The mutational landscape of Bacillus subtilis conditional hypermutators suggests how proofreading inherently skews polymerase error rates

Polymerase errors during DNA replication are a major source of point mutations in genomes. The resulting rate of spontaneous mutation also depends on the counteracting activity of DNA repair mechanisms, with mutator phenotypes appearing constantly and allowing for periods of rapid evolution in nature and in the laboratory. Here, we use the Gram-positive model bacterium Bacillus subtilis to disentangle the contributions of DNA polymerase initial nucleotide selectivity, DNA polymerase proofreading, and mismatch repair (MMR) to the mutation rate. To achieve this, we constructed several conditional hypermutators with a proofreading-deficient allele of polC and/or a deficient allele of mutL and performed mutation accumulation experiments. With their wide range of mutation rates and contrasting mutation profiles, these conditional hypermutators enrich the B. subtilis synthetic biology toolbox for directed evolution. Using mathematical models, we investigated how to interpret the apparent probabilities with which errors escape MMR and proofreading, highlighting the difficulties of working with counts that aggregate potentially heterogeneous mutations and with unknowns about the pathways leading to mutations in the wild-type. Aware of these difficulties, the analysis shows that proofreading prevents partial saturation of the MMR in B. subtilis and that an inherent drawback of proofreading is to skew the net polymerase error rates by amplifying intrinsic biases in nucleotide selectivity.