Replication of the genome is a basic process relevant to normal cell growth and division. Detailed knowledge of the mechanism of eukaryotic DNA replication is central to understanding how normal cells lose control over this process and become cancer cells. The goal of this project is to isolate and characterize protein factors involved in the initiation of eukaryotic DNA synthesis. The analysis of these proteins and their role in the initiation process is required in order to understand the mechanism and controls over the initiation of DNA replication . SV40 will be used as a model system because it depends on host cell proteins to replicate its DNA. The in vitro SV40 DAN replication system can also be exploited by genetic and biochemical techniques. The initiation process will be studied using three different approaches to the problem. The first is to analyze the interaction of the SV40-encoded replication protein, T antigen, with the sequences at the origin of replication. The ability of T antigen to alter the DNA structure by binding to the origin will be tested using assays for unwinding and bending of the DNA helix. Mutant T antigens will be used to correlate the ability to replicate DNA with other functional activities of T antigen. The second specific aim is to purify cellular initiation proteins by using the affinity of these proteins for the T antigen/origin complex. Preparative isolation of in vitro assembled initiation complexes by conventional techniques will be performed. Isolated proteins will be characterized by several criteria including funciton in DNA synthesis, enzymatic activity, physical parameters and affinity for either T anitgen or SV40 DNA.
The final aim of the project is to generate monoclonal antibodies directed against the cellular replication proteins. These antibodies will be used as tools to study the mechanism of initiation of DNA replication and ultimately to clone the genes encoding the cellular replication proteins. The cell-cycle dependent expression of these proteins will be studied in an effort to understand the controls over eukaryotic DNA replication.
