Characterization of Wild-type and Temperature Sensitive Mutants of HSV-1 DNA Polymerase
UNIFORMED SERVICES UNIV OF THE HEALTH SCIENCES BETHESDA MD
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This study was undertaken in order to gain a better understanding of the activities and functional domains of HSV DNA polymerase. HSV polymerases were partially purified from cells infected with HSV-1 wildtype strains mP and KOS, and temperature sensitive mutants, tsC4 and tsC7 derived from KOS whose mutations map within the HSV polymerase gene. The activities of these enzymes were characterized as to salt and pH optima, divalent cation optima, and DNA synthesis requirements. Differences between parental and mutant induced polymerases were observed in divalent cation use and sulfhydryl reagent requirements. The polymerases from tsC4 and tsC7 also showed decreased affinity for deoxynucleoside triphosphates as compared to the parental strain. The HSV mP, tsC4, and tsC7 HSV polymerases were more sensitive to phosphonoacetate than KOS polymerase. The rate of thermal inactivation of partially purified polymerases and crude extracts of infected cell including the polymerase from an additional ts mutant, tsD9 was determined. Little difference was seen between the wild-type and putative temperature sensitive enzymes with the exception of tsD9 polymerase in crude extracts. Velocity sedimentation of native and heat-treated nuclear extracts on neutral glycerol gradients revealed that the residual polymerase activity in the heat-treated extracts for all strains was consistently shifted to a slightly slower sedimentation value indicating the possible dissociation of a macromolecular complex. These studies did not uncover any obvious differences in the catalytic activity of the mutant HSV DNA polymerases with the exception of tsD9 that would account for the temperature sensitivity seen in vivo with the HSV-1 mutants. The temperature sensitive phenotype of these viruses is therefore likely du to some other interaction of the HSV replication system and not the basic deoxynucleotide polymerization activity.
- *DEOXYRIBONUCLEIC ACIDS
- HEAT TREATMENT
- IN VIVO ANALYSIS
- Medicine and Medical Research