The initiation of chromosomal DNA replication is likely to be a vital control point in the maintenance of normal cell proliferation. Significant insights into the mechanisms and controls involved in mammalian DNA synthesis have derived from analysis of a model system, the replication of the genome of the papovavirus SV40. Recent studies have indicated that the initiation of SV40 DNA replication in vitro may be controlled in a cell cycle regulated manner by the phosphorylation state of the viral initiator protein, the SV40 large T antigen. Chromosomal DNA replication may similarly be initiated by the cell cycle controlled modification of cellular initiator proteins. Therefore, cellular modification of T antigen phosphorylation may be a useful model for studying the control of DNA replication in mammalian cells. This project aims to achieve 1) an understanding of how the site-specific dephosphorylation of T antigen stimulates initiation of DNA replication, and 2) the identification of the cell cycle regulated factors which modify T antigen phosphorylation. The catalytic subunit of the cellular phosphoprotein phosphatase 2A (PP2Ac) stimulates the early stages in SV40 DNA replication and dephosphorylates a limited number of specific sites on T antigen. The critical phosphates removed by PP2Ac will be determined. Dephosphorylated T antigen will be re-isolated and assayed for activity in initiation of replication, to determine if dephosphorylation is both necessary and sufficient for stimulation of activity. Recently constructed point mutants in T antigen, lacking the specific phosphorylation sites, will also be assayed. Dephosphorylation- induced changes in the interaction of T antigen with the 64 base pair minimal origin of SV40 DNA replication will be assessed. Assays, performed under replication conditions, will measure changes in binding constants, changes in T antigen self-association, T antigen-induced changes in origin DNA structure, and alterations in T antigen helicase activity. Alterations in T antigen interactions with cellular proteins involved in cellular proliferation will be assessed by co-immunoprecipitation experiments. PP2Ac stimulates SV40 DNA replication in extracts from Gl phase cells to a much greater extent than extracts from S phase cells. This suggests the existence of a cell cycle regulated factor which modifies the phosphorylation state of T antigen. The cellular factor(s), most likely a kinase, which reversibly inactivates T antigen will be identified and purified. Activities of the T antigen phosphatase and kinase will be assessed through the cell cycle, and the mechanism which modulates these activities through the cell cycle will be defined. A detailed analysis of the control of initiation of SV40 DNA replication may provide insights into the control of cellular DNA replication, and its abnormalities in cancerous cells.
