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Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V

Escherichia coli pol V (UmuD′(2)C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in...

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Autores principales: McDonald, John P., Vaisman, Alexandra, Kuban, Wojciech, Goodman, Myron F., Woodgate, Roger
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493448/
https://www.ncbi.nlm.nih.gov/pubmed/23144626
http://dx.doi.org/10.1371/journal.pgen.1003030
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author McDonald, John P.
Vaisman, Alexandra
Kuban, Wojciech
Goodman, Myron F.
Woodgate, Roger
author_facet McDonald, John P.
Vaisman, Alexandra
Kuban, Wojciech
Goodman, Myron F.
Woodgate, Roger
author_sort McDonald, John P.
collection PubMed
description Escherichia coli pol V (UmuD′(2)C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs in vitro. Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A in vitro, strains expressing umuC_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by umuC_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV in vivo. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass in vitro. In a recA730 lexA(Def) ΔumuDC ΔdinB strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, umuC_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic ΔrnhB strain and ∼72% of wild-type levels in a ΔrnhA ΔrnhB double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the E. coli genome, but at the cost of higher levels of cellular mutagenesis.
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spelling pubmed-34934482012-11-09 Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V McDonald, John P. Vaisman, Alexandra Kuban, Wojciech Goodman, Myron F. Woodgate, Roger PLoS Genet Research Article Escherichia coli pol V (UmuD′(2)C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs in vitro. Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A in vitro, strains expressing umuC_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by umuC_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV in vivo. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass in vitro. In a recA730 lexA(Def) ΔumuDC ΔdinB strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, umuC_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic ΔrnhB strain and ∼72% of wild-type levels in a ΔrnhA ΔrnhB double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the E. coli genome, but at the cost of higher levels of cellular mutagenesis. Public Library of Science 2012-11-08 /pmc/articles/PMC3493448/ /pubmed/23144626 http://dx.doi.org/10.1371/journal.pgen.1003030 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
McDonald, John P.
Vaisman, Alexandra
Kuban, Wojciech
Goodman, Myron F.
Woodgate, Roger
Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
title Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
title_full Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
title_fullStr Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
title_full_unstemmed Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
title_short Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
title_sort mechanisms employed by escherichia coli to prevent ribonucleotide incorporation into genomic dna by pol v
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493448/
https://www.ncbi.nlm.nih.gov/pubmed/23144626
http://dx.doi.org/10.1371/journal.pgen.1003030
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