Zebularine: A Novel DNA Methylation Inhibitor that Forms a Covalent Complex with DNA Methyltransferases

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Abstract

Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

Introduction

Modulation of histone modification (acetylation, phosphorylation, and methylation) and DNA methylation are the principal driving forces behind the phenomenon of epigenetics.1., 2., 3. While histone modification is restricted to the eukarya, in organisms ranging from bacteriophage to man, differential DNA methylation has been co-opted for the regulation of genetic transactions, including transcription, imprinting, and recombination, and classically provides a barrier to host-specific restriction endonucleases.4., 5., 6., 7. Moreover, the recently demonstrated, close molecular similarity between the human DNMT2 protein and M.HhaI,8 a bacterial cytosine-[C5]-specific DNA methyltransferase (C5 MTase) lends strong support to the notion that bacterial DNA MTases represent a generic platform for understanding mechanistic aspects of biological DNA methylation.

The mechanism of DNA-C5 MTases involves the addition of a protein thiol group (from a cysteine residue in a highly conserved Pro-Cys motif) to the C6 position of the target dC, which activates the carbon atom at the 5 position allowing reaction with S-adenosyl-l-methionine (AdoMet) (Figure 1). Cheng & Roberts9 have recently surveyed the known C5 MTase nucleic acid-related inhibitors (which have been useful in elucidating mechanistic features), including the classical MTase inhibitor 5-azacytidine (AzaC),10 5-fluoro-cytosine (FdC),11 and 5,6-dihydro-5-azacytidine12 as well as compounds such as 4′-thio-2′-deoxycytidine13 in which the sugar moiety is modified. The substitution of the C5 proton by fluorine in FdC has proved to yield an invaluable reagent for studying methyl transfer reactions (Figure 1). Indeed, Chen et al.14 incorporated FdC into a specific duplex and were able to trap M.HaeIII in a covalent complex through the conserved Pro-Cys motif. Subsequently, the crystallization of a covalent ternary complex between M.HhaI, AdoMet, and an FdC oligonucleotide led to the discovery of the phenomenon of base flipping and provided a structural basis of this mechanism-based inhibition.15 Earlier suggestions that the catalytic mechanism of DNA methyltransfer involves transient disruption of the DNA duplex proved well founded.16., 17., 18. However, while the presence of a fluorine atom at the C5 position of the target base renders covalent attack irreversible, it does not significantly impair or stimulate initial complex formation or base flipping. In a similar manner, replacement of C5 by a nitrogen atom in AzaC does not influence initial binding events; rather, nucleophilic attack is facilitated at the C6 position19 and methyl transfer, although possible, is substantially retarded.20

The covalent attachment of a C5 MTase to its recognition sequences will presumably lead to persistent but aberrant nucleoprotein complexes throughout the genome.21., 22. This leads to a cumulative depletion of the enzyme from the nuclear pool, leading to the net demethylation of the genome: the repair of damage as a consequence of nucleoprotein adduct formation subsequently takes place and may be, in part, error-prone.22

The observation that oligodeoxynucleotide duplexes containing zebularine at the target dC form high-affinity, SDS-resistant complexes with M.MspI23., 24. and M.HgaI-225 suggests the zebularine-containing DNA could be an effective inhibitor.

Here, we describe the structure of a complex between the bacterial DNA MTase M.HhaI and an oligodeoxynucleotide duplex containing zebularine incorporated at the position normally occupied by the base targeted for methylation. The enzyme forms a covalent complex in the absence of methyl transfer from AdoMet, unlike that formed between a duplex similarly substituted with FdC. We present a generalized framework for the inhibitory properties of this and the other known C5 MTase inhibitors based on facilitated flipping and electrostatic properties of the flipped nucleotide.

Section snippets

Results

The structure of M.HhaI in a ternary complex with AdoHcy and a 13-mer non-palindromic DNA duplex containing a 5′-GZGC-3′-5′GCGC-3′ with zebularine as the target nucleotide on one strand was determined by X-ray crystallography. The target zebularine was flipped out of the DNA helix (Figure 2(a)), while the dG on the complementary strand remained stacked within the DNA helix. The absence of the amino (NH2) group of the zebularine was confirmed by the (FoFc, αc) electron density map, in which the

Discussion

The proposed increase in the reactivity of C6 in zebularine24 is coupled to the reduction in the barrier to base flipping as observed for the inhibitory duplexes containing an abasic site or a mismatch at the target dC.29., 30., 31. This increase in reactivity at C6 in zebularine-substituted DNA has been substantiated by the finding that replacement of the catalytic Cys in M.MspI by either Thr or Ser, but not by Tyr or any other side-chain, leads to the formation of high-affinity, SDS-resistant

Oligodeoxynucleotide synthesis and purification

Oligondeoxynucleotides were prepared as described23., 34. with minor modifications. Oligodeoxynucleotides were synthesized on a 1 μmol scale (×3) using NH3 labile (FOD) deoxynucleoside phosphoramidites. The phosphoramidite of 5′-dimethoxytrityl-2-pyrimidinone-1-β-d-2′-deoxyribofuranoside was dissolved in anhydrous acetonitrile at the usual concentration of 0.1 M. All syntheses were performed trityl-on. After synthesis, deblocking was performed at room temperature for four to five hours in 35%

Acknowledgements

We thank Susan Sunay and Aiping Dong for technical assistance during protein purification and crystallization. The study was supported, in part, by the US National Institutes of Health GM49245 to X.C. and through BBSRC and Wellcome studentships to P.J.H. and M.J.D., respectively: the Krebs Institute is a BBSRC designated centre for Biomolecular research. Work in the laboratory of B.A.C. was supported by the UK BBSRC, the EU and the Wellcome Trust.

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