DNA Replication-Transcription Conflicts Do Not Significantly Contribute to Spontaneous Mutations Due to Replication...

Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch repair (MMR)-defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base pair substitutions or small indels (i.e., insertion and deletions of ≤4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hot spots for indels. No additional orientation bias in mutation frequencies occurred when MMR^- strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus codirectional to replication. In addition, the data revealed a strong correlation of the frequency of base pair substitutions with gene length but no correlation with gene expression levels. IMPORTANCE Because DNA replication and transcription occur on the same DNA template, encounters between the two machines occur frequently. When these encounters are head-to-head, genomic disruption can occur. However, whether replication-transcription conflicts contribute to spontaneous mutations is debated. Analyzing in detail a large collection of mutations generated with mismatch repair-defective Escherichia coli strains, we found that across the genome there are no significant differences in mutation frequencies between genes oriented codirectionally and those oriented head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for small insertions and deletions in head-on-oriented genes, but this difference was almost entirely due to the asymmetrical locations of tRNA genes containing mononucleotide runs, which are hot spots for these mutations. Thus, biases in the positions of mutational target sequences can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on and codirectionally to replication.