Mechanistic insights on the inhibition of c5 DNA methyltransferases by zebularine

• Published in: PloS one Vol. 5; no. 8; p. e12388
• Main Authors: Champion, Christine, Guianvarc'h, Dominique, Sénamaud-Beaufort, Catherine, Jurkowska, Renata Z, Jeltsch, Albert, Ponger, Loïc, Arimondo, Paola B, Guieysse-Peugeot, Anne-Laure
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.08.2010; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Azacitidine - analogs & derivatives; Azacitidine - chemistry; Azacitidine - pharmacology; Azacytidine; Base Sequence; Binding; Biochemistry; Biochemistry/Biomacromolecule-Ligand Interactions; Biochemistry/Small Molecule Chemistry; Cancer; Cancer therapies; Chemical Sciences; Chromatography; Coordination compounds; CpG islands; Cytidine - analogs & derivatives; Cytidine - chemistry; Cytidine - pharmacology; Cytosine; Demethylation; Deoxyribonucleic acid; Dissociation; DNA; DNA (Cytosine-5-)-Methyltransferases - antagonists & inhibitors; DNA (Cytosine-5-)-Methyltransferases - chemistry; DNA (Cytosine-5-)-Methyltransferases - metabolism; DNA - genetics; DNA - metabolism; DNA methylation; Engineering schools; Enzyme Inhibitors - pharmacology; Enzymes; Epigenetics; FDA approval; Gene expression; Genes; Genetic research; Genomes; Health aspects; Humans; Inhibitors; Mammals; Mass spectrometry; Methylation; Methylation - drug effects; Methyltransferases; Mice; Organic chemistry; Pyrimidines; Scientific imaging; France
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0012388

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.