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Journal of Virology, March 2003, p. 2946-2955, Vol. 77, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.5.2946-2955.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Ying T. Hwang and Charles B. C. Hwang*

Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, New York 13210

Received 14 October 2002/ Accepted 27 November 2002


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ABSTRACT
 
The effect of exonuclease activity of the herpes simplex virus DNA polymerase (Pol) on DNA replication fidelity was examined by using the supF mutagenesis assay. The recombinants with exonuclease-deficient Pol, containing an integrated supF gene in the thymidine kinase locus (tk), exhibited supF mutation frequencies ranging from 0.14 to 5.6%, consistent with the tk mutation frequencies reported previously (Y. T. Hwang, B.-Y. Liu, D. M. Coen, and C. B. C. Hwang, J. Virol. 71:7791-7798, 1997). The increased mutation frequencies were 10- to 500-fold higher than those observed for wild-type Pol recombinants. The increased mutation frequencies also were significantly higher than those of supF mutant replicated by exonuclease-deficient Pols in the plasmid-borne assay. Furthermore, characterization of supF mutants demonstrated that recombinants with a defective exonuclease induced types and distributions of supF mutations different from those induced by wild-type Pol recombinants. The types of supF mutations induced by exonuclease-deficient recombinants differed between the plasmid- and genome-based assays. The spectra of supF mutations also differed between the two assays. In addition, exonuclease-defective viruses also induced different spectra of supF and tk mutations. Therefore, both the assay methods and the target genes used for mutagenesis studies can affect the repication fidelity of herpes simplex virus type 1 Pol with defective exonuclease activity.


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INTRODUCTION
 
Herpes simplex virus type 1 (HSV-1) is a well-characterized system used for studying DNA replication in mammalian cells (4). The genes encoded by HSV-1 can be manipulated in the laboratory to obtain specific mutations, including lethals, for genetic, molecular, and biochemical characterizations of gene function. For example, certain polymerase (Pol) mutations with altered amino acid residues critical for the exonuclease activity have been engineered (7, 9, 12, 19). To compensate for the lethal phenotype of certain mutations, recombinant viruses with the mutation can be constructed and propagated by using a cell line expressing the wild-type Pol enzyme (7). Further, the use of multiple approaches to characterize these mutants has revealed the biological significance of the exonuclease activity associated with Pol (7, 9, 12, 13, 15, 19).

As the pivotal enzyme involved in DNA replication, Pol is required for duplicating genomic DNA and maintaining the integrity of the genome. The catalytic subunit of HSV-1 Pol possesses both polymerase and exonuclease activities (17, 18, 23). The polymerase activity functions in the selection of correct deoxynucleoside triphosphates during the polymerization reaction, since mutations within conserved regions of the polymerase domain can result in altered binding affinity to certain nucleoside analogs (2, 6, 10, 11, 15, 21, 26). On the other hand, the significance of the intrinsic exonuclease activity on the polymerization reaction remains unclear. One study demonstrated that the exonuclease activity is critical for proper polymerase activity, since certain mutations within the conserved Exo II motif lead to a lethal phenotype (7). Attempts to construct recombinants bearing Exo I, II, and III mutations, which result in defective exonuclease activity while retaining polymerase activity in vitro, have been unsuccessful. These data suggest that exonuclease activity may be critical for virus replication, assuming that mutants with defective exonuclease activity may be unviable due to error catastrophe (9). However, recombinants harboring Exo III mutations that have defective exonuclease activity in vitro have been successfully constructed (12).

The roles of the exonucleolytic proofreading activity of HSV-1 Pol in DNA replication fidelity are poorly defined. For example, it was demonstrated that recombinants containing mutated Exo III Pol are extremely mutagenic and replicate the thymidine kinase (tk) gene at a mutation frequency approximately 300- to 800-fold higher than that of wild-type Pol (12). These recombinants, however, replicated with only a four- to fivefold increase in the mutation frequency of the supF gene in a plasmid-borne assay (13), supporting an earlier hypothesis that HSV-1-associated exonuclease activity has a negligible impact on DNA replication accuracy (1). However, the discrepancies between these results could be attributed to sequence context effects, including the sequence compositions of target genes, and the forms of replications.

The purpose of this study was to examine the contribution of exonuclease activity to DNA replication fidelity and to evaluate the influence of forms of DNA replication in the frequency and type of mutations generated. For this purpose, recombinants were constructed so as to have an altered sequence within the Exo III motif and an integrated supF gene in the tk locus of HSV-1. Mutagenesis studies were performed to examine the mutation frequencies and spectra of supF mutants replicated by these exonuclease-deficient recombinants. The results revealed that exonuclease-deficient Pol has a mutator phenotype in replicating the supF gene. However, the mutagenic ability of the defective Pol was higher in this system than in the plasmid-based assay system. Furthermore, the types and spectra of mutations, as well as the distributions of substituted bases in mutated supF genes replicated by these recombinants, were different from those induced by recombinants with wild-type Pol. Therefore, the assay method (the form of DNA replication) and the target genes can influence the replication fidelity of Pol, regardless of whether Pol lacks proofreading activity. Characterization of certain derivatives of exonuclease-deficient viruses suggested that other replicative proteins also could influence the fidelity of DNA replication.


