The goal of this research is to understand the molecular mechanisms of DNA replication and mutagenesis in mammalian cells, using the DNA polymerase of herpes simplex virus type 1 as a model. Genetic, biochemical and computer analysis will be used to define mechanisms of enzyme action, understand modifications of these mechanisms in mutant enzymes and model possible structures on the polymerase molecule. The first objective is to understand the mechanism of substrate recognition by analyzing the binding of nucleoside triphosphates to mutant and normal DNA polymerases. Amino acid changes in polymerases with altered substrate interactions will be identified and used to suggest interactions between residues in the polymerase and nucleoside triphosphates. Directed mutations will be made to test these predictions. A second objective is to understand the interactions between DNA polymerase and other proteins in the replication complex. Replication in vitro by DNA polymerase and other replication proteins (e.g. DNA binding protein) will be studied. Interactions between the DNA binding protein and DNA polymerase will also be investigated by isolating mutations within the binding protein gene which suppress polymerase defects. The third objective is to understand the role of DNA polymerase in viral mutagenesis by studying polymerases which produce mutator or antimutator phenotypes and analyzing the types of mutations made in each case. Finally, computer analysis of the DNA sequence will be used to search for similarities between the herpes simplex and other DNA polymerases and to predict secondary structures within the herpes simplex gene. Included in this analysis will be the mutant data above, possible structures of the related Epstein-Barr polymerases and possible similarities to the structure proposed for polymerase I of Escherichia coli.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032646-08
Application #
3281688
Study Section
Virology Study Section (VR)
Project Start
1983-04-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1992-03-31
Support Year
8
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
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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