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,...

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Bibliographic Details
Published in:The Journal of biological chemistry 1998-09, Vol.273 (37), p.24075-24082
Main Authors: Baker, Robert O., Hall, Jennifer D.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Animals
Base Sequence
Cell Line
Chromatography, Affinity
Chromatography, Ion Exchange
Conserved Sequence
DNA Primers
DNA Replication
DNA-Directed DNA Polymerase - chemistry
DNA-Directed DNA Polymerase - genetics
DNA-Directed DNA Polymerase - isolation & purification
DNA-Directed DNA Polymerase - metabolism
Exodeoxyribonuclease V
Exodeoxyribonucleases - chemistry
Exodeoxyribonucleases - genetics
Exodeoxyribonucleases - isolation & purification
Exodeoxyribonucleases - metabolism
Herpesvirus 1, Human - enzymology
Herpesvirus 1, Human - genetics
Kinetics
Molecular Sequence Data
Point Mutation
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Sequence Alignment
Spodoptera
Templates, Genetic
Transfection
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.273.37.24075