Mutations in the Exo III Motif of the Herpes Simplex Virus DNA Polymerase Gene Can Confer Altered Drug Sensitivities

Hwang, Ying T.; Smith, Jennifer F.; Gao, Li; Hwang, Charles B. C.

doi:10.1006/viro.1998.9201

Cited by 30 publications

(31 citation statements)

References 34 publications

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“…Moreover, most KOS-induced substitutions were not found in Y7-induced mutants, thereby refuting the assumption that most substitutions observed in KOS mutants would also be isolated in Y7-induced mutants, if Y7 Pol is defective only in proofreading activity. Nevertheless, this suggests that Y7 Pol may also exhibit altered Pol activity, consistent with our previous finding that Y7 exhibits altered binding affinities to certain nucleoside analogs (12). Finally, the HSV-1 Pol might evolve base substitution mainly by misinsertion during DNA chain elongation.…”

supporting

confidence: 88%

“…Although the lack of sufficient common mutations could be due to the limited number of samples analyzed, the observation that most Y7-induced substitutions are Y7 specific suggested that Y7 Pol might exhibit an altered Pol activity in addition to the loss of its exonuclease activity. Consistent with this proposition is the observation that the Y7 recombinant exhibited different binding affinities to certain nucleoside analogs (12). However, the extents of the altered activity contributing to the accuracy of Pol's activity and whether it is essential for retaining Y7 Pol's activity in vivo require further study.…”

mentioning

confidence: 73%

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Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

Lü

Hwang

2002

J Virol

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To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominately frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion of frameshifts (21%; P < 0.005). Furthermore, the majority of substitutions were transitional changes in both groups, although they distributed differently. The implications of these findings are discussed.The fidelity of DNA replication is regulated by several different mechanisms (reviewed in reference 14 and references therein). In particular, the selection of correct nucleotides by DNA polymerase (Pol) serves a major role in preserving the integrity of the genome. Furthermore, the excision of misinserted bases by the exonucleolytic proofreading activity of the Pol also directly contributes to the high level of fidelity. While exonucleolytic proofreading is important for the integrity of the genetic materials, its association with most Pols adds the complexity of the mechanisms by which Pol regulates the accuracy of nucleotide incorporation. For example, the polymerization reaction acts to extend the nascent primer strand in the 5Ј-to-3Ј direction, whereas the exonuclease activity reacts in an opposite direction to remove the newly incorporated nucleotide. Although it is known that each polymerization involves multistep reactions to achieve Pol selectivity against insertion of incorrect nucleotides, the contribution of each individual step to the fidelity requires further detailed characterizations. Exonucleolytic proofreading contributes on average about 100-fold to fidelity. However, the contribution of exonuclease activity to base substitution fidelity can vary over a wide range, and the degree of its contribution is enzyme and sequence specific. This is based on the studies demonstrating that the "frayed" single-stranded primer due to a terminal mispair will preferentially bind to the exonuclease site and that the doublestranded terminus will preferentially bind to the Pol active site. Therefore, the stability of the duplex region of the templateprimer will depend on its DNA sequence, and proofreading efficiency will vary in different sequence contexts. Furthermore, the degree of fraying required for single-stranded DNA to bind to the exonuclease active site may vary, depending on the distance between the Pol and exonuclease active sites. Therefore, it is also enzyme specific (for details see review in reference 19).Use of the herpes simplex virus (HSV) is advantageous, given that its genome can be altered for biological, biochemical, and genetic studies in vitro and in vivo. We previously engineered two exonucleolytic-proofreading-deficient HSV type 1 (HSV-1) recombinants, Y7 and YD12, which are nonetheless competent...

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confidence: 88%

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confidence: 73%

Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

Lü

Hwang

2002

J Virol

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“…Plaque reduction assays were performed to examine the drug sensitivity of each recombinant, as described previously (19). Since these recombinants are TK negative, only phosphonoacetic acid (PAA) and aphidicolin (APH) (Sigma) were used to analyze the Pol phenotype.…”

Section: Recombinant Virusesmentioning

confidence: 99%

Replication Fidelity of the supF Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1

Hwang

Liu

Hwang

2002

J Virol

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Recombinant viruses were constructed to have an Escherichia coli replicon containing a mutagenesis marker, the supF gene, integrated within the thymidine kinase locus (tk) of herpes simplex virus type 1. These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the supF gene. A mutation frequency of approximately 10 ؊4 was observed for wild-type strain KOS-derived recombinants in their replication of the supF gene. However, recombinants derived from the PAA r 5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the tk gene, had three-to fourfold increases in supF mutation frequency (P < 0.01), a result similar to that exhibited when the supF gene was induced to replicate as episomal DNA (Y. T. Hwang, B.-Y. Liu, C.-Y. Hong, E. J. Shillitoe, and C. B. C. Hwang, J. Virol. 73:5326-5332, 1999). Thus, the PAA r 5 Pol mutant had an antimutator function in replicating the tk gene and was less accurate in replicating the supF gene than was the wild-type strain. The spectra of mutations and distributions of substituted bases within the supF genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA. Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication. Furthermore, the replication mode of the target gene also affected the mutational spectrum.DNA replication in mammalian cells is a complicated process (reviewed in references 2 and 25). It requires intricate DNA replication machinery that both duplicates chromosomal DNA and maintains genomic integrity. As the pivotal enzymes, DNA polymerases (Pols) are critical for ensuring the accuracy of DNA synthesis via their roles in the selection of nucleotides to be incorporated during DNA replication (polymerization) and in the removal of misinserted nucleotides from the 3Ј-OH end of the primer strand (proofreading) (reviewed in references 20 and 23

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“…Theoretically, one would expect to observe more identical substitutions in both groups if the exonuclease Pol does not have altered polymerase activity. The observation that recombinant Y7 (Y577H) virus also exhibits altered drug sensitivities relative to wild-type virus further supports that the Exo III motif has a role on the polymerization activity (Hwang et al, 1998) and that the polymerase and exonuclease domains of HSV-1 Pol are interdependent structurally and functionally, also as suggested by the overlap between conserved -region C and Exo III motif (Fig. 2).…”

Section: Exonuclease Deficient Polymerase Induces Altered Mutation Spmentioning

confidence: 55%

DNA Replication Fidelity of Herpes Simplex Virus

Hwang¹

2011

DNA Replication and Related Cellular Processes

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Mutations in the Exo III Motif of the Herpes Simplex Virus DNA Polymerase Gene Can Confer Altered Drug Sensitivities

Cited by 30 publications

References 34 publications

Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

Replication Fidelity of the supF Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1

DNA Replication Fidelity of Herpes Simplex Virus

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