A MAJOR ROLE FOR BACTERIOPHAGE T4 DNA POLYMERASE IN FRAMESHIFT MUTAGENESIS

Genetics ◽  
1983 ◽  
Vol 103 (3) ◽  
pp. 353-366
Author(s):  
Lynn S Ripley ◽  
Nadja B Shoemaker
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Base Substitution ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Frameshift Mutagenesis ◽  
Substitution Mutations ◽  
Polymerase Mutants

ABSTRACT T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.

scholarly journals Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 213-220 ◽  
Author(s):  
L J Reha-Krantz
Keyword(s):  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Rnase H ◽  
Ribonuclease H ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Dna Polymerase I ◽  
E Coli ◽  
Intragenic Complementation ◽  
Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

The Spectrum of Acridine Resistant Mutants of Bacteriophage T4 Reveals Cryptic Effects of the tsL141 DNA Polymerase Allele on Spontaneous Mutagenesis

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1655-1665
Author(s):  
Fei Jun Wang ◽  
Lynn S Ripley
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Bacteriophage T4 ◽  
Resistance Mechanism ◽  
Base Substitution ◽  
Spontaneous Mutant ◽  
Spontaneous Mutagenesis ◽  
Compact Size ◽  
Nonsense Codons ◽  
The Impact

Abstract Mutations in the ac gene of bacteriophage T4 confer resistance to acridine-inhibition of phage development. Previous studies had localized the ac gene region; we show that inactivation of T4 Open Reading Frame 52.2 confers the Acr phenotype. Thus, 52.2 is ac. The resistance mechanism is unknown. The ac gene provides a convenient forward mutagenesis assay. Its compact size (156 bp) simplifies mutant sequencing and diverse mutant types are found: base substitutions leading to missense or nonsense codons, inframe deletions or duplications within the coding sequence, deletion or duplication frameshifts, insertions, complex mutations, and large deletions extending into neighboring sequences. Comparisons of spontaneous mutagenesis between phages bearing the wild-type or tsL141 alleles of DNA polymerase demonstrate that the impact of the mutant polymerase is cryptic when total spontaneous mutant frequencies are compared, but the DNA sequences of the ac mutants reveal a substantial alteration of fidelity by the mutant polymerase. The patterns of base substitution mutagenesis suggest that some site-specific mutation rate effects may reflect hotspots for mutagenesis arising by different mechanisms. A new class of spontaneous duplication mutations, having sequences inconsistent with misaligned pairing models, but consistent with nick-processing errors, has been identified at a hotspot in ac.

scholarly journals Identification of a Mutant DNA Polymerase δ in Saccharomyces cerevisiae With an Antimutator Phenotype for Frameshift Mutations

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 177-186 ◽  
Author(s):  
Michalis I Hadjimarcou ◽  
Robert J Kokoska ◽  
Thomas D Petes ◽  
Linda J Reha-Krantz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Base Substitution ◽  
Msh2 Gene ◽  
Frameshift Mutations ◽  
Frameshift Mutagenesis

Abstract We propose that a β-turn-β structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol δ. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative β-turn-β structure. The mutant DNA pol δ has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol δ was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol δ in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

scholarly journals A Mutation of the Yeast Gene Encoding PCNA Destabilizes Both Microsatellite and Minisatellite DNA Sequences

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 511-519 ◽  
Author(s):  
Robert J Kokoska ◽  
Lela Stefanovic ◽  
Andrew B Buermeyer ◽  
R Michael Liskay ◽  
Thomas D Petes
Keyword(s):  
Dna Polymerase ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Repeat Unit ◽  
Nuclear Antigen ◽  
Temperature Sensitive ◽  
Dinucleotide Repeats ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Polymerase Δ ◽  
Repeat Units

