Inhibition of HhaI DNA (Cytosine-C5) Methyltransferase by Oligodeoxyribonucleotides Containing 5-Aza-2′-deoxycytidine: Examination of the Intertwined Roles of Co-factor, Target, Transition State Structure and Enzyme Conformation

Abstract

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.

Endogenous Assays of DNA Methyltransferases: Evidence for Differential Activities of DNMT1, DNMT2, and DNMT3 in Mammalian Cells In Vivo

“…The method gives a snapshot of DNMT activity on the genome of any given cell or tissue that represents the physical interactions between DNMT and genomic DNA at the time of cell lysis. By analogy to well-characterized prokaryotic methyltransferases, aza-dC is a necessary in order to drive adduct formation (6). The fact that methylation can be also detected at aza-dC sites (6, 10) is mutually consistent with the notion that ICM detection of aza-dC DNA-DNMT adducts represents bona fide methylation sites.…”

“…(i) We note that there is a total dependence on preincorporation of aza-dC. That dependence, based upon collective data from a number of different labs (6,9,10,15,33,34), suggests that catalytically active methylases are being detected.…”

Mechanism of inhibition of DNA methyltransferases by cytidine analogs in cancer therapy

“…Therefore, a stable covalent complex between the MTase and the DNA is formed that can be detected by gel electrophoresis under denaturating conditions. [50][51][52][53][54] The higher reactivity of the 6-position in 5-aza-dC as compared to cytidine is illustrated by the finding that a C186S mutant of a methyltransferase M.EcoRII in which the attacking cysteine residue is replaced by a less reactive serine, also forms a stable covalent complex with 5-aza-dC substituted DNA. 53 If or die, which explains the dose dependent immediate toxicity of the drug.…”

“…55 Interestingly, in vitro stable covalent complexes of the MTase with 5-aza-dC modified DNA are also formed in the absence of cofactor. 54 In this case, protonation occurs at N5, a reaction that also takes place with unmodified DNA in the absence of cofactor. In the case of unmodified DNA the protonated complex can be resolved by deprotonation at C6 and elimination of the cysteine, whereas this is not possible with 5-aza-dC modified DNA.…”

“…14,[25][26][27]56,57 The mechanism of the inhibition of DNA MTases by 5-aza-C and 5-FCdR has been demonstrated by chemical, mutational and structural analyses mostly with the prokaryotic cytosine MTases. The formation of a covalent complex between the catalytic cysteine residue and 5-aza-dC or 5-F-dC incorporated DNA has been biochemically demonstrated in several experimental systems 14,[25][26][27][50][51][52][53][54]56,57 and observed in structures of complexes between DNA-(cytosine-C5)-MTases and DNA (Fig. 5).…”