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...

The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in an Escherichia coli strain defective in uracil repair (ung mutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome.uracil-DNA glycosylase | APOBEC3A | APOBEC3B | kataegis | cancer genome mutations P airing of complementary DNA strands protects the DNA bases against modification by a number of hydrolytic, oxidizing, and alkylating chemicals (1-4). For example, water reacts with cytosine, creating uracil, and the rate of this reaction in single-stranded DNA (ssDNA) is more than 100-fold the rate in double-stranded DNA [dsDNA (5-7)]. Uracil-DNA glycosylase (Ung) excises uracils created by cytosine deamination in both ssDNA and dsDNA, resulting in abasic (AP) sites. In dsDNA, the AP sites are replaced with cytosines as a result of copying of the guanine in the complementary strand during repair by the base-excision repair (BER) pathway (8). In contrast, cytosine deaminations occurring in ssDNA are problematic because the complementary strand is not available to the BER pathway. Uracils that escape repair create C:G to T:A mutations, and incomplete repair of uracils can result in persistent AP sites and strand breaks that can destabilize the genome. Hence, identifying ssDNA regions that are susceptible to damage will increase our understanding of causes of mutations and genome instability.The AID/APOBEC (activation-induced deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of DNA-cytosine deaminases are specific for ssDNA (9, 10). They are found only in vertebrates and are good probes of ssDNA in cells because of their relatively small size (about 190-amino acid catalytic domain). They are active in heterologous hosts such as Escherichia coli (11, 12) and yeast (13-15) and cause mutations in the same sequence context as in their known targets, such as Ig genes and the DNA copy of HIV-1 genome (11,16,17). In particular, the catalytic domain of human APOBEC3G (A3G-CTD) was expressed in an engineered yeast strain lacking the UNG gene and was shown to target ssDNA generated through aberrant resection of telomeric ends (13).To similarly probe ssDNA in E. coli, we expressed A3G-CTD on a plasmid (pA3G-CTD) in...

Mismatch repair (MMR) is a major contributor to replication fidelity, but its impact varies with sequence context and the nature of the mismatch. Mutation accumulation experiments followed by whole-genome sequencing of MMR-defective strains yielded ≈30,000 base-pair substitutions (BPSs), revealing mutational patterns across the entire chromosome. The BPS spectrum was dominated by A:T to G:C transitions, which occurred predominantly at the center base of 5'NC3'+5'GN3' triplets. Surprisingly, growth on minimal medium or at low temperature attenuated these mutations. Mononucleotide runs were also hotspots for BPSs, and the rate at which these occurred increased with run length. Comparison with ≈2000 BPSs accumulated in MMR-proficient strains revealed that both kinds of hotspots appeared in the wild-type spectrum and so are likely to be sites of frequent replication errors. In MMR-defective strains transitions were strand biased, occurring twice as often when A and C rather than T and G were on the lagging-strand template. Loss of nucleotide diphosphate kinase increases the cellular concentration of dCTP, which resulted in increased rates of mutations due to misinsertion of C opposite A and T. In an double mutant strain, these mutations were more frequent when the template A and T were on the leading strand, suggesting that lagging-strand synthesis was more error-prone, or less well corrected by proofreading, than was leading strand synthesis.

23Mismatch repair (MMR) is a major contributor to replication fidelity, but its impact 24 varies with sequence context and the nature of the mismatch. Mutation accumulation 25 experiments followed by whole-genome sequencing of MMR-defective E. coli strains yielded 26 ≈30,000 base-pair substitutions, revealing mutational patterns across the entire chromosome. 27The base-pair substitution spectrum was dominated by A:T > G:C transitions, which occurred 28 predominantly at the center base of 5′NAC3′+5′GTN3′ triplets. Surprisingly, growth on minimal 29 medium or at low temperature attenuated these mutations. Mononucleotide runs were also 30 hotspots for base-pair substitutions, and the rate at which these occurred increased with run 31 length. Comparison with ≈2000 base-pair substitutions accumulated in MMR-proficient strains 32 revealed that both kinds of hotspots appeared in the wild-type spectrum and so are likely to be 33 sites of frequent replication errors. In MMR-defective strains transitions were strand biased, 34 occurring twice as often when A and C rather than T and G were on the lagging-strand 35 template. Loss of nucleotide diphosphate kinase increases the cellular concentration of dCTP, 36 which resulted in increased rates of mutations due to misinsertion of C opposite A and T. In an 37 mmr ndk double mutant strain, these mutations were more frequent when the template A and 38 T were on the leading strand, suggesting that lagging-strand synthesis was more error-prone or 39 less well corrected by proofreading than was leading strand synthesis. 40 41 5 origin (FOSTER et al. 2013;DETTMAN et al. 2016;DILLON et al. 2017). Thus, there is much yet to 63 learn about what determines the mutability of any given DNA site. 64Another lesson learned from Benzer's classic paper is the importance of large numbers 65 when studying rare events such as mutations. Investigating mutational processes with mutation 66 accumulation (MA) protocols that exert minimal selective pressure has the advantage of 67 allowing mutations to accumulate in an unbiased manner. Coupling the MA protocol to whole-68 genome sequencing (WGS) eliminates the possibility that peculiarities of particular DNA 69 segments can bias the overall results, and also allows mutation rates across the genome to be 70 evaluated. However, the laboriousness of the MA procedure limits the numbers of mutations 71 that can be analyzed. To overcome this limitation, we and others have used mutator strains of 72 model microorganisms. 73 DNA replication in E. coli is performed by DNA polymerase III holoenzyme, a 74 multisubunit machine with high processivity and accuracy. The fidelity of replication, which, in 75 E. coli, is about 1 mistake in 1000 generations (LEE et al. 2012), is mainly due to three factors: 76 the intrinsic base-pairing fidelity of the DNA polymerase, error-correction by the exonuclease 77 activity of the proofreader, and correction of mismatches by the mismatch-repair system 78 (MMR). In E. coli, mismatch repair is accomplished by four major enzymes. ...