Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-β- d-arabinofuranosylcytosine triphosphate (ara-CTP)

Abstract

The antimetabolite 1-β- d-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-β- d-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases α, δ, and ϵ, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases α, δ, and ϵ are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase α increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase δ activity was not inhibited significantly by ara-CTP at 100 μM. Recent work has shown that the synthesome-associated DNA polymerase ϵ does not function in in vitro SV40 DNA replication, suggesting that only polymerases α and δ drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase α. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.