Determinants of base-pair substitution patterns revealed by whole-genome sequencing of DNA mismatch repair defectiveEscherichia coli

Abstract

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. ...

New complexities of SOS-induced “untargeted” mutagenesis in Escherichia coli as revealed by mutation accumulation and whole-genome sequencing

“…The parameters of the mutation accumulation (MA) experiments described in this paper are given in Table S2 in Appendix A. The MA protocol has been described in our previous papers [12,21,22]. Multiple parallel MA lines were initiated from one or two founder colonies, as described [22].…”

“…The MA protocol has been described in our previous papers [12,21,22]. Multiple parallel MA lines were initiated from one or two founder colonies, as described [22]. Every day each MA line was streaked for single colonies on LB-agar plates that were then incubated at 37 o C. The number of passes required to achieve a statistically meaningful number of mutations was estimated from preliminary fluctuation assays, as described [12,21,22].…”

“…Multiple parallel MA lines were initiated from one or two founder colonies, as described [22]. Every day each MA line was streaked for single colonies on LB-agar plates that were then incubated at 37 o C. The number of passes required to achieve a statistically meaningful number of mutations was estimated from preliminary fluctuation assays, as described [12,21,22]. At the end of the MA experiment, a single colony of each line was grown in LB broth overnight and a freezer stock made.…”

“…As described [12,21,22], the mutation rate for each MA experiment was calculated by dividing the total number of mutations accumulated by the total number of generations undergone by all the MA lines. For the conditional mutation rates, this value (which was not certified by peer review) is the author/funder.…”

“…was then divided by the appropriate number of sites (A:T, G:C, etc.). The mutation rates for each MA line in an experiment was used to compute the variance for statistical analysis, as described [22].…”