Perfect short inverted repeats (IRs) are known to be enriched in a variety of bacterial and eukaryotic genomes. Currently, it is unclear whether perfect IRs are conserved over evolutionary time scales. In this study, we aimed to characterize the prevalence and evolutionary conservation of IRs across 20 proteobacterial strains. We first identified IRs in Escherichia coli K-12 substr MG1655 and showed that they are overabundant. We next aimed to test whether this overabundance is reflected in the conservation of IRs over evolutionary time scales. To this end, for each perfect IR identified in E. coli MG1655, we collected orthologous sequences from related proteobacterial genomes. We next quantified the evolutionary conservation of these IRs, that is, the presence of the exact same IR across orthologous regions. We observed high conservation of perfect IRs: out of the 234 examined orthologous regions, 145 were more conserved than expected, which is statistically significant even after correcting for multiple testing. Our results together with previous experimental findings support a model in which imperfect IRs are corrected to perfect IRs in a preferential manner via a template switching mechanism.

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Okazaki fragments that are formed during lagging strand DNA synthesis include an initiating primer consisting of both RNA and DNA. The RNA fragment must be removed before the fragments are joined. In Saccharomyces cerevisiae, a key player in this process is the structure-specific flap endonuclease, Rad27p (human homolog FEN1). To obtain a genomic view of the mutational consequence of loss of RAD27, a S. cerevisiae rad27Δ strain was subcultured for 25 generations and sequenced using Illumina paired-end sequencing. Out of the 455 changes observed in 10 colonies isolated the two most common types of events were insertions or deletions (INDELs) in simple sequence repeats (SSRs) and INDELs mediated by short direct repeats. Surprisingly, we also detected a previously neglected class of 21 template-switching events. These events were presumably generated by quasi-palindrome to palindrome correction, as well as palindrome elongation. The formation of these events is best explained by folding back of the stalled nascent strand and resumption of DNA synthesis using the same nascent strand as a template. Evidence of quasi-palindrome to palindrome correction that could be generated by template switching appears also in yeast genome evolution. Out of the 455 events, 55 events appeared in multiple isolates; further analysis indicates that these loci are mutational hotspots. Since Rad27 acts on the lagging strand when the leading strand should not contain any gaps, we propose a mechanism favoring intramolecular strand switching over an intermolecular mechanism. We note that our results open new ways of understanding template switching that occurs during genome instability and evolution.

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