Herpes Simplex Virus Type 1 DNA Polymerase

Abstract

Like true DNA replicases, herpes simplex virus type 1 DNA polymerase is equipped with a proofreading 3'-5'-exonuclease. In order to assess the functional significance of conserved residues in the putative exonuclease domain, we introduced point mutations as well as deletions within and near the conserved motifs' exonuclease (Exo) I, II, and III of the DNA polymerase gene from a phosphonoacetic acid-resistant derivative of herpes simplex virus-1 strain ANG. We examined the catalytic activities of the partially purified enzymes after overexpression by recombinant baculovirus. Mutations of the motifs' Exo I (D368A, E370A) and Exo III (Y577F, D581A) yielded enzymes without detectable and severely impaired 3'-5'-exonuclease activities, respectively. Except for the Exo I mutations, all other Exo mutations examined affected both exonuclease and polymerization activities. Mutant enzymes D368A, E370A, Y557S, and D581A showed a significant ability to extend mispaired primer termini. Mutation Y557S resulted in a strong reduction of the 3'-5'-exonuclease activity and in a polymerase activity that was hyperresistant to phosphonoacetic acid. The results of the mutational analysis provide evidence for a tight linkage of polymerase and 3'-5'-exonuclease activity in the herpesviral enzyme.

Contribution of the 3′- to 5′-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis

“…In the absence of correct dNTP, the wild-type pol can engage in idling reactions to successively misincorporate and then remove another misincorporated nucleotide with its associated exo activity (9,33). The results in Fig.…”

“…However, the rate constant (k pol ) for polymerization by the D368A mutant pol was 199 Ϯ 26 s Ϫ1 , ϳ20% faster than the 157 Ϯ 31 s Ϫ1 reported for dTTP incorporation by the wild-type pol (13). The slower rate constant for the wild-type enzyme most probably reflects competition of the forward polymerization reaction with the competing excision reaction (9). Taken together, these results demonstrate that the D368A mutation destroys exo activity without significantly altering the polymerization domain or its inherent function.…”

“…Nucleotide Excision Activity-A mutation in the conserved exo site I domain of the HSV-1 pol (D368A) had been reported previously to lack significant exo activity (9,20). To confirm the complete absence of exo activity in D368A pol and to ensure that the mutation did not compromise the polymerizing activity of the enzyme, we purified the mutant and wild-type enzymes from Sf9 insect cells infected with recombinant baculoviruses, which expressed these genes as described under "Experimental Procedures."…”

“…Cells and Viruses-Recombinant baculoviruses were used to express the HSV-1 wild-type (gift of Dr. Robert Lehman, Stanford University) or exo-deficient (D368A) pol genes with or without the UL42 processivity factor gene and have been described elsewhere (9,23). Recombinant viruses were propagated in Sf9 insect cells at 27°C as described previously (24).…”

“…We selected for study the D368A mutant pol, which converts an aspartate to alanine in the conserved exo site I domain. This site is thought to be involved in the coordination of two divalent metal ions responsible for the exo catalytic activity (19), and previous studies demonstrated that the D368A mutant pol lacked any detectable exo activity (9,20). A previous study with this mutant pol used a limited set of primer/templates (P/Ts) and concluded that the enzyme possessed a defect in mismatch extension (11).…”