Mutagenic cost of ribonucleotides in bacterial DNA
Jeremy W. Schroeder a, 1,2, Justin R. Randall a, 1, William G. Hirst a, Michael E. O’Donnell b, 3, and Lyle A. Simmons a, 3
Author Affiliations
Contributed by Michael E. O’Donnell, September 18, 2017 (sent for review June 19, 2017; reviewed by Martin Marinus and Roger Woodgate)
Significance
DNA polymerases frequently incorporate ribonucleotides in place of deoxyribonucleotides during genome replication. RNase HII is responsible for initiating the removal of ribonucleotide errors across all three domains of life. Ribonucleotides that persist in genomic DNA due to defects in RNase HII result in strand breaks, mutagenesis, and neurodevelopmental disease in humans. Here, we define the proteins important for ribonucleotide excision repair in Bacillus subtilis and use genome-wide mutational profiling to determine the mutagenic cost of ribonucleotides in RNase HII-deficient cells. We show that the absence of RNase HII yields error-prone ribonucleotide correction via a pathway that relies on an essential DNA polymerase. We further demonstrate that error-prone ribonucleotide removal causes sequence context-dependent GC → AT transitions on the lagging strand.
Abstract
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.
ribonucleotide excision repair DNA polymerase mutagenesis RNase HII
Footnotes
1J.W.S. and J.R.R. contributed equally to this work.
2Present address: Department of Bacteriology, University of Wisconsin, Madison, WI 53706.
3To whom correspondence may be addressed. Email: odonnel@mail.rockefeller.edu or lasimm@umich.edu.
Author contributions: J.W.S., J.R.R., W.G.H., and L.A.S. designed research; J.W.S., J.R.R., W.G.H., and L.A.S. performed research; J.W.S. and J.R.R. contributed new reagents/analytic tools; J.W.S., J.R.R., W.G.H., M.E.O., and L.A.S. analyzed data; and J.W.S., J.R.R., M.E.O., and L.A.S. wrote the paper.
Reviewers: M.M., University of Massachusetts Medical School; and R.W., National Institute of Child Health and Human Development, National Institutes of Health.
The authors declare no conflict of interest.
Data deposition: The sequences reported in this paper have been deposited in the Sequence Read Archive database (accession no. SRP117359).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1710995114/-/DCSupplemental.
Copyright © 2017 the Author(s). Published by PNAS.
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