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MATERIALS AND METHODS
 
Cells and viruses. Vero cells were maintained as described previously (12). Recombinant viruses, including the pol-null mutant HP66 (24) and its derivative HPF11, that contained the pSupF1 amplicon within the tk locus were propagated in Pol A5 cells as described previously (12). Recombinants of Y7/F that contained a tyrosine (Tyr)-to-histidine (His) mutation at amino acid residue 577 (within the conserved Exo III motif) and the pSupF1 amplicon sequences within the tk locus were propagated in Vero cells.

Plasmid. The plasmid pSupF-tk-2 (Fig. 1A) was designed to eliminate the Moloney murine leukemia virus long terminal repeat sequences present in pSupF-tk-R (13), because of the difficulty of propagating the latter plasmid. To construct pSupF-tk-2, oligonucleotide primers TK10C (5'-GGGGGATCCTGCAGATACCGCACCG-3', corresponding to nucleotides (nt) 751 to 734 of the tk sequences, with the A nucleotide of the first ATG codon defined as nucleotide position 1) and TK101 (5'-CCGGATCCGGGCGGCGGGTCGT-3', corresponding to nt 750 to 763), each containing a BamHI site (underlined) at the 5' end of the sequences, were used to amplify by PCR the tk sequences present in pSupF-tk-R. The 1.8-kbp PCR products, which retained the PstI site (italic font in primer TK10C) within the tk sequences, were then digested with BamHI and ligated into BamHI-digested and dephosphorylated pSupF1 DNA (12). The resulting plasmid contained all components of pSupF-tk-R, except for the Moloney murine leukemia virus long terminal repeat sequences, allowing for homologous recombination between the tk sequences.




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  		FIG. 1. (A) Map of the plasmid pSupF-tk-2. (B) Restriction maps of sequences around the tk locus of recombinant Y7 (panel 1), recombinants Y7/F containing integrated pSupF1 sequences (panel 2), and the plasmid pSupF-tk-2 (panel 3). The relative positions of 300-bp tk and 2.4-kbp pSupF1 probes also are illustrated in panel 3. Enzymes shown in the figure include the following: B, BamHI; P, PstI; R, EcoRI; S, SpeI. Restriction enzymes shown in parentheses indicate that the cutting site is lost due to cloning. ori, ColE1; amp, ampicillin-resistant gene; F, supF gene.

Both the 3.3-kbp BamHI and the 4.0-kbp EcoRI DNA fragments were isolated from DNA prepared from virus-infected Vero cells and were cloned into BamHI- or EcoRI-digested pUC18. Colonies containing the correct inserts of the pol fragment were selected for DNA sequencing analyses as described previously (14). Oligonucleotide primers pol-5' (corresponding to nt -18 to -2 of the pol gene) and 2736L (nt 2156 to 2137) were used to amplify the 2.0-kbp 5' half of the pol gene; and primers 2564R (nt 1984 to 2002) and 3776L (nt 3776 to 3757) were used to amplify the 1.8-kbp 3' half of the pol gene. PCR products were sequenced as previously described (14).

Construction of recombinant viruses and marker rescue experiments. Recombinant HPF-11 was constructed as follows: 5 x 105 Pol A5 cells plated on 60-mm2 tissue culture plates were cotransfected with infectious virus DNA of HP66 and pSupF-tk-2 by using Lipofectamine (Gibco-BRL) as described previously (13). Recombinants containing the integrated pSupF1 sequence within the tk locus were selected as TK-negative plaques based on their ability to form plaques on Pol A5 cells in the presence of 10 µM ganciclovir. Recombinants were verified by PCR and Southern blot analyses. Each recombinant virus was plaque purified twice, and the identity of the recombinants was confirmed by Southern blotting (Y. Hwang, Q. Lu, and C. B. C. Hwang, unpublished data).

Recombinants derived from the Y7 mutant were constructed by cotransfection of HPF-11 infectious virus DNA and pGEM-Y7 plasmid DNA (12) into Vero (for Y7/F-1) or Pol A5 (for Y7/F-2, Y7/F-3, and Y7/F-4) cells by using Lipofectamine as described previously (12). The progeny of transfectants was screened for white plaques. White plaques were purified, and the viruses were propagated in Vero cells (Y7/F-1) or Pol A5 cells (Y7/F-2, Y7/F-3, and Y7/F-4). To confirm that the recombinants contained the Y7 mutations in the pol gene, virions were collected from supernatants of infected cells and subjected to a second round of plaque purification. DNA was extracted and amplified by PCR with primers 1200R (nt 1190 to 1209) and 2736L (nt 2156 to 2137), yielding a 967-bp fragment corresponding to nt 1190 to 2156 of the pol open reading frame, with the first A nucleotide of the first ATG codon defined as nucleotide position 1. This DNA fragment contains the Exo III motif, including nucleotides (TAC) coding for Tyr577 in the wild-type pol. The DNA fragment amplified from the Y7 pol contains CAC sequences (coding for His) at the corresponding position (12) and is resistant to ScaI digestion. Recombinants Y7/F containing the T-to-C mutation within the Exo III motif were first screened by ScaI digestion of the PCR products and later confirmed by sequencing (Hwang et al., unpublished).

