Herpes simplex virus type 1 DNA polymerase. Mutational analysis of the 3'-5'-exonuclease domain.
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.
Kühn
F J
FJ
Department of Genomforschung und Bioinformatik, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, D-69120 Heidelberg, Federal Republic of Germany. c.knopf@dkfz-heidelberg.de
Knopf
C W
CW
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D002852:Chromatography, Ion Exchange; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus; D043211:Exodeoxyribonuclease V