DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

20 pages, 7 figures, 1 table.-- PMID: 19467223 [PubMed].-- PMCID: PMC2756644.-- NIHMSID: NIHMS130041.-- Printed version published Sep 15, 2009.Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.Partial support for this work was provided by a grant from the NIH/NCI (R21CA91315) to J.K.C. and a fellowship from the Graduate College at UNMC to D.V.B. We are grateful to S. Kumar of New England Biolabs for providing us with Eschericia coli strain ER1727 containing the pUHE25HhaI plasmid. This research was also supported in part with funds from the Intramural Research Program of the NIH, Center for Cancer Research, NCI Frederick.Peer reviewe

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

20 pages, 7 figures, 1 table.-- PMID: 19467223 [PubMed].-- PMCID: PMC2756644.-- NIHMSID: NIHMS130041.-- Printed version published Sep 15, 2009.Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.Partial support for this work was provided by a grant from the NIH/NCI (R21CA91315) to J.K.C. and a fellowship from the Graduate College at UNMC to D.V.B. We are grateful to S. Kumar of New England Biolabs for providing us with Eschericia coli strain ER1727 containing the pUHE25HhaI plasmid. This research was also supported in part with funds from the Intramural Research Program of the NIH, Center for Cancer Research, NCI Frederick.Peer reviewe

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2′-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.a European Molecular Biology Laboratory, D-69117 Heidelberg, Germany b Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 69198-4525, United States c UNMC/Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 69198-4525, United States d Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, United States e Institut de Biología Molecular de Barcelona, CSIC, Jordi Girona 18-26, E-08034 Barcelona, SpainN

Inhibition of Hha I 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

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 Hha I methyltransferase (M.Hha I) and oligo-deoxyribonucleotides 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.Hha I. 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.Hha I that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.Hha I.Partial support for this work was provided by the DAMD Breast Cancer Program (DAMD 17-98-1-8215) to JKC and fellowship support from the Graduate College at UNMC to ASB. We are also greatful to Drs. X. Cheng and S. Kumar for their generous gifts of purified M.HhaI.Peer reviewe