To marker rescue the pol mutation within recombinant Y7/F-1, Vero cells were cotransfected with Y7/F-1 infectious virus DNA and different pol DNA fragments by using Lipofectamine. DNA fragments used to rescue the pol mutation included the 1.1-kbp KpnI fragment (nt 949 to 2089 of the pol gene), the 0.9-kbp EcoRI-Bsu36I fragment (nt 1328 to 2212), and the 3.8-kbp fragment of the entire pol open reading frame. Resulting progeny virions were selected and plaque purified on Vero cells based on their resistance to 150 µM arabinosyladenine (AraA), which inhibited the growth of the parent Y7 virus (15). Marker-rescued recombinants were examined by PCR amplification of corresponding DNA fragments and restriction digestion. For screening of rescued recombinants containing wild-type Exo III sequences, the PCRs and ScaI restrictions were performed as described above. For analysis of recombinants with rescued sequences coding for amino acid residue 1038, the PCR was applied to amplify a 1,032-bp fragment with primers 3325R (corresponding to nt 2745 to 2764) and 3776L (nt 3776 to 3757; located downstream of the stop codon). In this case, the PCR products of wild-type pol contain a DdeI site (5'-CTGAG-3') at nt 3109 to 3113, whereas a G-to-T change at nt 3113, causing a serine-to-isoleucine change at amino acid residue 1038, results in the loss of the DdeI site. Marker-rescued recombinants would regain both SacI and DdeI restriction sites at amino acids 577 and 1038, respectively.

Southern blots. Southern blot analysis was performed as described previously (14).

Mutagenesis assay. The mutagenesis assay and data analyses were performed as described previously (14). Briefly, 5 x 104 Vero cells were infected with recombinant virus at either 1,000 or 200 PFU for 5 or 72 h, respectively. Total DNA, including virion DNA present in the supernatants, was extracted, purified, and digested with BamHI followed by self-ligation with T4 DNA ligase. After phenol-chloroform extraction and ethanol precipitation, aliquots of DNA (one-third of samples from the 5-h infection and original viral stocks and one-fifth of samples from the 72-h infection) were electroporated into Escherichia coli host MBM7070. Transformants were plated onto Luria-Bertani agar plates containing X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), IPTG (isopropyl-ß-D-thiogalactopyranoside), and ampicillin. White and light blue colonies were identified, and the mutation frequency, defined as the ratio of the number of white and light blue colonies to the total number of colonies recovered, was determined.

Statistics. The statistical significance (P value) of the differences between the mutation frequencies induced by the viruses was examined by tests of differences between proportions (3). The chi-square values of goodness-of-fit tests (5) were also used to compare the patterns of the types of mutations induced by different viruses.


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RESULTS
 
Previous studies (12, 13) demonstrated that exonuclease-deficient Pol mutants of HSV-1 replicated the tk and supF genes with different degrees of accuracy. Since the supF mutagenesis was a plasmid-based assay and the tk mutagenesis examined the mutated tk gene within the viral genome (genome-based assay), a question arises regarding whether different forms of DNA replication contribute to the observed differences in replication fidelity. Therefore, we engineered recombinants of an exonuclease-deficient mutant, Y7, to harbor the supF gene integrated into the tk gene of the viral genome in order to examine the mutation frequencies and spectra of mutated supF genes. This allowed for the analysis of the effect of exonuclease activity in regulating the replication fidelity of different genes and a comparison of supF mutants obtained from two different replication systems.

Recombinant viruses with an integrated supF gene in the tk locus. Recombinant virus HPF-11 derived from HP66 (retaining the pol-null phenotype) was constructed so as to have the supF gene integrated into the tk locus as described in Materials and Methods. The presence of the integrated sequences was confirmed by PCR as well as Southern blot analysis (Hwang et al., unpublished). This virus offers an advantage for subsequent construction of other pol mutants by replacing the lacZ gene, located within the pol locus of HPF-11, with the desired pol mutation by homologous recombination. Using this approach, four independent Y7/F recombinants, namely Y7/F-1, Y7/F-2, Y7/F-3, and Y7/F-4, were constructed by cotransfection of HPF-11 infectious virus DNA and pGEM-Y7 DNA into Vero or Pol A5 cells as indicated. Recombinants were isolated and examined for the presence of integrated pSupF1 sequences and the Exo III mutation. Figure 2 is an example of a Southern blot used to examine the presence and the homogeneity of the integrated pSupF1 sequence in Y7/F-1.



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  		FIG. 2. Southern blots of recombinants containing integrated pSupF1 sequences. The blot was hybridized with the pSupF1 probe (A) and then stripped and reprobed with the 300-bp tk probe (B). The relative locations of probes used are shown in Fig. 1B (panel 3), and the relative sizes of restriction fragments of recombinants Y7 and Y7/F-1 and the plasmid pSupF-tk-2 are depicted in Fig. 1B. Sample orders in panels A and B are as follows: lanes 1 and 4, Y7; lanes 2 and 5, Y7/F-1; lanes 3 and 6, pSupF-tk-2. Samples in lanes 1 to 3 were digested with BamHI, and samples in lanes 4 to 6 were digested with EcoRI. 32P-labeled lambda DNA digested with HindIII was used as the molecular weight marker in this experiment (not shown), and the relative positions of marker bands are depicted on the left.

