Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase catalytic subunit (pol) is less faithful than for most replicative DNA polymerases, despite the presence of an associated 3 -to 5 -exonuclease (exo) activity. To determine the aspects of fidelity affected by the exo activity, nucleotide incorporation and mismatch extension frequency for purified wild-type and an exo-deficient mutant (D368A) pol were compared using primer/templates that varied at only a single position. For both enzymes, nucleotide discrimination during incorporation occurred predominantly at the level of K m for nucleotide and was the major contributor to fidelity. The contribution of the exo activity to reducing the efficiency of formation of half of all possible mispairs was 6-fold or less, and 30-fold when averaged for the formation of all possible mispairs. In steady-state reactions, mismatches imposed a significant kinetic barrier to extension independent of exo activity. However, during processive DNA synthesis in the presence of only three nucleotides, misincorporation and mismatch extension were efficient for both exo-deficient and wildtype pol catalytic subunits, although slower kinetics of mismatch extension by the exo-deficient pol were observed. The UL42 processivity factor decreased the extent of misincorporation by both the wild-type and the exo-deficient pol to similar levels, but mismatch extension by the wild-type pol⅐UL42 complex was much less efficient than by the mutant pol⅐UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol⅐UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by the kinetic barrier to extending a mismatch. The kinetic barrier also increases the probability that a mismatched primer terminus will be transferred to the exo site where it can be excised by the associated exo activity and subsequently extended with correct nucleotide.Herpes simplex virus type 1 (HSV-1) 1 is the best characterized member of the large family of Herpesviridae pathogenic to humans, which also includes Epstein-Barr virus, varicella-zoster virus, human cytomegalovirus, and Kaposi sarcoma-associated herpes virus (reviewed in Ref. 1). Viruses in this family encode most of the proteins essential for and directly involved in DNA replication (2-4), including a well conserved DNA polymerase catalytic subunit (pol), which is a member of the polymerase B family (5, 6). HSV-1 pol possesses 5Ј-to 3Ј-polymerizing and 3Ј-to 5Ј-exonuclease (exo) activities (7,8), the latter of which is involved in the removal of incorrectly incorporated deoxyribonucleoside triphosphates (9 -12). The importance of this proofreading activity for maintaining fidelity of DNA replication was suggested by studies from our laboratory that demonstrated the relatively poor ability of HSV-1 pol to discriminate between the correct and incorrect nucleotide for incorporation in single turnover experiments (13). That study (13) reported that selectivity of correct o...
The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5' -3'-exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i. e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5' -3' direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5' -3'-exonucleolytic activities of the DNase and the polymerase-associated 3' -5' exonuclease.The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3'-5' exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.Herpes simplex virus (HSV) DNA polymerase is the major essential function of the viral DNA synthesis machinery [I]. The availability of well-characterized temperature-sensitive [l -31 and drug-resistant 14-61 DNA polymerase mutants and recent sequencing information [7 -91 render the viral polymerase an attractive model enzyme for studies of eukaryotic DNA polymerases. As a further powerful tool for the biochemical analysis we have recently established polyclonal antibodies directed against selected domains of the polymerase polypeptide. With these monospecific antibodies it was possible to demonstrate [lo], in agreement with the potential coding sequences and the mapping limits for the catalytic domain [9], that HSV type-1 (HSV-1) DNA
Polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the herpes simplex virus type I (HSV-1) DNA polymerase of strain Angelotti were generated. Each of the five different rabbit antisera rexted specifically with a viral 132 5-kDa polypeptide as shown by immunoblot analysis. Enzyme binding and inhibition studies revealed that antibodies raised to the central and the C-terminal domains of the protein inhibited the polymerizing activity by 70 -90%, respectively, which is well in line with the proposed site of the catalytic center of the enzyme and with the possible involvement of these polypeptide chains in DNA -protein interactions. In agreement with this, antibodies directed towards the N-terminal domain bound to the enzyme without effecting the enzymatic activity. The strong binding but low inhibitory properties of antibodies directed to the polypeptide region between residues 1072 and 1146 confirms previous suggestions that these C-terminal sequences, which share no homology to the Epstein-Barr virus DNA polymerase, are less likely involved in the building of the polymerase catalytic site. Antibodies, raised to the very C terminus of the polymerase (EX3), were successfully used to identify a single 132 f 5-kDa polypeptide, which coeluted with the HSV DNA polymerase activity during DEAE-cellulose chromatography, and were further shown to precipitate a major viral polypeptide of identical size. From the presented data it can be concluded that the native enzyme consists of a single polypeptide with a size predicted from the long open reading frame of the HSV-1 DNA polymerase gene.The herpes simplex virus type I (HSV-1) DNA polymerase represents an attractive model enzyme for studies of eukaryotic DNA polymerases. Like the M: polymerase, the key enzyme of cellular DNA synthesis, the HSV DNA polymerase is of central importance for successful viral replication. The viral enzyme has been involved in the initiating events of the gene amplification processes [l], has been shown to play a key role in viral mutagenesis [2] and is one of the major target enzymes of modern antiviral chemotherapy. The analysis of HSV mutants, which induce a drug-resistant and/or a temperature-sensitive polymerase, by marker rescue as well as by sequencing studies has allowed us to identify the gene encoding the viral enzyme (Fig.
We present evidence that mutation frequencies in a mammalian system can vary according to the replication fidelity of the DNA polymerase. We demonstrated previously that several derivatives ofherpes simplex virus type 1 that encode polymerases resistant to various antiviral drugs (e.g., nucleotide analogues) also produce reduced numbers of spontaneous mutants. Here we show that the DNA polymerase from one antimutator virus exhibits enhanced replication fidelity. First, the antimutator virus showed a reduced response to known mutagens that promote base misparinng during DNA replication (N-methyl-N'-nitro-N-nitrosoguanidine, 5-bromodeoxyuridine). Second, purified DNA polymerase from the antimutator produced fewer replication errors in vitro, based on incorporation of mispaired nucleotides or analogues with abnormal sugar rings. We have investigated possible mechanisms for the enhanced fidelity of the antimutator polymerase. We show that the mutant enzyme has altered interactions with nucleoside triphosphates, as indicated by its resistance to nucleotide analogues and elevated K. values for normal nucleoside triphosphates. We present evidence against increased proofreading by an associated 3',5' exonuclease (as seen for T4 bacteriophage antimutator polymerases), based on nuclease levels in the mutant polymerase. We propose that reduced affinity of the polymerase for nucleoside triphosphates accounts for the antimutator phenotype by accentuating differences in base-pair stability, thus facilitating selection of correct nucleotides.Accurate DNA replication is a major mechanism for maintaining the integrity of chromosomal information (1). Since the frequency ofreplication errors is much less than expected from differences in stabilities between correctly and incorrectly paired bases, other components (e.g., replication complex, nucleotide pools, mismatch repair) must contribute to these low frequencies. DNA polymerases in Escherichia coli and T4 bacteriophage control replication errors by modulating the selection of nucleoside triphosphates for polymerization and the levels of an associated 3',5' exonuclease able to remove incorrectly inserted nucleotides (2-5).In mammalian cells, certain evidence suggests that DNA polymerase a (the major replication enzyme) plays a role in controlling mutation frequencies. Such control likely involves changes in specificities of nucleotide selection, since polymerase a lacks a proofreading nuclease. For example, a mutator Chinese hamster cell line produces an altered (aphidicolin-resistant) polymerase a (6), which may contribute to the high cellular mutation rates. Also, polymerases from certain aging and cancer-related cells are error-prone in vitro (7-9).
