Determinants of spontaneous mutation in the bacteriumEscherichia colias revealed by whole-genome sequencing

Abstract

A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1-2 × 10 −3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold.A complete understanding of the evolution and stability of the genome requires that the determinants of spontaneous mutation be identified and characterized. Among the variety of mistakes that can occur during DNA transactions, four sources of sequence variation appear to dominate in prokaryotes: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage induced by exogenous agents, and the activities of error-prone polymerases. This conclusion is based on changes in the rates and spectra of mutations that occur when genes affecting these processes are deleted or amplified. In particular, loss of a DNA repair pathway often gives a mutator phenotype, indicating that the pathway of interest exerts an important limitation on spontaneous mutation (1). However, investigations of the mutagenic impact of various DNA repair pathways have relied almost exclusively on reporter genes, leaving open the possibility that the results are biased by the particular features of the selected loci. This concern can be avoided by allowing mutations to accumulate nonselectively in DNA repair-defective strains and identifying the resulting sequence changes by whole-genome sequencing (WGS). Although this approach may miss rare but interesting mutational processes, it can reveal the overall threats to genomic stability and identify features, such as local sequence context, that influence mutational frequencies. Surprisingly, this technique has been used with the eukaryote Caenorhabditis elegans (2) but has not been extensively applied to prokary...

Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli

“…As observed previously with the WT strain (18,19), when normalized to the number of nucleotides in coding versus noncoding DNA, all three ung mutant strains showed a twofold bias against mutations occurring in coding sequences (Table S1). In ung + strains, this bias was shown to be a result of better mismatch repair of the coding regions than of noncoding regions (19).…”

“…This weak mutator phenotype contrasts with the 160-fold increase in the overall mutation rate that occurred when a pathway for the repair of 8-oxoguanines was blocked in the same genetic background (18) and shows that cytosine deamination is not the major form of endogenous DNA damage. The mutator phenotype of the ung mutant strain was almost entirely the result Significance C:G to T:A mutations constitute the largest class of spontaneous base substitutions in all organisms.…”

“…The ung mutant E. coli K12 strain used in this study, PFM16, was derived from WT strain PFM2 (18,19). The Δung::Kn allele was moved into PFM2 from the Keio strain JW2564 (55) via P1 bacteriophage transduction, and the kanamycin-resistance gene was then removed using FLP recombination (56), leaving an in-frame scar sequence that encodes a 34-amino acid peptide.…”

“…These results were compared with results from similar experiments performed using ung mutant cells without a plasmid, ung mutant cells expressing catalytically inactive A3G-CTD (pA3G-CTDmut), and published results from the ung + wild-type (WT) E. coli strain (18,19).…”

Antibiotic treatment enhances the genome-wide mutation rate of target cells

“…2B), we conducted MA experiments with a ΔmutY strain to determine whether antibiotic treatment affects DNA oxidativedamage repair. The ΔmutY strain is dysfunctional in recognizing adenines mispaired with 8-oxo-guanines (49), and it is known that the failure to recognize and remove 8-oxo-guanines residing in DNA elevates the postreplication G:C→T:A transversion mutation rate (50)(51)(52).…”