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

Abstract

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 PAAr5, Y7, and YD12. The pHOS1 shuttle plasmid, which contained the SupF mutagenesis marker gene and the HSV oris sequence, was used for analysis of the mutation frequency and the mutation spectrum. All three Pol mutants induced significant increases in the mutation frequencies of the target gene, despite the fact that PAAr5 was previously shown to have an antimutator phenotype by the thymidine kinase mutagenesis assay (J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. Gelep, and P. A. Schaffer, Virology 132:26-37, 1984; C. B. C. Hwang and J.-H. Chen, Gene 152:191-193, 1995). Altered spectra of mutated target genes induced by these three mutants were also observed. The relative frequencies of both deletion and complex mutations found in mutants induced by exonuclease-proficient Pols were significantly higher than those induced by exonuclease-deficient Pols. On the other hand, the exonuclease-deficient Pols induced significant increases in the frequency of base substitutions, which comprised predominantly G. C-to-T. A transversions, as well as mutations at additional hot spots. These results suggest that the HSV-1 DNA Pol can incorporate purine-purine or pyrimidine-pyrimidine mispaired bases which may be preferentially proofread by its intrinsic exonuclease activity. Furthermore, the effects of the sequence context of the target gene and the assay method should also be considered carefully in any analysis of replication fidelity.

FIG. 1

HSV pol-dependent replication of the pHOS1 plasmid. (A) Aliquots of the total DNA recovered from mock (lanes 1 to 3)- or KOS (lanes 4 to 6)-infected cells were either untreated (lanes 1 and 4) or treated with HindIII alone (lanes 2 and 5) or HindIII plus DpnI (lanes 3 and 6) and subjected to Southern blot analysis. (B) DNA samples isolated from KOS (lane 1)-, HP66 (lane 2)-, and PAA^r5 (lane 3)-infected Vero cells were digested with both HindIII and DpnI and subjected to Southern blot analysis. The arrows indicate the positions of the linearized pHOS1.

FIG. 2

Schematic of mutations within the SupF gene. The top line shows the coding sequence of the SupF gene. Mutations induced by the KOS, PAA^r5, P5Aph⁺K2, Y7, and YD12 pol genes are shown beneath the sequence for each group. Each letter indicates the changed base found in single mutations, while multiple changes found in the same mutant are underlined. The number in parentheses for one mutant isolated from the PAA^r5 group indicates the change at position 77. Symbols: ▵, nucleotide(s) deleted; +, nucleotide inserted at the corresponding location.