coordination of DNA replication and cell cycle, post-replicative mismatch repair, and regulation of the expression of several genes (4). Recently, the structure of the EcoDam enzyme in complex with specific DNA was solved illustrating the mechanism of sequence-specific DNA interaction of this enzyme (10). The protei...

Section: * This Work Was Supported By Deutsche Forschungsgemeinschaftmentioning

confidence: 99%

The M.EcoRV DNA-(Adenine N6)-methyltransferase Uses DNA Bending for Recognition of an Expanded EcoDam Recognition Site

Jurkowski

Anspach

Kulishova

et al. 2007

Self Cite

“…Binding of AdoMet in the absence of substrate DNA has been confirmed, in particular by co-crystal structures (5,19,23,28) and by copurification of MTase with tightly bound AdoMet (45)(46)(47). On the other hand, MTases are capable of recognizing and binding at specific DNA sequence(s) and flipping the target base in the absence of AdoMet or its nonreactive analogs (17,34,48,49); although, as a rule, AdoMet (or its non-reactive analogs) increases the enzyme's affinity for substrate DNA (13, 17, 21, 50 -52). However, DNA MTases can also form stable, non-functional (dead-end) enzyme-productsubstrate ternary complexes, as has been observed for the MTase-AdoHcy-DNA complex of HhaI (53).…”

Section: Thementioning

confidence: 99%

“…Such homology may, in turn, indicate that a considerable amount of similarity exists in their three-dimensional structures, as well as in their mechanism of action. Recently, more progress has been made in the study of this family of enzymes; the first crystal structure for the GATC-specific DpnM MTase complexed with AdoMet was reported (28), and an extensive biochemical investigation of the GATATC-specific EcoRV MTase was performed (15,(32)(33)(34)(35).…”

mentioning

confidence: 99%

Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

Evdokimov¹,

Zinoviev²,

Malygin³

et al. 2002

. After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.

“…Flipping of the target deoxynucleoside has not been shown directly with other (Ade-N 6 )-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-N 6 )-MTases (13)(14)(15)(16)(17)(18). 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.…”

mentioning

confidence: 99%

Bacteriophage T4Dam (DNA-(Adenine-N)-methyltransferase)

Malygin

Lindstrom

Zinoviev

et al. 2003

We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N 6 )-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 AdoMetmethyl 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) 1 usually recognize short (4 -6 bp) palindromic sequences and catalyze methyl group transfer from donor S-adenosyl-L-methionine (AdoMet) to position N 6 of adenine (Ade) and either the C5 or N 4 of cytosine (Cyt) (1); 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 (2), but also three-dimensional structures of enzyme-substrate complexes we...