Characterizations of DNA replication proteins from herpes simplex virus type 1.
AdvisorHall, Jennifer D.
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PublisherThe University of Arizona.
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AbstractThis study was designed to characterize two DNA replication proteins from herpes simplex virus type 1 (HSV-1): the major DNA binding protein (ICP8) and the DNA polymerase. The two proteins are among the seven proteins required for viral DNA synthesis. Four areas were investigated. First, the ICP8 protein was purified from an overproducing cell and showed to behave the same as the viral protein in interacting with DNA. To define the DNA-binding domain of the protein, a proteolytic fragment with the same DNA-binding specificity as the intact protein was identified by a protein blotting assay. N-terminal protein sequencing located the fragment between residues 300 and about 849 in the intact protein. This fragment contains some features important for DNA binding. Second, the DNA polymerase was found to replicate non-processively in vitro. The ICP8 protein slightly stimulated the polymerase activity at lower concentrations and inhibited its activity as the ICP8 concentration increased. Third, to define the functional domain of the DNA polymerase, a fragment of the polymerase predicted to contain both the polymerase and 3$\sp\prime$-5$\sp\prime$ exonuclease activities was overexpressed in Escherichia coli. The fragment was partially purified and its enzymatic activity was examined. Finally, to identify polymerase residues involved in substrate binding, three amino acid changes in polymerase mutants with altered drug sensitivities have been identified by DNA sequencing. Two mutations, affecting nucleotide binding, occur near two highly conserved regions which constitute part of a putative nucleotide binding site. This result indicates that two more residues are important for substrate binding. The third mutation, affecting pyrophosphate binding, occurs upstream of any previously known mutations. This result may indicate a new region involved in substrate binding.
Degree ProgramMolecular and Cellular Biology