DNA cytosine methylation: structural and thermodynamic characterization of the epigenetic marking mechanism.

The DNA methyl transferases play critical roles in many biological functions. They are key players in regulating gene expression1: in embryonic development2, 3, in X-chromosome inactivation4, in genomic imprinting5 and, overall, in epigenetic mechanisms that transmit genetic information without altering the actual base sequence of the DNA through regulation of the methylation status6-8. Aberrant methylation is a feature of cancers and other diseases9-11. Methyl transferase activity is impaired by DNA damage resulting from environmental carcinogens, notably benzo[a]pyrene12, 13 and methylation status has an important impact on the reactivity of DNA with benzo[a]pyrene metabolites14. The design of methylation inhibitors is an active current research frontier15-19. Understanding the mechanism for the methyl transfer reaction in atomistic detail would advance drug design and could provide novel opportunities for regulating gene expression through control of the methylation process. We focus here on the very well-studied prokaryotic cytosine methyltransferase HhaI (M.HhaI) which carries out the enzymatic process for methylation of cytosine C5 in DNA. Like all methyltransferases it uses S-adenosyl-L-methionine (AdoMet) as the methyl donor, and flipping of the target base into a pocket of the enzyme. It has been established based on numerous investigations that the catalytic process entails nucleophilic attack of cysteine 81 (Cys81) of this enzyme on the cytosine C6 to form a covalent adduct via Michael addition; this adduct promotes the nucleophilicity of the cytosine C5 for attack on the AdoMet methyl group20, and subsequently, the C5 proton is abstracted via a β-elimination reaction6, 21. The mammalian enzymes employ a similar mechanism6, 8, 22, which has recently been substantiated with a crystal structure of a productive covalent DNMT1-DNA complex23. It is notable that this crystal structure reveals a covalent adduct between a conserved cysteine and C6 of the target cytosine, as had been observed crystallographically with the prokaryotic enzyme, including some examples containing incorporated nucleoside inhibitor drugs in place of the cytosine24-26. It is also worth noting that DNMT1 is a maintenance methyltransferase, and in this case the mechanism for maintenance and de novo methyltransferases such as M.HhaI are similar; hence, new mechanistic insights could be relevant to drug design for inhibition of both methyltransferases. Current DNA methyltransferase inhibitors generally fall into two categories: nucleoside analogues and non-nucleoside inhibitors. The nucleoside analogues become incorporated in the DNA and may function by inhibiting methylation or β-elimination, but they form the Michael adduct and thus acting as covalent inhibitors. Two nucleoside analogue inhibitors are currently in use, azacytidine and decitabine, employed to treat myelodysplastic syndrome. The non-nucleoside inhibitors bind DNA methyltransferases and exert their inhibitory effects through a variety of different mechanisms27. At present, a full atomistic, thermodynamic and dynamic characterization of the chemical reaction process remains to be determined. Questions that remain not fully resolved include whether the methylation reaction is concerted or stepwise, the specific roles of certain key amino acids in the active site, the nature of the base that abstracts the C5 proton, the role of waters in the chemical process, and the energetics and dynamics of the bond forming and breaking events. Previous computational studies28, 29 for this enzyme system did not obtain free energies or take the enzyme dynamics into account, and either used the semi-empirical DFTB approach as the quantum mechanical method28 or treated the heterogeneous enzyme environment with an implicit continuum solvent model29. We have utilized Born-Oppenheimer ab initio QM/MM-MD calculations to investigate the mechanism for the methyl transfer reaction in M.HhaI; the chemically reacting moieties are described by the ab initio QM method, the surrounding enzyme and aqueous environment are treated explicitly by classical MM, and the enzyme active site dynamics and those of the surroundings are simulated on an equal footing. This approach now has the power to robustly elucidate the full course of the chemical steps in enzyme mechanisms30-36. We explored many mechanistic issues that remain under consideration, and thoroughly investigated a variety of plausible reaction schemes. Our extensive computational investigations resulted in a preferred methyl transfer mechanism for M.HhaI. This mechanism involves nucleophilic attack of deprotonated Cys81 thiolate on the cytosine C6, concerted with methyl transfer. Subsequently, an OH−, derived through a proton wire to surface water and involving crystal and solvent waters, serves as the base to abstract the C5 proton. Our results provide complete free energy profiles for the reaction mechanism, reaction barriers and transition state and intermediate structures for both methylation and proton abstraction steps; our extensive investigations of possible bases support the previous mechanistic suggestion28 that the OH− is likely to be the proton-abstracting base, and that proton abstraction is chemically rate-determining. We obtain atomistic and dynamic views of the bond breaking and forming processes, which reveal the intricate dynamic interplay between formation of the Michael adduct and the methyl transfer step. We also resolve uncertainties in the role of Glu119 in the active site: it forms a hydrogen bond with cytosine N3 at the reactant and at the transition state where the proton spontaneously and reversibly oscillates between being bonded to Glu and to cytosine N3. Hence, the chemical participation of the Glu in the reaction mechanism is manifested. Thus, our study for the first time fully characterizes the HhaI methyltransferase reaction, provides new molecular insights on experimental data20, 24, 37, and more broadly is very likely applicable to the critical human cytosine methylation enzymes that are key in governing epigenetic inheritance, since mammalian DNMT1 relies on a similar chemical mechanism23.