We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the
DNA synthesome, from human
breast cancer cells and biopsied human
breast tumor tissue. The purified
DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell
DNA synthesome, in which
DNA polymerases alpha, delta, and epsilon,
DNA primase, and
replication factor C (RF-C) represent members of the core component, or tightly associated,
proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these
proteins. We report here that
poly(ADP-ribose)polymerase (PARP) and
DNA ligase 1 are also members of the breast cell
DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the
protein-
protein interactions between several members of the core component of the
DNA synthesome. Consistent with our proposed model for the breast cell
DNA synthesome, our data indicate that
DNA polymerases alpha and delta,
DNA primase, RF-C, as well as
proliferating cell nuclear antigen (
PCNA), tightly associate with each other in the complex, whereas
DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only
PCNA or
DNA polymerase alpha. The association of PARP with the synthesome core suggests that this
protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three
DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell
DNA. To our knowledge this is the first report ever to describe the close physical association of
polypeptides constituting the intact human breast cell DNA replication apparatus.