| Vaccinia virus displays both physical and genetic autonomy from the host cell. All of the functions required for viral DNA replication are encoded within its own genome. The centerpiece of the replication apparatus is the vaccinia DNA polymerase. Although this enzyme is intrinsically distributive, polymerase activity in cytoplasmic extracts of infected cells is highly processive. This dichotomy is seen in virtually all bacterial, viral and eukaryotic replication systems. In these systems the catalytic subunit attains processivity through interaction with another protein, a processivity factor. In vaccinia, the product of the A20 gene has been identified as a stoichiometric component of the processive form of the polymerase. To complement biochemical analyses of the vaccinia processivity factor, a genetic approach was undertaken to study the structure and function of the A20 protein. Initially, clustered charge-to-alanine mutagenesis was performed on seven regions of the A20 gene; four more regions were selected for analysis as part of this dissertation. Eight mutant viruses containing altered A20 alleles were isolated via transient dominant selection, and two, tsA20-6 and tsA20-ER5, displayed tight temperature-sensitive (ts) phenotypes. At the nonpermissive temperature, plaque formation was abrogated in both viruses and the yield of infectious virus was reduced 100-fold relative to that obtained at permissive temperature.{09}Both viruses showed a profound defect in the accumulation of viral DNA at the non-permissive temperature, despite the accumulation of both A20 and DNA polymerase proteins to wt levels. (Abstract shortened by UMI.)... |
