Reduced in vivo mutagenesis by mutant herpes simplex DNA polymerase involves improved nucleotide selection

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Mutation frequencies can be influenced by DNA polymerase replication fidelity.
• Herpes simplex virus type 1 (HSV-1) derivatives encoding drug-resistant polymerases show reduced spontaneous mutants.
• Antimutator polymerases are key to understanding DNA replication accuracy.

Purpose of the Study:

• To investigate the enhanced replication fidelity of a specific antimutator virus DNA polymerase.
• To elucidate the molecular mechanisms underlying this enhanced fidelity.
• To determine if altered polymerase interactions with nucleotides contribute to the antimutator phenotype.

Main Methods:

• Assessing antimutator virus response to base-mispairing mutagens (e.g., N-methyl-N'-nitro-N-nitrosoguanidine).
• In vitro replication error assays using purified antimutator DNA polymerase.
• Enzyme kinetics (Km values) and nucleotide analogue resistance assays.
• Analysis of associated 3',5' exonuclease activity.

Main Results:

• The antimutator virus demonstrated reduced sensitivity to mutagens that induce base mispairing.
• Purified antimutator DNA polymerase produced fewer in vitro replication errors.
• Mutant polymerase showed altered nucleoside triphosphate interactions, including resistance to analogues and elevated Km values.
• Evidence against increased proofreading activity by an associated exonuclease was found.

Conclusions:

• The antimutator DNA polymerase exhibits significantly enhanced replication fidelity.
• Altered interactions with nucleoside triphosphates, specifically reduced affinity, likely explain the antimutator phenotype.
• This reduced affinity accentuates base-pair stability differences, favoring correct nucleotide selection and reducing mutations.