9728474 Roseman 1. Technical Vaccinia replication occurs in the cytoplasm of infected cells with a high degree of independence from host functions, since virus encodes most, if not all, of the enzymes required for DNA metabolism and replication, including the deoxyuridine triphosphatase (dUTPase). dUTPase helps maintain the fidelity of DNA replication by lowering the dUTP pool, high levels of dU incorporation into replicating DNA is a lethal event due to the excision repair process which causes double-strand breaks. It provides also the precursor for TMP production by thymidylate synthetase. dUTPase deficient viruses exhibit reduced replication and/or pathogenicity. The vaccinia enzyme has high amino acid identity to its human homologue (63%). This study explores the role of phosphorylation in the regulation of both viral and human dUTPase activities. This is achieved by identifying the phosphorylated site(s) by peptide mapping and phosphoamino acid analysis. The effect of mutation at these sites on enzyme activity is analyzed in vitro and in vivo. Also studied is whether the vaccinia enzyme is a substrate for either of two virally encoded protein kinases. The function of this enzyme in DNA synthesis and replication fidelity is assessed by using dUTPase deficient virus with -galactosidase as a marker. The role of a critical amino acid in the uracil binding motif that differs from that in the vaccinia protein is assessed by mutagenesis. Nickel affinity chromatography in conjunction with His-tagged is used in enzyme purification. 2. Non-technical Vaccinia virus replication occurs in the cytoplasm of infected cells with a high degree of independence from host functions. This is in part due to the fact that the virus encodes most, if not all, of the enzymes required for DNA metabolism and replication, including the deoxyuridine triphosphatase (dUTPase) enzyme. This enzyme helps maintain the fidelity of DNA replication and provides a precursor for DNA replication. It is essential in a number of organisms, and dUTPase deficient viruses exhibit reduced replication and/or pathogenicity. The vaccinia enzyme has a high amino acid identity to its human homologue (63%). This study thus explores the regulation of both viral and human dUTPase activities and the role of other viral enzymes in this regulation. Mutagenesis approach is used to assess the role of the enzyme in viral DNA synthesis and replication fidelity, and of critical amino acids in the uracil binding site that differs between the human and the vaccinia UTPase.