Figure 1B illustrates the restriction map of sequences around the tk locus of Y7, Y7/F recombinants, and the plasmid pSupF-tk-2. Panels A and B of Fig. 2 show the results of Southern blots using the 2.4-kb probe of pSupF1 and the probe of a 300-bp SstI-PstI fragment of the tk gene, respectively. The pSupF1 probe (Fig. 2A) hybridized to a 2.4-kb fragment of BamHI-digested DNA isolated from Y7/F-1-infected cells (lane 2) and pSupF-tk-2 DNA (lane 3), but not that of Y7-infected cell DNA (lane 1). The pSupF1 probe also hybridized to both 2.4- and 2.5-kb fragments of EcoRI-digested DNA prepared from Y7/F-1-infected cells (lane 5), as well as to a linearized 4.3-kbp EcoRI fragment of pSupF-tk-2 (lane 6), but not to Y7-infected cell DNA (lane 4). The 300-bp tk probe (Fig. 2B) hybridized to a 3.6-kbp fragment of BamHI-digested DNA of Y7-infected cells (lane 1), a 1.6-kbp fragment of BamHI-digested DNA of Y7/F-1-infected cells (lane 2), and a 1.8-kbp fragment of pSupF-tk-2 DNA (lane 3). This probe also hybridized to corresponding DNA fragments of the following samples digested by EcoRI: a 2.4-kbp fragment in Y7-infected cell DNA (lane 4), a 2.5-kbp fragment in Y7/F-1-infected cell DNA (lane 5), and a 4.3-kbp fragment of pSupF-tk-2 DNA (lane 6). The lack of hybridization to the 3.6-kbp BamHI (lane 2) and 2.4-kbp EcoRI (lane 5) fragments in Y7/F-1-infected cell DNA demonstrated the homogeneity of Y7/F-1 viruses in terms of the presence of integrated pSupF1 sequences within the tk locus.

Furthermore, the presence of the Y7 mutation within the pol gene of Y7/F-1 was confirmed by the lack of a ScaI site within the 967-bp fragment of PCR products amplified by using primers 1200R and 2736L (Fig. 3, lane 3), whereas the corresponding PCR products of the wild-type sequences were digested by ScaI, resulting in 540- and 427-bp fragments (Fig. 3, lane 2).



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  		FIG. 3. Analysis of PCR products of the pol fragments of Y7/F recombinants. PCRs were performed to amplify a 967-bp fragment (lanes 1 to 7) encompassing the Exo III motif and a 1,032-bp fragment (lanes 8 to 14) overlapping the C-terminal 1.0-kbp sequence of the pol gene. PCR products then were digested with either ScaI (lanes 1 to 7) or DdeI (lanes 8 to 14) to determine the presence or absence of the corresponding restriction site. The Exo III mutation present in Y7 pol results in the loss of the ScaI recognition (lane2). Mutation at the nucleotides coding for amino acid 1038 results in the resistance to DdeI restriction (lanes 11 and 12). Heterogeneity was found in PCR products in lane 9 in which approximately 10% of samples were resistant to DdeI digestion. BstEII-digested DNA was used as the molecular weight marker, with corresponding sizes depicted on the left. Lanes: 1 and 8, PCR products from wild-type pol gene; 2 and 9, Y7/F-1; 3 and 10, Y7/F-10; 4 and 11, Y7/F-11; 5 and 12, Y7/F-13; 6 and 13, Y7/F-12; 7 and 14, Y7/F-41.

Mutation frequency of the integrated supF gene replicated by Y7 Pol. The mutagenesis assay was performed to measure the mutation frequencies of the supF genes replicated by independent exonuclease-deficient Y7/F recombinants. The amount of input virus was <200 PFU to prevent the likelihood of introducing preexisting mutants; total DNA was recovered at 72 h postinoculation (p.i.) and subjected to the mutagenesis assay. Mutagenesis assays for both KOS/F-A and KOS/F-B recombinants were performed in parallel with those of the Y7/F-3 and Y7/F-4 recombinants. The supF gene replicated by wild-type Pol in recombinants KOS/F-A and KOS/F-B had mutation frequencies of 0.012 and 0.011%, respectively (Table 1), consistent with previous results (14). Y7/F recombinants induced the supF mutations at frequencies ranging from 0.14 to 5.6%, which was 10- to 500-fold higher than that of wild-type Pol (P < 0.001).


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  		TABLE 1. supF mutation frequencies

The supF mutation frequencies were examined at 5 h p.i. In this case, 5- and 10-fold higher amounts of input KOS and Y7 recombinant viruses, respectively, were used to infect Vero cells. Total DNA was recovered and tested in the supF mutagenesis assay. While only a small amount of the supF genes was recovered, the results revealed that both wild-type and Y7 recombinants had much higher supF mutation frequencies at 5 h p.i. than at 72 h p.i. and in the original viral stocks (Table 1). Although the higher supF mutation frequencies observed at 5 h p.i. could have resulted from the inoculation with and subsequent amplification of preexisting mutants (due to the higher input of viruses; see below), this possibility is difficult to exclude because of the relatively small number of supF genes recovered from these samples. Nevertheless, Y7 Pol exhibited a mutagenic effect manifested by at least a 10-fold-higher mutation frequency of the supF gene when replicated as genomic DNA.

