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

The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent: fur the treatment of leukemia. The active meta bolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible fdr inhibiting intact cell DNA synthesis. We have shown that a multiprotei n complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissue s and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon 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 differenti ally by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase or increased in a concentration-dependent manner, and was correlated close ly with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta act ivity was not inhibited significantly by ara-CTP at 100 mu M. Recent work h as shown that the synthesome-associated DNA polymerase epsilon does not fun ction in in vitro SV40 DNA replication, suggesting that only polymerases al pha and delta drive the DNA replication fork. Therefore, our results sugges t that inhibition of the activity of the mammalian cell DNA synthesome by a ra-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target spe cific phases of the DNA synthetic process in human cells. BIOCHEM PHARMACOL 60;3: 403-411, 2000. (C) 2000 Elsevier Science Inc.