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.