The Accelerated Evolution of Lagging Strand Genes Is Independent of Sequence Context

We previously discovered that lagging strand genes evolve faster in Bacillus subtilis (and potentially other bacteria). Lagging strand genes are transcribed in the head-on orientation with respect to DNA replication, leading to collisions between the two machineries that stall replication and can destabilize genomes. Our previous work indicated that the increased mutagenesis of head-on genes depends on transcription-coupled repair and the activity of an error prone polymerase which is likely activated in response to these collisions. Recently, it was proposed that sequence context is a major contributor to the increased mutagenesis and evolution of head-on genes. These models are based on laboratory-based evolution experiments performed in B. subtilis. However, critical evolutionary analyses of naturally occurring single nucleotide polymorphisms (SNPs) in wild strains were not performed. Using the genomic sequences from nine closely related wild B. subtilis strains, we analyzed over 200,000 naturally occurring SNPs as a proxy for natural mutation patterns for all genes and in particular, head-on genes. Our analysis suggests that (frame-independent) triplet sequence context can impact mutation rates: certain triplet sequences (TAG, CCC, CTA, and ACC) accumulate SNPs at a higher rate and are depleted from the genome. However, the triplet sequences previously identified as mutagenic in laboratory experiments (CCG, GCG, and CAC) do not have an elevated rate of SNP accumulation and are not depleted from the genome. Importantly, dN/dS analyses indicate that the accelerated evolution of head-on genes is not dependent on any particular triplet sequence. Thus, in agreement with our previous results, mutagenic transcription-coupled repair, rather than sequence context, is sufficient to explain the accelerated evolution of head-on genes.