Whole Genome Sequence Analysis of Mutations Accumulated inrad27ΔYeast Strains with Defects in the Processing of Okazaki Fragments Indicates Template-Switching Events

Abstract

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.

Large-scale contractions of Friedreich’s ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability

“…The original model postulated that a long flap that forms in the absence of Rad27 cleavage gets incorporated into the newly synthesized Okazaki fragment, resulting in a repeat expansion (91). This model cannot explain, however, how the persistence of long flaps in rad27 mutants could lead to repeat contractions, as observed by us and others (38,40,41,(43)(44)(45)(48)(49)(50)(51). Our current data show no direct correlation between flap-endonuclease activity and the repeat contraction rate in various rad27 mutants.…”

“…It was long known that the yeast Rad27 flap-endonuclease protein protects various DNA repetitive sequences from instability (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). The original model postulated that a long flap that forms in the absence of Rad27 cleavage gets incorporated into the newly synthesized Okazaki fragment, resulting in a repeat expansion (91).…”

“…Overall, these data show that long GAA repeats are prone to large-scale contractions in yeast that seem to occur in one step. Impaired Okazaki fragment processing is known to destabilize repetitive DNA sequences (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). We looked at whether this was true for our system by testing major yeast endonucleases implicated in the processing of single-stranded flaps during Okazaki fragment maturation: Rad27, Exo1, and Dna2.…”

On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability

“…In yeast, two endonucleases do the bulk of the job: Rad27, which only cuts short flaps (300 -302), and Dna2, which cuts longer flaps (301)(302)(303)(304)(305)(306). Mutations in either of these nucleases lead to dramatic increases in the expansions of various repeats, including CAG, GAA, CGG, G, GT, CAGT, CAACG, CAATCGGT, yeast telomeric repeats, and several minisatellites (142,289,(307)(308)(309)(310)(311)(312)(313)(314)(315)(316)(317)(318). In other words, the effect of rad27 or dna2 mutations is virtually independent of the repeat sequence.…”

The Prevalence and Evolutionary Conservation of Inverted Repeats in Proteobacteria

“…Fork replication problems as well as DNA repair proteins affect the rate of IR template switching. For example, the loss of the yeast RAD27 gene, a key player in Okazaki fragment maturation, increases the rate of template switching ( Omer et al. 2017 ).…”