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.