Impaired mismatch extension by a herpes simplex DNA polymerase mutant with an editing nuclease defect.
The D368A mutation within the 3'-5'-exonuclease domain of the herpes simplex type 1 DNA polymerase inactivates this nuclease and severely interferes with virus viability. Compared with the wild type enzyme, the D368A mutant exhibits substantially elevated rates of incorrect nucleotide incorporation, as measured in a LacZ reversion assay. This high rate occurs in the presence of high levels of dNTPs, a condition that forces the enzyme to extend mismatched primers. Hence, the mutant fails to correct many misincorporations that are removed in the wild type. In addition, the mutant shows a much reduced ability to replicate DNA templates primed with a 3'-mismatch as compared with wild type. This extension defect also appears more severe than observed for replicases which naturally lack editing nucleases. Based on these findings, we suggest that the inability of the D368A herpes simplex mutant polymerase to replicate beyond a mismatched base pair severely inhibits viral replication.
Baker
R O
RO
Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721-0106, USA.
Hall
J D
JD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence; D017931:DNA Primers; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D013698:Templates, Genetic; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V