Although we have learned a great deal about the proteins involved in eukaryotic DNA replication, we know relatively little about how they communicate with one another to initiate DNA synthesis and to carry out the elongation reaction. Nearly everything we know about this process in high eukaryotes has come from the study of SV40. This virus codes for only one protein directly involved in DNA synthesis; the remaining factors come from the cell. T antigen associates with a number of these proteins, including DNA polymerase alpha, RPA and topoisomerase I (topoI) and there is accumulating evidence that the interaction of T antigen with these cellular proteins is required for the replication of SV40 DNA. We have concentrated on the association between T antigen and topoI and have obtained evidence that these two proteins may form the first functional complex at the origin of replication. Our goal is to build on these observations to obtain a clearer picture of the order in which cellular proteins associated with one another to form the initiation complex and to determine the function of each protein during initiation and elongation. In particular, we would like to better understand the relationship between the T antigen helicase and topoisomerase to better appreciate how these two enzymes communicate with one another during DNA unwinding. Since this complex must deal with the single strands that are generated during unwinding, we would also like to characterize the single-stranded DNA binding domain of T antigen which we have recently shown to be completely separate from the origin- binding domain. We therefore want to analyze the role of topoisomerase I in SV40 DNA replication, study the composition of the complex(es) that participates in SV40 DNA replication and determine the function of each protein in the complex, and characterize the function of the single- stranded DNA binding domain of T antigen in an effort to gain insights into the mechanism of helicase action.
Showing the most recent 10 out of 34 publications
