Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair
Open accessAbstract
Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8(th) phosphodiester bond 5' and 4(th)-5(th) phosphodiester bonds 3' of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER.
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Both replicative and translesion DNA polymerases make sugar errors.
“Both replicative and translesion DNA polymerases make sugar errors”
From Ribonucleotide errors, RNase HII, and ribonucleotide excision repair (2026) · cites this paper as Vaisman et al. 2013
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“In the absence of RER, nucleotide excision repair (NER) may enact repair of single misincorporated rNMPs in bacteria, albeit leading to mutagenic resynthesis (Figure 5C) (Vaisman et al. 2013, 2014; Wozniak and Simmons 2022).”
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- Authors
- Vaisman, A., McDonald, J. P., Huston, D., Kuban, W., Liu, L., Van Houten, B., & Woodgate, R.
- Affiliations
- National Institutes of Health (US)
- UPMC Hillman Cancer Center (US)
- Corresponding author
- Woodgate, Roger
- Venue
- PLoS Genetics
- Publisher
- Public Library of Science (PLoS)
- Published
- 2013-11-07
- Volume
- 9
- Issue
- 11
- Pages
- e1003878
- ISSN
- 1553-7390 (ISSN-L)
- 1553-7404
- PMC
- PMC3820734
- OpenAlex
- W2144683765
- Cited by
- 70
- References
- 71
- Citations by year
- 2026: 3 · 2025: 1 · 2024: 1 · 2023: 6 · 2022: 5 · 2021: 8 · 2020: 1 · 2019: 8 · 2018: 3 · 2017: 8 · 2016: 4 · 2015: 10 · 2014: 11 · 2013: 1
- FWCI
- 3.88
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS Genetics (publishedVersion, public-domain)
- Europe PMC (PubMed Central) (publishedVersion, public-domain)
- PubMed Central (publishedVersion, public-domain)
- PubMed (publishedVersion)
- D-Scholarship@Pitt (University of Pittsburgh) (acceptedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- National Institutes of Health
- National Institute of Environmental Health Sciences (R01ES019566)
- National Institute of Child Health and Human Development
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- nucleotide excision repair, ribonucleotide excision repair, DNA polymerase V, UmuC, RNase HII, mutagenesis, DNA mismatch repair, base excision repair, Escherichia coli, prokaryotic genomes
- MeSH terms
- DNA, DNA-Directed DNA Polymerase, DNA Repair, Endodeoxyribonucleases, Escherichia coli, Mutation, Prokaryotic Cells, Ribonucleotides, Genome, Bacterial, Ribonuclease H, Amino Acid Substitution, Escherichia coli Proteins
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