The goal of this application is to study the mechanism from herpes simplex virus type 1 (HSV-l). This virus is related to other herpes viruses which cause severe human diseases (neoplasia, mononucleosis, chicken pox, etc.) and which are an increasing problem in immuno-suppressed patients who receive organ transplants or who have AIDS (acquired immune deficiency syndrome). HSV-1 is a useful model for understanding the infection strategy used by this virus group. Furthermore, the herpes polymerase is related to cellular polymerases (e.g. human DNA polymerase alpha) and, hence, is an important model for these enzymes. Since herpes infections are often treated with antiviral drugs targeted against the viral DNA polymerase (e.g. acyclovir), a better understanding of the molecular mechanism utilized by this enzyme should aid in designing more effective drugs. New drugs are needed since development of drug resistance during therapy is a problem. A primary goal of this application is to identify regions within the HSV-1 polymerase required for enzyme activity. Toward this end, site-directed changes will be made in five highly conserved sites within the polymerase. Mutant proteins will be overexpressed and screened for enzymatic defects in DNA template binding, deoxynucleoside triphosphate (dNTP) binding, or polymerase catalysis. The function associated with each site will be extrapolated from the mutant phenotypes. A second goal is to investigate how the polymerase controls replication fidelity. The function of the polymerase-associated 3'-5' proofreading exonuclease will be investigated by constructing an exonuclease-deficient mutant and determining whether this mutant has a mutator phenotype. Two new antimutator mutations (Su1, Su2) will be evaluated to establish whether these mutations affect the 3'-5' exonuclease or dNTP insertion steps of polymerization. And finally, the mutagenesis patterns of wild type and antimutator polymerases will be studied in vitro using defined DNA templates.

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National Institute of General Medical Sciences (NIGMS)
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Virology Study Section (VR)
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University of Arizona
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Wang, Y S; Woodward, S; Hall, J D (1992) Use of suppressor analysis to identify DNA polymerase mutations in herpes simplex virus which affect deoxynucleoside triphosphate substrate specificity. J Virol 66:1814-6
Wang, Y S; Hall, J D (1990) Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8). J Virol 64:2082-9
Hall, J D; Wang, Y S; Pierpont, J et al. (1989) Aphidicolin resistance in herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site. Nucleic Acids Res 17:9231-44
Hall, J D; Woodward, S (1989) Aphidicolin resistance in herpes simplex virus type 1 appears to alter substrate specificity in the DNA polymerase. J Virol 63:2874-6
Hall, J D; Myers, E W (1988) A software tool for finding locally optimal alignments in protein and nucleic acid sequences. Comput Appl Biosci 4:35-40
Hall, J D (1988) Modeling functional sites in DNA polymerases. Trends Genet 4:42-6
Chatterjee, S; Petzold, S J; Berger, S J et al. (1987) Strategy for selection of cell variants deficient in poly(ADP-ribose) polymerase. Exp Cell Res 172:245-57
Hall, J D; Gibbs, J S; Coen, D M et al. (1986) Structural organization and unusual codon usage in the DNA polymerase gene from herpes simplex virus type 1. DNA 5:281-8
Hall, J D; Furman, P A; St Clair, M H et al. (1985) Reduced in vivo mutagenesis by mutant herpes simplex DNA polymerase involves improved nucleotide selection. Proc Natl Acad Sci U S A 82:3889-93
Gibbs, J S; Chiou, H C; Hall, J D et al. (1985) Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain. Proc Natl Acad Sci U S A 82:7969-73

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