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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI031657-04
Application #
2066644
Study Section
Virology Study Section (VR)
Project Start
1991-03-01
Project End
1996-02-29
Budget Start
1994-03-01
Budget End
1995-02-28
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Utah
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112