AbstractThe POL30 gene of the yeast Saccharomyces cerevisiae encodes the proliferating cell nuclear antigen (PCNA), a protein required for processive DNA synthesis by DNA polymerase δ and ϵ. We examined the effects of the pol30-52 mutation on the stability of microsatellite (1- to 8-bp repeat units) and minisatellite (20-bp repeat units) DNA sequences. It had previously been shown that this mutation destabilizes dinucleotide repeats 150-fold and that this effect is primarily due to defects in DNA mismatch repair. From our analysis of the effects of pol30-52 on classes of repetitive DNA with longer repeat unit lengths, we conclude that this mutation may also elevate the rate of DNA polymerase slippage. The effect of pol30-52 on tracts of repetitive DNA with large repeat unit lengths was similar, but not identical, to that observed previously for pol3-t, a temperature-sensitive mutation affecting DNA polymerase δ. Strains with both pol30-52 and pol3-t mutations grew extremely slowly and had minisatellite mutation rates considerably greater than those observed in either single mutant strain.

scholarly journals Characterization of the DNA polymerase gene of varicella-zoster viruses resistant to acyclovir

2001 ◽  
Vol 82 (11) ◽  
pp. 2761-2765 ◽  
Author(s):  
Tomoko Kamiyama ◽  
Masahiko Kurokawa ◽  
Kimiyasu Shiraki
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Sequence Similarity ◽  
Polymerase Gene ◽  
Varicella Zoster ◽  
Dna Polymerase Gene ◽  
Simplex Virus ◽  
Polymerase Mutants ◽  
Hsv 1

The nucleotide changes of the DNA polymerase gene and the susceptibility of acyclovir (ACV)-resistant varicella-zoster virus (VZV) mutants to anti-herpetic drugs were determined and compared to those of herpes simplex virus type 1 (HSV-1) mutants. The seven ACV-resistant VZV mutants were classified into three groups, N779S, G805C and V855M, according to the sequences of their DNA polymerase genes. The amino acid substitutions N779S and G805C were identical in position to the N815S and G814C mutations in the HSV-1 DNA polymerase mutants, respectively, and the V855M amino acid substitution was similar to the HSV-1 V892M mutation. All three groups of VZV mutants were susceptible to ACV, phosphonoacetic acid, vidarabine and aphidicolin, at levels similar to those seen with the respective HSV-1 mutants, except for subtle differences that were due possibly to the non-conserved regions in their sequences. Although both the HSV-1 and the VZV DNA polymerase genes show 53% sequence similarity, both viruses essentially show a similar biochemical behaviour.

scholarly journals Role of the dinB Gene Product in Spontaneous Mutation in Escherichia coli with an Impaired Replicative Polymerase

2000 ◽  
Vol 182 (23) ◽  
pp. 6742-6750 ◽  
Author(s):  
B. S. Strauss ◽  
R. Roberts ◽  
L. Francis ◽  
P. Pouryazdanparast
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Spontaneous Mutation ◽  
Luria Bertani ◽  
Base Substitution ◽  
Temperature Sensitive ◽  
Selection Scheme ◽  
Mutator Effect ◽  
Substitution Mutations

ABSTRACT We isolated several new mutator mutations of the Escherichia coli replicative polymerase dnaE subunit alpha and used them and a previously reported dnaE mutation to study spontaneous frameshift and base substitution mutations. Two of thesednaE strains produce many more mutants when grown on rich (Luria-Bertani) than on minimal medium. A differential effect of the medium was not observed when these dnaE mutations were combined with a mismatch repair mutation. The selection scheme for thednaE mutations required that they be able to complement a temperature-sensitive strain. However, the ability to complement is not related to the mutator effect for at least one of the mutants. Comparison of the mutation rates for frameshift and base substitution mutations in mutS and dnaE mutS strains suggests that the mismatch repair proteins respond differently to the two types of change. Deletion of dinB from both chromosome and plasmid resulted in a four- to fivefold decrease in the rate of frameshift and base substitution mutations in a dnaE mutSdouble mutant background. This reduction indicates that most mistakes in replication occur as a result of the action of the auxiliary rather than the replicative polymerase in this dnaE mutant. Deletion of dinB from strains carrying a wild-typednaE had a measurable effect, suggesting that a fraction of spontaneous mutations occur as a result of dinB polymerase action even in cells with a normal replicative polymerase.

