THE HUMAN BREAST CELL-DNA SYNTHESOME - ITS PURIFICATION FROM TUMOR-TISSUE AND CELL-CULTURE

In this report, we describe for the first time the isolation and purif ication of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA sy nthesome, fully supports the in vitro replication of simian virus 40 ( SV40) origin-containing DNA in the presence of the viral large T-antig en. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DN A replication proteins constitute the DNA synthesome: DNA polymerase a lpha, DNA primase, DNA polymerase delta, proliferating cell nuclear an tigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isola ted the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell l ine MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replicatio n in vitro and in vivo. Copyright (C) 1996 Elsevier Science Inc.