The long term goals of this proposal are to understand the fundamental mechanisms of eukaryotic chromosomal DNA replication and to determine how these mechanisms are disrupted in transformed cells. Since chromosomal DNA replicationis deregulated in transformed cells, this proposal may provide information on steps of eukaryotic DNA replication that act as regulatory checkpoints for the cell. Simian Virus 40 (SV40) will be used as a model system in vitro to explore the mechanisms of eukaryotic DNA replication. The investigator will examine how the SV40 origin of replication (ori) becomes denatured during the initiation phase of viral DNA replication. Critical contacts on ori that are used by SV40 large T antigen and human single-stranded DNA-binding protein (hRPA) to denature ori will be determined. The ori region contains three essential domains, the early palindrome, a 17bp AT region, and four pentanucleotides (GAGGC) to which T antigen directly binds.
A second aim i s to characterize the function of these essential domains within the ori region. Populations of SV40 origins will be prepared in which the early palindrome and AT region are replaced by random sequences, followed by selection for functional origins using either viral DNA replication(in vivo or in vitro) or the T-antigen dependent DNA unwinding reaction.Thirdly, the mechanism by which the DNA helicase activity of T antigen unwinds DNA will be examined. The unwinding of chemically-modified or ribose-substituted DNA forks by T antigen will be examined to determine the DNA helicase step size. The essential features of the DNA helicase mechanism will be examined bycharacterizing the binding of the T antigen RNA helicase to RNA forks as a comparative tool.Finally, the role of hRPA during SV40 DNA replication will be examined. Studies in the investigator's laboratory and in other labs have found that hRPA can form two markedly different complexes with single-stranded DNA. The interaction of hRPA in these complexes with modified single stranded DNA binding substrates will be individually characterized.
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