Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.

Abstract

The properties of the methyl-iected DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that ntains patterns of methylation in the human genome. Proposals for the enzyme's mhanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynuceotide containg 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine,, suggesting that it comprises an enzymelinked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transi mutation are epially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to searchfor thymidine that might be generated by hydrolysis during the methyl triaser reaction. Despite the potential for deamination ient in the formation of the intermedite, the methylferae did not produce detectable amounts of thymidine. The data suggest that the alit of the human methylter to preserve genetic information when copying a methylation pattern (i.e., its fdelity) is comparable to the ability of a amm DNA polymerase to preserve genetic information when copying a DNA sequence.Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.Proposals for the mechanism of action of DNA methyltransferases (6) suggest production ofa dihydrocytosine intermediate (Fig. 2). These derivatives are extremely susceptible to hydrolytic deamination, since saturation ofthe 5-6 bond increases the PKa of the ring significantly (7). This increase will facilitate protonation ofdihydrocytosine and thereby increase the rate of subsequent hydrolytic deamination in a form of the additionelimination mechanism (8, 9) depicted in Fig. 2A. Although the expected enzyme-DNA intermediate has been detected with a prokaryotic methyltransferase (10), prior to the experiments described here, direct evidence for its formation by a eukaryotic methyltransferase had not, to our knowledge, been reported. Even so, we have pointed out that the production of this nonplanar intermediate in DNA would be consistent with the observed preference of the human enzyme for unusual DNA structures (11).Since the putative intermediate (Fig. 2) might also be susceptible to deamination during catalysis (12), the enzymatic maintenance of methylation patterns could contribute to the high frequency of C-to-T transitions at the CG dinucleotide pair that are thought to be the result of deamination of methylcytosine to thymidine (13-15). Since the C-to-T mutation frequency observed in vivo at 5-methylcytosine relative to that a...

Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a

“…Structural studies with bacterial cytosine methylases have shown that the cytosine base is extruded from the DNA double helix into the active site of the enzyme where it can be reacted upon without steric hindrance (23). The active site cysteine attacks the 6-position of the pyrimidine ring and then activates the 5-carbon for nucleophilic attack of the methyl group donor S-adenosyl-L-methionine (AdoMet) (24).…”

Nucleoprotein-based nanoscale assembly

“…They have well characterized DNA sequence specificities (11) and they form abortive covalent complexes (Fig. 1) between an active-site cysteine and 5-fluorocytosine in their DNA recognition sequences (12)(13)(14)(15). In this report, we demonstrate this approach to macromolecular assembly using two representative methyltransferases: M⅐HhaI and M⅐MspI.…”

“…NaCl (50 mM) was used when both were incubated together. The products were separated on native protein gels poured with an exponential gradient (6-20% arcylamide) and run at 25 mA for 3 hr (14). The gel was then exposed to x-ray film directly for autoradiography.…”

“…Activity was determined using trichloroacetic acid-precipitable counts retained on glass fiber filters as described (14). M⅐HhaI and M⅐HhaI-dod were assayed in a buffer containing 50 mM Tris⅐HCl (pH 7.5), 10 mM EDTA, 5 mM 2-mercaptoethanol, 6 M S-adenosyl-L-[ 3 H-methyl] methionine (Amersham) and 20 M oligodeoxynucleotide duplex 30-mer as described below.…”

Incorporation of Zebularine from its 2′-Deoxyribonucleoside Triphosphate Derivative and Activity as a Template-Coding Nucleobase

“…Methylation of cytosine is catalyzed by DNA methyltransferase enzymes, all of which work through the same catalytic mechanism (2). The addition of a methyl group at the 5-position of cytosine is dependent on formation of a covalent intermediate between a conserved cysteine moiety of the enzyme and the 6-position of the cytosine ring ( Figure 1A&B) (3)(4)(5).…”