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.
Bibliographical noteFunding Information:
We thank Margaret Dominska, Patricia W. Greenwell, Marina Druseikis, and Aseelnor Abedalkhal for their help with yeast subculturing. This project started at the laboratory of Thomas D. Petes at Duke University and was supported by National Institutes of Health grants GM24110 and GM52319. P.A.M. is funded by the University of North Carolina at Chapel Hill University Cancer Research Fund. S.C. is supported by the Research Career Development Award of the Israel Cancer Research Foundation. E.H.-C. was supported by the Israel Cancer Association grant 20150038 and by the Open University of Israel Research fund.
© 2017 Omer et al.
- Inverted repeats
- Okazaki fragments
- Template switching