Like true DNA replicases, herpes simplex virus type 1 DNA polymerase is equipped with a proofreading 3'-5'-exonuclease. In order to assess the functional significance of conserved residues in the putative exonuclease domain, we introduced point mutations as well as deletions within and near the conserved motifs' exonuclease (Exo) I, II, and III of the DNA polymerase gene from a phosphonoacetic acid-resistant derivative of herpes simplex virus-1 strain ANG. We examined the catalytic activities of the partially purified enzymes after overexpression by recombinant baculovirus. Mutations of the motifs' Exo I (D368A, E370A) and Exo III (Y577F, D581A) yielded enzymes without detectable and severely impaired 3'-5'-exonuclease activities, respectively. Except for the Exo I mutations, all other Exo mutations examined affected both exonuclease and polymerization activities. Mutant enzymes D368A, E370A, Y557S, and D581A showed a significant ability to extend mispaired primer termini. Mutation Y557S resulted in a strong reduction of the 3'-5'-exonuclease activity and in a polymerase activity that was hyperresistant to phosphonoacetic acid. The results of the mutational analysis provide evidence for a tight linkage of polymerase and 3'-5'-exonuclease activity in the herpesviral enzyme.
SUMMARYBy comparative sequence analysis of the herpes simplex virus type 1 DNA polymerase gene of strain Angelotti and a phosphonoacetic acid-resistant (PAA9 derivative, the site of the PAA r mutation was identified as a single nucleotide (C --, T) conversion within the mapping limits of the known PAA r mutations of strains KOS and 17. The conservative amino acid change at residue 719 from alanine to valine results in a radical change in the properties of the polymerase, rendering the mutant enzyme resistant to PAA and various antiviral compounds. Amino acid homologies as well as secondary structure analysis reveal that the PAA ~ mutation is contained in a 14 amino acid sequence which is highly conserved, and detected in the central domain of prokaryotic and eukaryotic DNA polymerases.The herpes simplex virus (HSV) DNA polymerase represents one of the major target enzymes of modern antiviral chemotherapy. The unique response of the enzyme towards antiherpetic drugs, and the ease by which drug-resistant variants with altered enzymic properties are isolated, have rendered the HSV polymerase gene an excellent model system for correlating genetic changes with altered enzymic functions. Biochemical studies on the polymerase activity of HSV type 1 (HSV-1) and type 2 variants and intertypic recombinants reveal that mutations causing resistance to the pyrophosphate analogue phosphonoacetic acid (PAA) also render the enzyme resistant to nucleoside analogues (Furman et al., 1981 ;Knopf et al., 1981 ;Coen et al., 1982). The mutually exclusive inhibition of the wild-type enzyme by pyrophosphate and nucleoside analogues suggests that the binding sites for dNTP and PP~ are kinetically overlapping (Frank & Cheng, 1985). By marker rescue studies (Chartrand et al., 1979;Coen et al., 1984) and sequence analysis (Gibbs et al., 1985), the limits for the PAA resistance (PAA r) mutations of strains KOS and 17 were narrowed to a region between amino acid residues 535 and 924. Owing to the extended homologies noted between the predicted DNA polymerase proteins of herpesviruses, poxviruses and adenoviruses (Quinn & McGeoch, 1985;Gibbs et al., 1985;Earl et al., 1986), this C terminal domain has been proposed to participate in the organization of the catalytic site of the enzyme.In the absence of crystallographic data, one way to obtain information on the structure of the catalytic site is to determine the sequence changes in mutants that alter the enzyme's behaviour towards substrate and substrate analogues. In this report, we present an analysis of the differences in structure and function of the DNA polymerase genes of wild-type HSV-1 strain Angelotti (ANG) and of a PAA ~ variant. The latter virus was selected for resistance to PAA by sequential passages ofHSV-1 ANG in Rita cells (RC-37) at a PAA concentration of 300 ~tg/ml, and was plaque-purified three times. This PAA r variant (isolate number III-2) was equally resistant to PAA (e.o.p. + 100 lag/ml PAA, 1.04 + 0-2) and acyclovir (ACV; e.o.p. + 50 ~tM-ACV, 1.0 + 0.15), and otherwise ...
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