Incorporation of 5-Aza-2'-deoxycytidine-5'-triphosphate into DNA. Interactions with mammalian DNA polymerase alpha and DNA methylase.
In order to understand further the molecular mode of action of 5-Aza-2'-deoxycytidine (5-AZA-dCyd), a potent antileukemic agent, we prepared enzymatically 5-Aza-2'-deoxycytidine 5'-triphosphate (5-AZA-dCTP) and performed studies with purified DNA polymerase alpha and DNA methylase from mammalian cells. DNA polymerase alpha catalyzed the incorporation of 5-AZA-dCTP into DNA. The apparent Km value for 5-AZA-dCTP was estimated to be 3.0 microM; the Km of dCTP was 2.0 microM. The apparent Vmax of 5-AZA-dCTP was slightly lower than that for dCTP. 5-AZA-dCTP was a weak competitive inhibitor (Ki 4.3 microM) with respect to dCTP. Template studies with 5-AZA-dCTP showed that this nucleotide analogue was incorporated into poly(dIC), but not into poly(dAT), suggesting that the incorporation follows the rules of Watson-Crick base pairing. Incorporation of 5-AZA-dCTP into hemimethylated DNA produced a significant inhibition of DNA methylase. These results show that 5-AZA-dCTP is a very good substrate for DNA polymerase alpha and that its incorporation into DNA inhibits DNA methylation.
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000378:metabolism; D002417:Cattle; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D003597:Cytosine Nucleotides; D004247:DNA / Q000096:biosynthesis*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D013698:Templates, Genetic
D000970:Antineoplastic Agents; D003597:Cytosine Nucleotides; D003570:Cytidine Triphosphate; C038673:5-aza-2'-deoxycytidine-5'-triphosphate; D004247:DNA; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D001374:Azacitidine