Site-specific mutagenesis of a highly conserved region of the herpes simplex virus type 1 DNA polymerase gene.
Seven point mutations were introduced into region I of the herpes simplex virus type 1 DNA polymerase, which is most highly conserved among DNA polymerases and has no drug sensitivity markers mapped to it. The functional consequences of these mutations were studied in an in vitro transcription-translation system in which T7 transcripts of cloned polymerase genes were used to generate enzymatically active polypeptides in reticulocyte lysate. Analysis of labeled polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to show any alterations of stability caused by these mutations. The mutations G885R, D886N, T887K, D888A, and G896V lacked polymerase activity and failed to be stimulated by cotranslation of the herpes simplex virus 65-kilodalton DNA-binding protein, whereas R881G and S889A retained both polymerase activity and the capacity to be stimulated by the 65-kilodalton DNA-binding protein.
Dorsky
D I
DI
Division of Infectious Diseases, University of Connecticut Health Center, Farmington 06032.
Crumpacker
C S
CS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013604:T-Phages / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics*
D015345:Oligonucleotide Probes; D015678:Viral Structural Proteins; D004259:DNA-Directed DNA Polymerase