Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine

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.

DNA Hypomethylating Drugs in Cancer Therapy

“…Zebularine is a cytidine analog similar to decitabine and azacitidine that incorporates into DNA and forms a reversible complex with DNMTs, effectively preventing methylation (Zhou et al 2002;Champion et al 2010). RG108 is a small molecular inhibitor discovered through virtual screening that binds to the catalytic site of DNMT1 and shows reactivation of silenced tumor-suppressor genes such as P16, SFRP1, and TIMP3 (Brueckner et al 2005).…”

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis

“…In vitro experiments with synthetic oligonucleotides revealed that DNMTs covalently bind to zebularine-containing DNA molecules (Champion et al, 2010). Since we could not detect zebularine directly in DNA, we tested whether the NPAs could cause DNA damaging effects by reducing the amount of available DNMTs.…”

“…Both drugs are bound by DNA METHYLTRANSFERASEs (DNMTs) and form nucleoprotein adducts (NPAs), which effectively deplete the DNMT pool (Egger et al, 2004). In vitro studies using synthetic oligonucleotides containing 5-azacytidine or zebularine revealed higher stability of NPAs when compared with DNMT bound to 5-methyl-deoxycytosine (Champion et al, 2010;Kiianitsa and Maizels, 2013). The data generated using 5-azacytidine and 5-azadeoxicytidine suggest that NPAs represent a physical barrier for enzymes sliding along the DNA molecule and are repaired by HR coupled with replication restart and nucleotide excision repair (Kuo et al, 2007;Salem et al, 2009).…”

DNA Repair Molecules and Cancer Therapeutical Responses

“…Inactivation of ATM give rise to cell cycle defects in response to irradiation and radiosensitise cancer cells [35]. In this way, Zebularine and 5-aza-2'-deoxycytidine are employed as radiosensitizing agents [36,37]. Histone deacetylase inhibitors such as LBH589 and MS-275 have been shown to enhance radiosensitivity through the similar mechanisms [38].…”