The mutational landscape ofBacillus subtilisconditional hypermutators shows how proofreading skews DNA polymerase error rates

AbstractPolymerase 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 bacteriumBacillus subtilisto 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 ofpolCand/or a deficient allele ofmutLand performed mutation accumulation experiments. With their wide range of mutation rates and contrasting mutation profiles, these conditional hypermutators enrich theB. subtilissynthetic 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 inB. subtilisand that an inherent drawback of proofreading is to skew the net polymerase error rates by amplifying intrinsic biases in nucleotide selectivity.