Research will be in the area of herpesvirus molecular biology. This system allows sophisticated genetic and molecular biological analysis. Analysis of these viruses, as stated in this proposal, may lead to improved understanding and possibly treatment of diseases caused by these common pathogens that can present to a dentist or physician. To a dental scientist interested in the molecular aspects of craniofacial development, herpesviruses are known to perturb development and provide a relatively simple system for probing aspects of eukaryotic gene expression and DNA replication. The proposed experiments will focus on aspects of DNA replication, a process that is important for development and growth. The functional relatedness of the DNA polymerases of CMV, EBV, VZV, and Vaccinia virus to the HSV-1 DNA polymerase will be examined. Plasmids bearing these non-HSV1 DNA polymerase genes (pol), under the control of an HSV promoter, will be tested for their ability to complement HSV DNA polymerase function using both transient and stable cell line assays. Chimeric nonHSV/HSV pol genes will be constructed using both in vitro and in vivo methods and tested for function. Plasmids with the pol genes (HSV wildtype, mutants, and chimeras) under the control of a strong E.coli promoter will also be constructed. The genes will be overproduced in E.coli, purified, and characterized biochemically. This characterization will include kinetic analysis and binding studies using appropriate substrates and substrate analogs and the ability of the major HSV binding protein to stimulate polymerase activities.
