THE ISOLATION OF A DNA SYNTHESOME FROM HUMAN LEUKEMIA-CELLS

In this report we describe, for the first time, the purification and c haracterization of a replication-competent multiprotein form of DNA po lymerase (designated the DNA synthesome) from the human leukemia cell line (HL-60) using a series of centrifugation, ion-exchange chromatogr aphy and velocity sedimentation steps. The proteins and enzymatic acti vities thus far identified to co-purify with the leukemia cell DNA syn thesome include the DNA polymerases alpha and delta, DNA primase, prol iferating cell nuclear antigen (PCNA), replication factor C (RF-C), re plication protein A (RP-A), and DNA topoisomerases I and II. We have d emonstrated that the DNA synthesome is fully competent to replicate si mian virus 40 (SV40) replication origin containing DNA in vitro in the presence of the viral large T-antigen. This result implies that all o f the cellular activities required for large T-antigen-dependent in vi tro 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 ind icate that the isolated leukemia cell DNA synthesome may play a role n ot only in viral DNA synthesis but also in human leukemia cell DNA rep lication. 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 f urther study of the molecular mechanisms required to carry out human l eukemia cell DNA replication, but may also provide a tool for eventual ly dissecting some of the regulatory controls of the cell's DNA synthe tic machinery. (C) 1997 Elsevier Science Ltd.