Herpes Simplex Virus Type 1 DNA Polymerase: MUTATIONAL ANALYSIS OF THE 3′-5′-EXONUCLEASE DOMAIN

Frank J.P Kühn,Charles W Knopf

doi:10.1074/jbc.271.46.29245

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.

Highlights

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
Mutation Y557S resulted in a strong reduction of the 3؅-5؅-exonuclease activity and in a polymerase activity that was hyperresistant to phosphonoacetic acid

Summary

EXPERIMENTAL PROCEDURES

Materials—Radiochemicals were obtained from Amersham Life Sciences (Braunschweig). Nucleotides were purchased from Pharmacia LKB (Freiburg). Recombinant baculovirus POL and the corresponding pVL1393 transfer vector (pPOL) containing the large open reading frame (LORF) of a PAA-resistant (PAAr) variant of HSV-1 strain ANG [33] from nucleotide position 326 to 4194 [32] were constructed following the BEVS protocol (34 –36). Preparation of Exonuclease Substrate—Reaction mixtures (50 ␮l) containing 50 mM Tris-Cl, pH 7.8, 1 mM dithiothreitol, 5 mM MgCl2, 0.4 mM each of dCTP, dGTP, and dTTP, 20 ␮Ci of [␣-32P]dATP (3000 Ci/mmol), 500 ␮g/ml activated calf thymus DNA, and 10 units of Klenow enzyme were incubated at 37 °C for 30 min, quenched by the addition of 2.5 ␮l of 0.5 M EDTA, and chilled in ice. DNA substrate was extracted twice with phenol/chloroform/isoamyl alcohol and purified by gel filtration using TE buffer

New restriction site

RESULTS

DISCUSSION

Sepharose chromatography

Activitya Exonuclease Polymerase