Types and distribution of supF mutations replicated by Y7 Pol. To characterize the types of mutations and the distribution of substituted bases within the mutated supF genes induced by the Y7/F recombinants, mutants were chosen randomly for sequencing analysis. Table 2 summarizes the types of supF mutants replicated by Y7/F recombinants in comparison to those induced by KOS/F viruses, including those reported in a previous study (14). Among 277 Y7/F-replicated supF mutants analyzed, there were only 12 complex mutations (4.3%), including deletions or insertions of more than three bases and rearrangements. On the other hand, 15.3% of KOS/F recombinants induced supF mutants containing complex changes. Furthermore, KOS/F viruses replicated a relatively lower percentage (84.5%) of base substitutions, including those with fewer than three substituted bases, compared to the 95.7% of such changes induced by Y7 Pol. Therefore, the Y7 Pol replicated different types of supF mutations (P < 0.005).


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  		TABLE 2. Classification of supF mutants

Examination of the distribution of substituted bases provided information regarding the potential hot spot(s) of mutations replicated by Pol. Figure 4 depicts the locations of all simple mutations characterized in this study. For the purpose of comparing and identifying the different mutational hot spot(s) generated by wild-type and Y7 Pol, this figure also includes the simple mutations replicated by wild-type Pol shown in a previous report (see Fig. 4 in reference 14). Table 3 also lists the numbers of base changes at mutational hot spots induced by each Y7/F recombinant. Mutational hot spots at nt 113, 133, 139, and 152 were identified only in Y7/F-induced mutants. Although we could not exclude the possibility that the relatively high prevalence of these substitutions resulted from amplification of preexisting mutations or from redundancy due to the isolation of sisters, each of these changes was found in at least two independent Y7/F recombinants (Table 3). Furthermore, mutants with changes of 2 nt (113 plus 152, and 113 plus deletion of 1 C at nt 172 to 176) were isolated from three independent Y7 recombinants (Table 3). These data suggested that Y7/F recombinants could replicate specific types of mutations within the supF gene. Since Y7 Pol is highly mutagenic (also being capable of creating one or more additional change in the pol gene [see below]) and not all Y7 recombinants replicated these mutations in the supF gene, it is also possible that these hot spots could be the result of heterogeneity in Y7 Pol.



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  		FIG. 4. Distribution of substituted and deleted bases found in supF mutants isolated from KOS/F recombinants including those reported in Fig. 4 of reference 14 (upper panel) and Y7/F-1 (lower panel). The coding sequence of the supF gene is shown at the top line of each panel from the 5' end of the sequence. Numbers shown in parentheses refer to the number of mutants identified. Multiple substitutions shown as the shapes of a pyramid or a half-pyramid refer to the same substitution at that particular nucleotide, {Delta}, base deleted at the nucleotide position shown; underlined bases, mutants containing two or three altered bases.


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  		TABLE 3. Prevalence of mutations found at hot spots induced by Y7 recombinants

Other mutational hot spots induced by Y7 recombinants were found at nt 159, 160, and 169. At least two independent Y7 recombinants replicated mutations at such bases. KOS/F recombinants, however, also replicated mutations at these positions, but of different types. For example, while most mutations at nt 160 were G-to-A changes that were replicated by both Y7/F and KOS/F recombinants, G-to-T changes were induced only by Y7/F. Both C-to-A and C-to-T changes at nt 169 were induced by both Y7/F and KOS/F recombinants; C-to-A changes were predominant in Y7/F-induced mutations. Another difference in the supF mutations induced by Y7/F and KOS/F recombinants was that Y7/F recombinants induced significant numbers of changes between nt 133 and 139, while KOS/F recombinants replicated only a few base substitutions within these sequences. It also was noted that recombinants derived from an exonuclease-proficient Pol mutant, PAAr5, rarely replicated mutations within these bases (14).

Altered spectra of mutations induced by Y7 Pol. Substituted bases generated from KOS/F and Y7/F recombinants were classified further based on the types of base substitutions. Table 4 summarizes the different transitions and transversions replicated by these recombinants. KOS/F recombinants replicated predominately the G:C to A:T transitions (76%) with the remaining mutants containing either G:C to A:T or A:T to G:C transversions (24%). On the other hand, Y7/F recombinants generated significantly more G:C to T:A transversions (46%) and less G:C to A:T transitions (37%). Furthermore, Y7/F, but not KOS/F, also replicated A:T to G:C transitions, and G:C to C:G and A:T to T:A transversions. These results demonstrate that wild-type and Y7 Pol possess distinct properties for maintaining the fidelity of DNA replication. However, mutants isolated from Y7/F recombinants could have been generated by additional changes in the genes involved in viral DNA replication, including the pol gene (see below).


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  		TABLE 4. Classification of substituted bases in the supF gene recovered at 72 h p.i.

