The Symmetrical Wave Pattern of Base-Pair Substitution Rates across the Escherichia coli Chromosome Has Multiple Causes.

Mutation accumulation experiments followed by whole-genome sequencing have revealed that, for several bacterial species, the rate of base-pair substitutions (BPSs) is not constant across the chromosome but varies in a wave-like pattern that is symmetrical about the origin of replication. The experiments reported here demonstrated that, in Escherichia coli, several interacting factors determine the wave. The origin is a major driver of BPS rates. When it is relocated, the BPS rates in a 1,000-kb region surrounding the new origin reproduce the pattern that surrounds the normal origin. However, the pattern across distant regions of the chromosome is unaltered and thus must be determined by other factors. Increasing the deoxynucleoside triphosphate (dNTP) concentration shifts the wave pattern away from the origin, supporting the hypothesis that fluctuations in dNTP pools coincident with replication firing contribute to the variations in the mutation rate. The nucleoid binding proteins (HU and Fis) and the terminus organizing protein (MatP) are also major factors. These proteins alter the three-dimensional structure of the DNA, and results suggest that mutation rates increase when highly structured DNA is replicated. Biases in error correction by proofreading and mismatch repair, both of which may be responsive to dNTP concentrations and DNA structure, also are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.IMPORTANCE It has been found in several species of bacteria that the rate at which single base pairs are mutated is not constant across the genome but varies in a wave-like pattern that is symmetrical about the origin of replication. Using Escherichia coli as our model system, we show that this pattern is the result of several interconnected factors. First, the timing and progression of replication are important in determining the wave pattern. Second, the three-dimensional structure of the DNA is also a factor, and the results suggest that mutation rates increase when highly structured DNA is replicated. Finally, biases in error correction, which may be responsive both to the progression of DNA synthesis and to DNA structure, are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair Defective Escherichia coli.

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 Escherichia coli 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'NAC3'+5'GTN3' 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 mmr ndk 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.

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

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.