Sequence-specific effects of ara-5-aza-CTP and ara-CTP on DNA synthesis by purified human DNA polymerases in vitro: visualization of chain elongation on a defined template

Abstract

1-beta-D-arabinofuranosyl-5-aza-cytosine (ara-5-aza-Cyd) is an analog of 1-beta-D-arabinofuranosylcytosine (ara-C), which resembles ara-C in anabolic metabolism, incorporation into DNA, and inhibition of DNA replication. Human T-lymphoblastic cells (Molt-4) incorporate three- to fivefold more ara-5-aza-Cyd than ara-C into DNA during 5-8 hr exposure. Although ara-5-aza-Cyd and its triphosphate metabolite are unstable in aqueous solution, the aza-analog was much more stable in solution when incorporated into native DNA isolated from Molt-4 cells. By using gapped duplex DNA as a substrate for purified human DNA polymerases alpha and beta, inhibition of [3H]-dCTP incorporation by ara-5-aza-CTP and ara-CTP was competitive, with Ki values for alpha of 11 and 1.5 microM, respectively. Ki values for polymerase beta were 39 and 7.6 microM, respectively. A DNA elongation assay was adapted from DNA sequencing technology, using singly primed bacteriophage M13mp19 or M13mp9 (+)-DNA. Elongation of 5'-[32P]-labeled primer by polymerase alpha is slowed considerably by incorporation of one ara-CMP and to a lesser extent after incorporation of one ara-5-aza-CMP. Neither analog significantly affected elongation by polymerase beta after a single incorporation. However, neither polymerase alone could appreciably extend the growing chain if two consecutive ara-5-aza-CMP or ara-CMP analogs were incorporated. Thus, if similar mechanisms are operant in intact cells, the greater incorporation of ara-5-aza-Cyd than ara-C into DNA may be due to a more facile elongation of the nascent DNA strand by polymerase alpha after incorporation of a single analog. The effect in vitro of incorporation of either analog on DNA chain elongation is widely variable, depending on the identity of the polymerase involved and the sequence of the DNA template being copied.