Abstract
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-(β-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.
Highlights
Cancer cells show a highly disturbed epigenetic landscape, which often features a global hypomethylation of the genome that induces abnormal expression of genes and a local hypermethylation of promotors that silences tumor suppressor genes (TSG) [1,2]
Zebularine increases the DNA binding of DNA methyltransferases (DNMTs) We first analyze the capacity of zebularine-containing DNA
The duplexes are incubated with M.SssI (10 and 25 fold excess) in the buffer supplied by New England Biolabs (NEB), supplemented with 300 mM of AdoMet and 100 mg/mL of bovine serum albumin (BSA)
Summary
Cancer cells show a highly disturbed epigenetic landscape, which often features a global hypomethylation of the genome that induces abnormal expression of genes and a local hypermethylation of promotors that silences tumor suppressor genes (TSG) [1,2]. A key feature of the catalytic mechanism of DNMTs is a nucleophilic attack of the enzyme on the carbon-6 of the target cytosine This attack is performed by the thiol group of the cysteine residue of a conserved Proline-Cysteine-Glutamine (PCQ) motif in the active site of DNMTs and is coupled with protonation of N3 to yield an activated enamine intermediate [4,5]. This electron flow back into the pyrimidine ring leads to activation of the C5 atom towards electrophilic attack and to the addition of the methyl group from the cofactor AdoMet to the cytosine. DNMT3L, required for the establishment of maternal genomic imprints, lacks the catalytic activity and participates in de novo methylation through stimulation of DNMT3a [6]
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