Nucleosome Dynamics Around a DNA Double Stranded Break During Repair by Gene Conversion.

• DOI: 10.1096/fasebj.29.1_supplement.709.8
• Publication Date: 2021-06-21T18:40:16Z
• Authors: Michael Tsabar; Wade Hicks; James Haber
• Keywords: 0301 basic medicine; 03 medical and health sciences

In eukaryotes, double‐stranded break repair (DSB) takes place in the context of chromatin. Saccharomyces cerevisiae mating type (MAT) switching provides an opportunity to investigate nucleosome dynamics around a well‐characterized DSB. MAT switching is initiated by synchronous induction of a DSB by HO endonuclease at the MAT locus following which the DSB ends are resected and the Rad51 recombinase is loaded on the 3‐ended single strand. Rad51 facilitates strand invasion with a homologous donor sequence, which is required for initiation of new DNA synthesis and the resulting repair.A hurdle in studying nucleosome positioning around the MAT locus has been the weak nucleosome positioning around the MAT locus relative to the heterochromatic donors HML and HMR. To address this hurdle we designed a novel system in which we inserted the Xenopus laevis 5S nucleosome positioning sequence adjacent to the MAT locus. Using this system we have, for the first time, monitored the loss of chromatin protection following resection of the DSB end and the re‐establishment of chromatin structure following repair. We show that nucleosome protection is lost around a DSB in a resection‐dependent manner. Further, we show that nucleosome protection is reestablished following repair of a DSB but not to the level of protection seen prior to induction of a DSB. This reestablishment depends on repair of the DSB and not on passage through the cell cycle. We show that complete protection is reestablished after several cell cycles, suggesting that there is a mechanistic difference between repair‐dependent and replication‐dependent nucleosome positioning.