Substitution of Cytosine with Guanylurea Decreases the Stability of i-Motif DNA.

Both 5-aza-2'-deoxycytidine (decitabine) and its primary breakdown product, 2'-deoxyriboguanylurea (GuaUre-dR), have been shown to act as mutagens and epimutagens that cause replication stress and alter both DNA methylation and gene expression patterns. As cytosine analogues, both are expected to be preferentially incorporated into regions of GC skew where runs of cytosine residues are sequestered on one strand and guanine residues on the other. Given that such regions have been identified as sites with the potential for effects on gene expression and replication stress linked to formation of alternative DNA secondary structures, it is of interest to determine the influence that these base analogues might have on the stability of structures of this kind. Here we report that incorporation of GuaUre-dR into an i-motif-forming sequence decreases both the thermal and pH stability of an i-motif despite the apparent ability of GuaUre-dR to base pair with cytosine.

2'-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2'-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites.

5-Aza-2'-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2'-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product.