The coordinated execution of faithful DNA replication is among the most basic and crucial of all biological processes. Comparative analysis of the cis- and trans-acting components of the replication machinery in diverse organisms has been invaluable in gaining an understanding of how DNA replication initiates and proceeds. Our laboratory has been engaged in a molecular genetic and biochemical analysis of vaccinia virus DNA replication. The vaccinia genome is a linear duplex of 192 kb with covalently closed hairpin termini. We have recently developed a minichromosome assay which has enabled us to demonstrate that linear plasmid sequences capped with viral telomeres of >150 bp replicated efficiently within infected cells. We have also shown that infected cells and virions contain proteins which can form specific protein/DNA complexes with the viral telomeres. In the first aim of this grant, we propose extensive analyses designed to reveal the precise features of the sequence and structure of the telomeres that are required for directing template replication. We also propose to purify the telomere-binding proteins and more fully characterize their interaction with the viral DNA. Identification of the genes encoding these proteins will allow us to determine the role(s) that these protein play in the viral life cycle. Vaccinia virus displays an unusual degree of physical and genetic autonomy from the host cell. It replicates solely within the cytoplasm, and is thought to encode most of the functions necessary for transcription, DNA replication, morphogenesis of the viral particle, and numerous interactions with the host's immune/ inflammatory system. Among the virus 200 genes are many with known or proposed roles in DNA replication. In the second aim of this grant, we will utilize genetic and biochemical approaches to more fully define the function of the B1 protein kinase, the D4 uracil DNA glycosylase, the I3 single stranded binding protein, the D5 DNA-independent NTPase, the A20 processivity factor, and the E9 DNA polymerase. We will also initiate studies to characterize protein:protein interactions between these replication proteins and other, as yet unknown, components of the replication apparatus. Finally, we will refine our in vitro replication system with goal of reconstructing many of the steps involved in vaccinia replication. Although smallpox has been eradicated, poxviruses remain of significant biomedical importance. Molluscum contagiosum is found as an opportunistic infection in 20 percent of AIDS patients, and vaccinia now plays an important role as a recombinant vaccine for many pathogens. Thus, gaining an understanding of poxvirus replication is of significant practical importance and well as intellectual interest.
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