Ara-C differentially affects multiprotein forms of human cell DNA polymerase

Pu,pose: The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has p roven to be one of the most effective agents available for the treatment of acute leukemia although the precise mechanism by which ara-C induces cytot oxicity remains unclear. Our laboratory has previously isolated from human cells a DNA replication complex, termed the DNA synthesome, which is fully competent to orchestrate, in vitro, all of the reactions required to effici ently and faithfully replicate DNA. Using this system and the active metabo lite of ara-C, ara-CTP, we demonstrated that the human DNA synthesome can e fficiently incorporate ara-CTP into internucleotide positions of newly repl icated DNA in vitro mimicking results obtained using intact cells and isola ted nuclei. We then hypothesized that DNA polymerase auxiliary proteins, pr esent within the DNA synthesome, may aid in incorporating this nucleotide a nalog into DNA, Methods: To test this hypothesis, we utilized three distinc t multiprotein complexes each of which contained human DNA polymerase cc an d examined with standard in vitro polymerase assays the effectiveness of ar a-C in inhibiting various aspects of their polymerase function. Results and conclusion: These polymerase-mediated elongation assays, which included ar a-CTP- or ara-C-containing primers in the reaction mixture, showed that the rate of DNA elongation in the presence of ara-CTP was significantly enhanc ed when the DNA polymerase was associated with its auxiliary proteins, and that the elongation resulted in the formation of internucleotide ara-CMP. N evertheless, the enhanced activities resulting from the association of thes e auxiliary proteins with polymerase alpha did not fully account for the re markable efficiency with which the DNA synthesome incorporated ara-C into i nternucleotide positions during DNA replication.