Complete and faithful duplication of the genome is a fundamental prerequisite for cell division in development and tissue renewal. Errors and malfunctions in the replication process can result in loss of cell viability and are responsible in large part for genetic diseases such as cancer, but our understanding of how the mammalian genome is duplicated, how this process is regulated, and how malfunctions are corrected remains incomplete. The long-term goal of the proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Replication of the simian virus 40 mini-chromosome in infected cells and in cell-free reactions has served as a simple model system to identify and characterize ten human proteins that, together with the viral protein T antigen, are necessary and sufficient to reconstitute SV40 DNA replication in vitro. These ten human proteins are conserved among eukaryotes and essential for cell DNA replication, suggesting that the virus and its host use similar mechanisms to replicate their genomes. However, the mechanism of viral DNA replication differs from that of the host in several key features. These differences suggest that viral DNA replication mechanisms may resemble those of host pathways that are activated by DNA damage signaling to rescue or restart stalled replication forks. The proposed research program is designed to explore this possibility by first determining the detailed molecular mechanisms of the early steps in viral DNA replication and then applying this knowledge to elucidate the role of a novel human DNA helicase (HDHB) in DNA damage repair.
Specific Aim 1 combines molecular genetics, biochemistry, and structural biology to determine the detailed interactions of T antigen with human DNA polymerase alpha-primase in initiation of SV40 replication in a cell-free system and to characterize host proteins that interact similarly with the polymerase-primase.
Specific Aim 2 uses the same approaches to investigate the interaction of the single-strand DNA-binding protein replication protein A with the polymerase-primase in primer synthesis and elongation.
Specific Aim 3 uses molecular genetics and biochemistry to determine the functional domains of HDHB, characterize in detail its interactions with DNA and other proteins, and elucidate its roles in genotoxin-regulated chromatin-binding, primosome activity, and DNA repair.
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