Heterogeneity of the pol gene in Y7/F-1 recombinant. The data presented above clearly demonstrated that Y7/F recombinants were different from KOS/F recombinants with respect to generating supF mutations. Factors contributing to this difference include the lack of proofreading activity (12) as well as the altered affinities of nucleotide binding possessed by the polymerase (15). Although each Y7/F recombinant was plaque purified and confirmed to have both the integrated pSupF1 sequences and the altered residue coding for amino acid Tyr577, these recombinants also might generate unknown mutation(s) within the viral genome, including the pol gene, due to the lack of proofreading activity and the associated highly mutagenic effect. To test this possibility, a 3.3-kbp BamHI fragment and a 4.0-kbp EcoRI fragment, which cover the entire pol open reading frame with an overlapping region between nt 1328 and 3212, of the Y7/F-1 recombinant were subcloned into pUC18 and sequenced. Results for the BamHI clone revealed a T-to-C change at nt 1729, resulting in a change from Tyr577 to His577, which was expected for the Y7 Pol mutation. However, a G-to-T mutation also was found at nt 3113 within the BamHI clone. This mutation results in a serine (Ser)-to-isoleucine (Ile) mutation at residue 1038. Interestingly, this mutation was not found in the 4.0-kbp EcoRI clone. This suggested that the Y7/F-1 recombinant contained heterogeneous pol genes and that only a fraction of Y7/F-1 viruses contained mutations at nt 3113. The ability of the Y7 virus to evolve heterogeneous mutants also was supported by a subsequent analysis of the original Y7 viral stock (Hwang et al., unpublished), in which a small fraction of viruses contained an additional mutation within the conserved region III of the pol gene.

Fidelity of marker-rescued recombinants. The heterogeneity of the Y7/F-1 pol gene has complicated the interpretation of the results of supF mutagenesis described above. To examine and confirm that Y7 Pol could affect the accuracy of supF gene replication, marker rescue experiments were performed by using the DNA fragments containing either the entire open reading frame or different portions of the wild-type pol gene. Progeny of marker rescuers were selected as AraA-resistant plaques, since Y7 recombinants were sensitive to this drug (15). Five representative rescued recombinants are listed in Table 5. The genotypes of marker-rescued recombinants were analyzed further by using restriction enzyme digestion (Fig. 3) and/or sequencing (Hwang et al., unpublished) of corresponding DNA fragments amplified by PCR. Examples of restriction enzyme analysis are shown in Fig. 3, and results of the genotypic characterization of these recombinants are summarized in Table 5.


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  		TABLE 5. Mutation frequency of the integrated supF gene

It is important to note that the 1.1-kbp KpnI and 0.9-kbp EcoRI-Bsu36I fragments contained sequences between nt 949 and 2088 and nt 1318 and 2212, respectively, which overlap with the Exo III motif. However, they did not include sequences coding for amino acid residue 1038, which was the unexpected mutation found in the BamHI clone of the pol gene of recombinant Y7/F-1. Therefore, recombinants rescued from these two DNA fragments would allow the rescue of only the Y7 mutation at residue 577, but not the sequence coding for residue 1038. Recombinants Y7/F-11 and Y7/F-12 were isolated from two independent marker-rescued experiments using the 1.1-kbp KpnI fragment, and Y7/F-10 and Y7/F-13 were obtained from two independent experiments using the 0.9-kbp Bsu36I fragment. While Y7/F-11 and Y7/F-13 retained the change at residue 1038, as determined by resistance to DdeI digestion (Fig. 3) and sequencing analyses (Hwang et al., unpublished), recombinants Y7/F-10 and Y7/F-12 contained the DdeI-sensitive wild-type sequence at this position (Fig. 3). These results further indicated that recombinant Y7/F-1 contained heterogeneous viruses, including those having either the wild-type or a mutated sequence at residue 1038. Approximately 10% of the 1,032-bp PCR products amplified from Y7/F-1 recombinant were resistant to DdeI (Fig. 3, lane 9), and sequencing of the 4.0-kbp EcoRI clone but not the 3.3 kbp BamHI clone revealed the wild-type sequence at this position (Hwang et al., unpublished). Nevertheless, recombinant Y7/F-41, which was rescued by using the 3.8-kbp pol DNA, was confirmed by both sequencing (Hwang et al., unpublished) and restriction analyses of PCR products to contain the wild-type sequences at both the Exo III motif and residue 1038 (Fig. 3).

We continued to examine the mutation frequency of the supF gene induced by these five independent marker rescuers. Although Y7/F-41 had rescued the highly mutagenic activity of Y7 Pol to a level identical to that of wild-type Pol (a change from 0.48 to 0.018%), it was striking that the remaining four rescuers exhibited a 5- to 10-fold-lower mutation frequency (Table 5). The higher fidelity of these rescuers was independent of the presence or absence of mutations at residue 1038, since only two of them contained the mutated residue. Furthermore, sequencing of PCR products amplified from recombinants Y7/F-10 and Y7/F-13 did not identify any additional mutations in the entire pol gene (Hwang et al., unpublished). These results imply that these Pols do not contribute to the higher accuracy of DNA replication observed in this system, suggesting that other genes involved in viral DNA replication may be responsible for the observed higher fidelity.


