Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions

Glick, Eithan; Vigna, Kellie L.; Loeb, Lawrence A.

doi:10.1093/emboj/20.24.7303

Cited by 48 publications

(45 citation statements)

References 47 publications

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“…1B) revealed greater killing than that observed for the wild-type enzyme at all doses examined. These data provide a direct demonstration of decreased biological activity conferred by substitution at Tyr-52 and substantiate the inability to recover replacements for this amino acid by functional complementation of UV sensitivity (26). The findings thus emphasize the importance of Tyr-52 in the bypass of UV-induced DNA damage in vivo.…”

Section: Resultssupporting

confidence: 62%

“…Twenty site-directed reactions were performed with pYEX-hPol (26) to create all 19 amino acid substitutions for Tyr-52 and the amber mutant, yielding pYEX-hPol Y52Xs (Y52V and Y52N exhibited low expression in the in vitro synthesis system (see below) and were not further characterized). M59 Saccharomyces cerevisiae rad30rad52 cells were transformed with 19 pYEX-hPol -Y52X plasmids (including Tyr-52 3 Stop) and exposed to 50 J/m 2 of UV-C radiation as described (26).…”

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

“…M59 Saccharomyces cerevisiae rad30rad52 cells were transformed with 19 pYEX-hPol -Y52X plasmids (including Tyr-52 3 Stop) and exposed to 50 J/m 2 of UV-C radiation as described (26). Each survival experiment was performed at least three times.…”

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

“…Five nM 32 P-5Ј-end-labeled template-primer (26) was incubated at 30°C for 15 min (or as indicated) in 10-l reaction mixtures containing 2 l of a 1:20 dilution of in vitrosynthesized enzyme (26) or 0.1 nM purified enzyme, 100 M each of the four dNTPs in Pol buffer (40 mM Tris-HCl (pH 8.0), 5 mM MgCl 2 , 60 mM KCl, 10 mM dithiothreitol, 250 g bovine serum albumin, 2.5% glycerol). Reactions were terminated by the addition of 20 l of 98% formamide, 10 mM EDTA; 5 l of each product was analyzed by electrophoresis through 14% denaturing polyacrylamide gels and quantified by phosphorimaging analysis.…”

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

“…Expression conditions and purification were as described (26). The predicted fusion protein sequence was verified by DNA sequencing.…”

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

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Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Glick

Chau

Vigna

et al. 2003

Journal of Biological Chemistry

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DNA polymerase (Pol ) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase (hPol ) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to noncomplementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol , one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.Accurate DNA replication is required for the maintenance of a species. The accuracy of cellular DNA replication results from the exceptionally high fidelity of DNA synthesis by replicative DNA polymerases and multiple mechanisms for the repair of DNA damage. As a result, the mutation rate in human cells has been estimated to be as low as 1 ϫ 10 Ϫ10 mutations/nucleotide/ cell division (1). Replicational accuracy is maintained despite the large number of lesions caused by both exogenous and endogenous DNA damaging agents. For example, evidence suggests that spontaneous depurination of DNA can result in 10,000 abasic sites per cell per day (2), and an even larger number of lesions are produced as a result of DNA damage by reactive oxygen species (3). Many of the lesions are likely to escape DNA repair mechanisms and therefore are present in DNA templates during copying by replicative DNA polymerases. Bulky lesions can block the progression of DNA replication, forcing the cell to employ additional mechanisms to complete replication in the presence of unrepaired lesions (4, 5).Recently, a new family of DNA polymerases, referred to as the Y-family, has been identified and categorized as translesion synthesis DNA polymerases. The Y-family of DNA polymerases is highly conserved through evolution. Thus far, four Y-family DNA polymerases have been identified in human cells: DNA polymerase (hPol ) 1 (6, 7), DNA polymerase (hPol ) (8), DNA polymerase (hPol ) (9), and the DNA-dependent dCMP transferase Rev1 (10). In contrast to replicative polymerases, ...

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Section: Resultssupporting

confidence: 62%

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

“…Expression conditions and purification were as described (26). The predicted fusion protein sequence was verified by DNA sequencing.…”

Section: Construction and Selection Of Active Hpol Tyr-52 3 X Mutants-mentioning

confidence: 99%

See 3 more Smart Citations

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Glick

Chau

Vigna

et al. 2003

Journal of Biological Chemistry

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Components of nucleotide excision repair and DNA damage tolerance in Arabidopsis thaliana

Kunz

Anderson

Osmond

et al. 2005

Environ and Mol Mutagen

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As obligate phototrophs, and despite shielding strategies, plants sustain DNA damage caused by UV radiation in sunlight. By inhibiting DNA replication and transcription, such damage may contribute to the detrimental effects of UV radiation on the growth, productivity, and genetic stability of higher plants. However, there is evidence that plants can reverse UV-induced DNA damage by photoreactivation or remove it via nucleotide excision repair. In addition, plants may have mechanisms for tolerating UV photoproducts as a means of avoiding replicative arrest. Recently, phenotypic characterization of plant mutants, functional complementation studies, and cDNA analysis have implicated genes isolated from the model plant Arabidopsis thaliana in nucleotide excision repair or tolerance of UV-induced DNA damage. Here, we briefly review features of these processes in human cells, collate information on Arabidopsis homologs of the relevant genes, and summarize the experimental findings that link certain of these plant genes to nucleotide excision repair or damage tolerance.

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Cloning and characterization of DNA polymerase η from Trypanosoma cruzi: Roles for translesion bypass of oxidative damage

Moura

Schamber-Reis

Silva

et al. 2009

Environ and Mol Mutagen

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We report the cloning and characterization of the DNA polymerase eta gene from Trypanosoma cruzi (TcPoleta), the causative agent of Chagas disease. This protein, which can bypass cyclobutane pyrimidine dimers, contains motifs that are conserved between Y family polymerases. In vitro assays showed that the recombinant protein is capable of synthesizing DNA in undamaged primer-templates. Intriguingly, T. cruzi overexpressing TcPoleta does not increase its resistance to UV-light (with or without caffeine) or cisplatin, despite the ability of the protein to enhance UV resistance in a RAD30 mutant of Saccharomyces cerevisiae. Parasites overexpressing TcPoleta are also unable to restore growth after treatment with zeocin or gamma irradiation. T. cruzi overexpressing TcPoleta are more resistant to treatment with hydrogen peroxide (H(2)O(2)) compared to nontransfected cells. The observed H(2)O(2) resistance could be associated with its ability to bypass 8-oxoguanine lesions in vitro. The results presented here suggest that TcPoleta is able to bypass UV and oxidative lesions. However the overexpression of the gene only interferes in response to oxidative lesions, possibly due to the presence of these lesions during the S phase.

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Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions

Cited by 48 publications

References 47 publications

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Components of nucleotide excision repair and DNA damage tolerance in Arabidopsis thaliana

Cloning and characterization of DNA polymerase η from Trypanosoma cruzi: Roles for translesion bypass of oxidative damage

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