Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

Abstract

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...

Exonuclease-Deficient Polymerase Mutant of Herpes Simplex Virus Type 1 Induces Altered Spectra of Mutations

“…These results suggested that HSV-1 Pol could preferentially misinsert nucleotides at these positions that were subsequently proofread by the intrinsic exonuclease activity. Conversely, different types of substitutions identified at other locations among wild-type and Y7 Pol-induced mutants suggest that Y7 Pol might possess an altered polymerase activity, consistent with a recent study of the mutation spectra of tk mutants derived from both wild-type and Y7 Pols (22). This is supported by the fact that Y7 Pol exhibits altered binding affinities for nucleoside analogs (15) and indicates that the exonuclease domain has critical structural and functional roles in the catalytic subunit of the HSV-1 Pol holoenzyme.…”

Contribution of the 3′- to 5′-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis

“…That study (13) reported that selectivity of correct over incorrect dNTP was ϳ300, whereas bacteriophage T4 and T7 DNA polymerases discriminate among dNTPs by a factor of Ͼ1000 and Ͼ100,000, respectively (14 -16). Previous studies suggested that the exo activity of HSV-1 pol contributes substantially to the fidelity of HSV replication in vivo, because mutation of the conserved exo site III of the HSV-1 pol catalytic subunit was associated with a strong mutator phenotype (10,17,18). Moreover, such pol mutants are genetically unstable and revert or recombine with high frequency (10), suggesting strong in vivo selection against an HSV-1 pol lacking exo activity.…”

DNA Replication Fidelity of Herpes Simplex Virus

“…Furthermore, the Y577H mutation not only loses exonuclease activity but also alters the polymerase activity as it replicates misinsertions at positions distinct from those of wild-type Pol. Only 3 identical base substitutions are found among 15 and 52 tk mutations replicated by wild-type and exonuclease negative Pol, respectively (Lu, Hwang, and Hwang, 2002b). Theoretically, one would expect to observe more identical substitutions in both groups if the exonuclease Pol does not have altered polymerase activity.…”

“…In contrast, the Y7 recombinant virus, which contains the Y577H mutation in the Exo III motif, replicates the tk gene with significantly less frameshift mutations (21%; p < 0.005) relative to wild-type virus (Lu, Hwang, and Hwang, 2002b). Furthermore, although the majority of substitutions are transitional changes in mutants derived from both wild-type and exonuclease-deficient viruses, they distribute differently (Lu, Hwang, and Hwang, 2002b). These findings imply that HSV Pol can incorporate mispaired nucleotides, and most of the misinsertions are corrected by the proofreading activity during DNA synthesis.…”

“…The higher magnitude of the mutation frequencies observed in the tk mutagenesis study may be theoretically explained by the fact that the tk mutation frequency could be amplified due to the increases of lethal mutations that reduces the total number of progeny viruses been analyzed on cell cultures. Nevertheless, the exonuclease-deficient Pol mutants replicate both target genes with significant difference of mutation spectra compared to those induced by the wild-type Pol Lu, Hwang, and Hwang, 2002b).…”