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DISCUSSION
 
A previous study demonstrated that recombinants with defective exonuclease activity are extremely mutagenic, replicating the tk gene at a mutation frequency 300- to 800-fold higher than that of the wild-type virus (12). In another system, these exonuclease-deficient recombinants replicated the supF gene with only a four- to fivefold increase in mutation frequency (13). These differences may be due to the effects of sequence content between the tk and supF genes, since such effects have been observed in other Pols by in vitro assays (reviewed in references 8 and 20). Furthermore, this inconsistency may also be the result of the context effect of these genes. The supF gene is replicated as episomal DNA, while the tk gene is replicated as part of the genomic DNA. The supF gene replicated in the plasmid form has its local context near (900 bp or 1.5 kbp, depending on the orientation) the oriS sequences, while the tk gene in the genomic DNA is located within the UL region approximately 14 kbp upstream from the oriL sequences. To address whether HSV-1 DNA replication fidelity can be affected by the sequence contexts, we engineered Pol recombinants to harbor an integrated supF gene within the tk locus for mutagenesis analysis (14; this study). Results of the supF mutagenesis assay obtained from supF-containing recombinants were then compared with mutation spectra obtained from other assays to elucidate the role of sequence contexts and of the exonuclease activity in the fidelity of DNA replication.

Exonuclease-deficient Pol is highly mutagenic. Results demonstrated that supF-containing recombinants with exonuclease-deficient Pol replicated the supF gene at significantly higher mutation frequencies than those replicated by wild-type Pol (Table 1). Although the possibility cannot be excluded that other replicative proteins may affect the replication fidelity, marker-rescued experiments demonstrated that the Exo III mutation in the pol gene was critical to the highly mutagenic activity. This was supported by the evidence that only mutants containing altered sequences within the Exo III motif replicated the supF genes with higher mutation frequencies, whereas rescuers lacking the Exo III mutation had levels similar to or significantly lower than that of wild-type Pol (Table 5).

In this study we observed that recombinants with the Exo III mutation could induce dramatically different supF mutation frequencies that varied from 0.14 to 7.5%. This type of fluctuation could result from preexisting mutations in the virus inoculum. This possibility was reduced in these experiments by the inoculation of low amounts of viruses. However, sequencing data for the viral inoculum indicated that a few changes could be derived from preexisting mutants, including single base deletions and the change at nt 160 (Table 3). However, most of the changes found at hot spots were not identified in samples collected from viral stock of the corresponding virus. Alternatively, analysis of the mutation frequencies could have been biased by the amplification of mutants that evolved early after infection. Although this was found to be the case in certain hot spot mutations generated by some recombinants, it could not account for most of the hot spot mutations generated by all Exo III mutant viruses (Table 3). Therefore, examination of the mutation frequencies and spectra of the supF mutants from several independent Y7/F recombinants and the inoculation of small amounts of virus are necessary for reducing bias to a minimum.

Altered spectra of supF mutations induced by Y7/F recombinants. Y7/F recombinants replicated more simple changes and transversions in the supF gene than did those induced by KOS/F (Tables 2 and 4) and another exonuclease-proficient recombinant, PAAr5/F (see Tables 3 and 5 in reference 14). Certain hot spots replicated by Y7/F recombinants were not found in mutants generated by KOS/F (Fig. 3) and PAAr5/F (see Fig. 4 in reference 14). These hot spots were found in mutants derived from at least two independent Y7/F recombinants and contributed, at least in part, to the differences in mutation spectra, supporting the conclusion that HSV-1 Pol with defective exonuclease activity could influence the spectra of supF mutations.

The outcome of the fidelity of DNA replication is assay dependent. In the plasmid-based assay, the Y7 recombinant induced a fivefold increase in the mutation frequency of the supF gene compared to that of the wild-type KOS virus (13). Y7 also induced types of supF mutations that were different from those induced by the KOS virus. In this study, supF mutants induced by Y7/F recombinants contained only 4.3% of complex changes that differed from the 25% of such changes observed in the plasmid-based assay. Furthermore, the spectra of substituted bases in supF mutants replicated as the plasmid DNA also differed from those of the genomic DNA. Only 24% of the plasmid-based supF substitutions were transitions (13), while 43% of supF substitutions recovered from the genomic component were transitions (Table 4). Different assay methods also identified a different hot spot of mutation: a C-to-T transversion at nt 152 was identified only from supF-containing recombinants (compare Fig. 4 with Fig. 2 in reference 13). These differences could be related to the assay method employed, especially for those applied to examine the effects of a mutator Pol. It is possible that the mutator Pol virus could replicate many mutations, including the lethal hits, and generated a bias population of progeny that could contribute to the differences in mutation frequencies observed in different assays, at different times of virus infections, and in different virus isolates. However, it was unlikely that a virus would encounter a selection pressure for viability during the early time of infection. Our data seem to support these notions. The fold increases in mutation frequencies of the supF genes in the plasmid-based assay (13), where there is no selection for viability, are more similar to those of the virus-based assay at the early time (5 h p.i.) (Table 1), at which no progeny virus is made. Similarly, the fold increases in mutation frequency are higher for the tk genes (12) and supF genes at 72 h p.i. (Table 1), a time at which they share similar survival pressures, contributing to the bias population of viruses and the much lower supF mutation frequencies than those isolated at the early time of infection (Table 1).

