Effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus DNA polymerase on the fidelity of DNA replication in vivo

A mutagenesis system was developed for the in vivo study of the fidelity of DNA replication mediated by wild-type herpes simplex virus type 1 (HSV-1) strain KOS and its polymerase (Pol) mutant derivatives PAA(r)5, Y7, and YD1 2. The pHOS1 shuttle plasmid, which contained the SupF mutagenesis marker g ene and the HSV ori, sequence, was used for analysis of the mutation freque ncy and the mutation spectrum. All three Pol mutants induced significant in creases in the mutation frequencies of the target gene, despite the fact th at PAA(r)5 was previously shown to have an antimutator phenotype by the thy midine kinase mutagenesis assay (J. D. Hall, D. M, Coen, B. L. Fisher, M. W eisslitz, S. Randall, R, E. Almy, P. Gelep, and P. A. Schaffer, Virology 13 2:26-37, 1984; C. B, C. Hwang and J.-H. Chen, Gene 152:191-193, 1995). Alte red spectra of mutated target genes induced by these three mutants were als o observed. The relative frequencies of both deletion and complex mutations found in mutants induced by exonuclease-proficient PoIs were significantly higher than those induced by exonuclease-deficient PoIs. On the other hand , the exonuclease-deficient Pols induced significant increases in the frequ ency of base substitutions, which comprised predominantly G . C-to-T . A tr ansversions, as well as mutations at additional hot spots. These results su ggest that the HSV-1 DNA Pol can incorporate purine-purine or pyrimidine-py rimidine mispaired bases which may be preferentially proofread by its intri nsic exonuclease activity, Furthermore, the effects of the sequence context of the target gene and the assay method should also be considered carefull y in any analysis of replication fidelity.