The Spectrum of Replication Errors in the Absence of Error Correction Assayed Across the Whole Genome of Escherichia coli

Abstract

When the DNA polymerase that replicates the Escherichia coli chromosome, DNA polymerase III, makes an error, there are two primary defenses against mutation: proofreading by the ϵ subunit of the holoenzyme and mismatch repair. In proofreading-deficient strains, mismatch repair is partially saturated and the cell's response to DNA damage, the SOS response, may be partially induced. To investigate the nature of replication errors, we used mutation accumulation experiments and whole-genome sequencing to determine mutation rates and mutational spectra across the entire chromosome of strains deficient in proofreading, mismatch repair, and the SOS response. We report that a proofreading-deficient strain has a mutation rate 4000-fold greater than wild-type strains. While the SOS response may be induced in these cells, it does not contribute to the mutational load. Inactivating mismatch repair in a proofreading-deficient strain increases the mutation rate another 1.5-fold. DNA polymerase has a bias for converting G:C to A:T base pairs, but proofreading reduces the impact of these mutations, helping to maintain the genomic G:C content. These findings give an unprecedented view of how polymerase and error-correction pathways work together to maintain E. coli's low mutation rate of 1 per 1000 generations.

Keywords: DNA proofreading; DNA replication fidelity; mismatch repair; mutation accumulation; mutation hotspots.

Figure 1

The conditional BPS rates and spectra accumulated by the mutD5 and mutD5 mutL-mutant strains. The bars represent the BPSs per generation per number of A:T or G:C base pairs in the genome; the error bars are 95% CLs. (A) Cells were grown on LB medium; mutD5, PFM163; mutD5 mutL, PFM165/397/399. (B) Cells were grown on minimal glucose medium; mutD5, PFM163; mutD5 mutL, PFM165. The data in (B) for the mutD5 strain on minimal medium is presented with an expanded axis in Figure S4A. BPS, base pair substitution; CL, confidence limit.

Figure 2

The context bias of the base pair substitutions accumulated by the mutD5 and mutD5 mutL strains. The x-axis labels are the 32 nonredundant triplets oriented 5′NMN3′ with the mutated base in the center. The bars represent the BPS per generation per triplet in the genome; the error bars are 95% CLs. (A) Cells were grown on LB medium; mutD5, PFM163; mutD5 mutL, PFM165/397/399. (B) cells were grown on minimal glucose medium; mutD5, PFM163; mutD5 mutL, PFM165. The data in (B) for the mutD5 strain on minimal medium is presented with an expanded axis in Figure S4B. BPS, base pair substitution; CL, confidence limit.

Figure 3

The rates of the indels in homopolymeric runs accumulated by the mutD5 and mutD5 mutL mutant strains. The bars represent the indels per generation per number of base pairs in each run of nt length in the genome. The error bars are 95% CLs, some of which are smaller than the symbols. (A) Cells were grown on LB medium; mutD5, PFM163; mutD5 mutL, PFM165/397/399. (B) Cells were grown on minimal glucose medium; mutD5, PFM163; mutD5 mutL, PFM165. CL, confidence limit; indel, insertion/deletion.

Figure 4

The conditional rates and spectra of the indels accumulated by the mutD5 and mutD5 mutL⁻ mutant strains. The bars represent the indels per generation per number of relevant base pairs in the genome; the error bars are 95% CLs. (A) Cells were grown on LB medium; mutD5, PFM163; mutD5 mutL, PFM165/397/399. (B) Cells were grown on minimal glucose medium; mutD5, PFM163; mutD5 mutL, PFM165. The data in (B) for the mutD5 strain on minimal medium is presented with an expanded axis in Figure S5. CL, confidence limit; indel, insertion/deletion.