The contexts of target sequences relative to the entire DNA molecule being replicated also could be an important factor. Studies of lacZ mutations revealed different results when the target gene was inserted at different locations within the viral genome. For example, HSV-1 replicated the lacZ gene with a mutation frequency of ~1.5 x 10-4 to 2.7 x 10-4 when it was inserted within the tk locus (16, 25). Conversely, a 0.5% mutation frequency was detected when it was located near the terminal repeat sequences (25). This implied that the position of the target gene could lead to differences in the fidelity of DNA replication, although other factors also could contribute to these differences. Nevertheless, the context effect of the target gene was observed in both exonuclease-proficient HSV-1 (12, 14) and exonuclease-deficient HSV-1 (12; this study).

Spectra of mutations are target gene dependent. Recently we characterized 66 independent tk mutants derived from exonuclease-deficient Y7 recombinants. Among these mutants, 63% of base substitutions were transitions and the remaining 37% were transversions (22), which was not significantly different from results observed with the wild-type KOS strain. However, they differed significantly from those of supF mutants (Table 4), which contained 43% transitions and 57% transversions. These demonstrated that the exonuclease-deficient Pol of HSV-1 also could induce different mutational spectra in different target genes.

Results of examinations of tk mutants derived from the Y7 virus also suggest that Y7 Pol may have acquired a structural change associated with altered kinetics, which may contribute to the induction of different spectra of mutations (22). However, since the Y7 Pol was highly mutagenic due to a lack of proofreading activity, the possibility that the differences in mutation spectra were due to the newly evolved mutation(s) in the viral genome, including the pol, could not be ruled out. Results from this study provided evidence to support the possibility that Y7-related viruses were highly heterogeneous.

Heterogeneity of Y7/F recombinants. Sequencing analyses of several pol subfragments derived from the Y7/F-1 recombinant and its marker rescuers demonstrated that the viral stock of Y7/F-1 contained heterogeneous populations. The heterogeneity was found not only in the pol gene but also in other genes involved in DNA replication. The former included the detection of a sequence coding for a Ser-to-Ile change at amino acid 1038 that was identified in a fraction of PCR products amplified from Y7/F-1 DNA (Fig. 4, lane 9) and from DNA prepared from Y7/F-11- and Y7/F-12-infected cells (lanes 11 and 12). Indirectly, the latter was evident by the higher fidelity of the supF genes found in marker rescuers Y7/F-10, Y7/F-11, Y7/F-12, and Y7/F-13 (Table 5). Direct evidence included the detection of different mutations within the UL42 gene, coding for the accessory protein of the Pol subunit, of these rescuers (Hwang et al., unpublished). Although the effects of these mutations on DNA replication fidelity remain to be tested, our data demonstrated that the heterogeneity of Y7/F recombinants could complicate the interpretation of results induced by these recombinants. Nevertheless, it is unlikely that additional mutations in either pol or other replicative genes will cause the exonuclease-negative phenotype of the Pol to revert to that of the proofreading-proficient Pol. Indeed, examination of a specific Pol mutant that contained changes within the Exo III motif and the conserved region VI of Pol revealed that it retained the exonuclease deficiency but exhibited altered kinetics of polymerase activity (Hwang et al., unpublished). Therefore, supF mutants observed in this study would be considered to be products of Pol lacking the proofreading activity directly or indirectly.

Implication of different spectra of supF mutations induced by wild-type and Y7 Pols. Y7/F recombinants induced spectra and distributions of supF mutations different from those of the wild-type Pol. This information is potentially very useful for determining whether wild-type Pol preferentially misincorporates certain nucleotides at particular positions of the template. It also would be useful for predicting bias in the exonuclease activity leading to proofreading of incorrect bases at specific position(s) of the template. However, the prerequisite is that the exonuclease-defective Pol must not have altered Pol activity. Under such circumstances, common hot spots should be found among wild-type and Y7 Pol-induced mutants, while unique mutations should only be found among Y7-induced mutants. The former would represent those generated by Pol without proofreading, whereas the latter would represent those processed normally by the exonuclease activity intrinsic to wild-type Pol.

Although most base substitutions identified in mutants induced by wild-type and Y7 Pol share common locations, certain hot spots, such as mutations at nt 113, 133, 139, and 152, were found only among Y7/F-induced mutants. Y7/F recombinants also induced mutations clustering between nt 133 and 139 that were not identified among KOS/F-induced mutants, with the exception of an A-to-C change at nt 136 induced by wild-type Pol. The A-to-C change at nt 136 also differed from the A-to-T mutation induced by Y7 Pol. 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. However, additional mutation(s) in pol and other genes could contribute to these differences. Further studies are required in order to answer these questions.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grant DE-10051.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY 13210. Phone: (315) 464-8739. Fax: (315) 464-7680. E-mail: hwangc{at}mail.upstate.edu. Back


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Journal of Virology, March 2003, p. 2946-2955, Vol. 77, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.5.2946-2955.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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