scholarly journals Processing of a single ribonucleotide embedded into DNA by human nucleotide excision repair and DNA polymerase η

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Akira Sassa ◽  
Haruto Tada ◽  
Ayuna Takeishi ◽  
Kaho Harada ◽  
Megumi Suzuki ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Large Deletion ◽  
Base Substitution ◽  
Mutagenic Potential ◽  
Large Deletions ◽  
Nucleotide Excision ◽  
Substitution Mutations

Abstract DNA polymerases often incorporate non-canonical nucleotide, i.e., ribonucleoside triphosphates into the genomic DNA. Aberrant accumulation of ribonucleotides in the genome causes various cellular abnormalities. Here, we show the possible role of human nucleotide excision repair (NER) and DNA polymerase η (Pol η) in processing of a single ribonucleotide embedded into DNA. We found that the reconstituted NER system can excise the oxidized ribonucleotide on the plasmid DNA. Taken together with the evidence that Pol η accurately bypasses a ribonucleotide, i.e., riboguanosine (rG) or its oxidized derivative (8-oxo-rG) in vitro, we further assessed the mutagenic potential of the embedded ribonucleotide in human cells lacking NER or Pol η. A single rG on the supF reporter gene predominantly induced large deletion mutations. An embedded 8-oxo-rG caused base substitution mutations at the 3′-neighboring base rather than large deletions in wild-type cells. The disruption of XPA, an essential factor for NER, or Pol η leads to the increased mutant frequency of 8-oxo-rG. Furthermore, the frequency of 8-oxo-rG-mediated large deletions was increased by the loss of Pol η, but not XPA. Collectively, our results suggest that base oxidation of the embedded ribonucleotide enables processing of the ribonucleotide via alternative DNA repair and damage tolerance pathways.

scholarly journals Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

1991 ◽  
Vol 19 (18) ◽  
pp. 4967-4973 ◽  
Author(s):  
P. Mattila ◽  
J. Korpela ◽  
T. Tenkanen ◽  
K. Pitkämem
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Total Concentration ◽  
Dna Amplification ◽  
Base Substitution ◽  
Thermococcus Litoralis ◽  
Heat Stable ◽  
Original Target

Abstract We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3′—5′ proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30×10−6. These values were 5–10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15−35×10−4 being 2 – 4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis In vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

scholarly journals MUTATOR MUTATIONS IN BACTERIOPHAGE T4 GENE 42 (dHMC HYDROXYMETHYLASE)

Genetics ◽  
1977 ◽  
Vol 86 (3) ◽  
pp. 501-511
Author(s):  
Warren E Williams ◽  
John W Drake
Keyword(s):  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Spontaneous Mutation ◽  
Mutation Rates ◽  
Temperature Sensitive ◽  
Repair System ◽  
Transition Rates ◽  
Error Prone Repair ◽  
Complex Manner

ABSTRACT Temperature-sensitive mutations of bacteriophage T4 gene 42 produce diverse effects upon spontaneous mutation rates. G:C → A:T transition rates are increased, often strongly; frameshift mutation rates are weakly increased; A:T → G:C transition rates (and perhaps also A:T → Py:Pu transversion rates) are decreased; and one G:C → Py:Pu transversion rate is also decreased. These results, together with certain interactions between gene-42 mutator effects and both base analogue mutagenesis and the viral error-prone repair system, suggest that the dHMC hydroxymethylase coded by gene 42 affects mutation rates in a more complex manner than by the simple regulation of the concentration of the DNA precursor dHMCTP.

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