In this report we describe, for the first time, the purification and characterization of a replication-competent multiprotein form of
DNA polymerase (designated the
DNA synthesome) from the human
leukemia cell line (HL-60) using a series of centrifugation, ion-exchange chromatography and velocity sedimentation steps. The
proteins and enzymatic activities thus far identified to co-purify with the
leukemia cell
DNA synthesome include the
DNA polymerases alpha and delta,
DNA primase,
proliferating cell nuclear antigen (
PCNA),
replication factor C (RF-C),
replication protein A (RP-A), and
DNA topoisomerases I and II. We have demonstrated that the
DNA synthesome is fully competent to replicate simian virus 40 (SV40) replication origin containing
DNA in vitro in the presence of the viral
large T-antigen. This result implies that all of the cellular activities required for
large T-antigen-dependent in vitro SV40
DNA synthesis are present in the isolated human
leukemia cell
DNA synthesome. Since SV40 is extensively dependent on the host cell's
DNA synthetic machinery for its own DNA replication, our results indicate that the isolated
leukemia cell
DNA synthesome may play a role not only in
viral DNA synthesis but also in human
leukemia cell DNA replication. We recently proposed a model to represent the
DNA synthesome that was isolated from HeLa and murine cells. Our data indicate that the organization of the
DNA synthesome from HL-60 cells also fits this proposed model. The purified
DNA synthesome will not only allow the further study of the molecular mechanisms required to carry out human
leukemia cell DNA replication, but may also provide a tool for eventually dissecting some of the regulatory controls of the cell's
DNA synthetic machinery.