Mutagenic cost of ribonucleotides in bacterial DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 2017-10, Vol.114 (44), p.11733-11738
• Main Authors: Schroeder, Jeremy W., Randall, Justin R., Hirst, William G., O’Donnell, Michael E., Simmons, Lyle A.
• Format: Article
• Language: English
• Subjects: Bacteria; Base pairs; Biological Sciences; Deoxyribonucleic acid; DNA; DNA polymerase; DNA-directed DNA polymerase; Errors; Genomes; Integrity; Mutagenesis; Nucleotide excision repair; Proteins; Repair; Ribonuclease H2; Ribonucleases; Ribonucleotides
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1710995114

Replicative DNA polymerases misincorporate ribonucleoside triphosphates (rNTPs) into DNA approximately once every 2,000 base pairs synthesized. Ribonucleotide excision repair (RER) removes ribonucleoside monophosphates (rNMPs) from genomic DNA, replacing the error with the appropriate deoxyribonucleoside triphosphate (dNTP). Ribonucleotides represent a major threat to genome integrity with the potential to cause strand breaks. Furthermore, it has been shown in the bacterium Bacillus subtilis that loss of RER increases spontaneous mutagenesis. Despite the high rNTP error rate and the effect on genome integrity, the mechanism underlying mutagenesis in RER-deficient bacterial cells remains unknown. We performed mutation accumulation lines and genome-wide mutational profiling of B. subtilis lacking RNase HII, the enzyme that incises at single rNMP residues initiating RER. We show that loss of RER in B. subtilis causes strand- and sequence-context–dependent GC → AT transitions. Using purified proteins, we show that the replicative polymerase DnaE is mutagenic within the sequence context identified in RER-deficient cells. We also found that DnaE does not perform strand displacement synthesis. Given the use of nucleotide excision repair (NER) as a backup pathway for RER in RNase HII-deficient cells and the known mutagenic profile of DnaE, we propose that misincorporated ribonucleotides are removed by NER followed by error-prone resynthesis with DnaE.