Re-establishment of nucleosome occupancy during double-strand break repair in budding yeast

Abstract

Homologous recombination (HR) is an evolutionary conserved pathway in eukaryotes that repairs a double-strand break (DSB) by copying homologous sequences from a sister chromatid, a homologous chromosome or an ectopic location. Recombination is challenged by the packaging of DNA into nucleosomes, which may impair the process at many steps, from resection of the DSB ends to the re-establishement of nucleosomes after repair. However, nucleosome dynamics during DSB repair have not been well described, primarily because of a lack of well-ordered nucleosomes around a DSB. We designed a system in budding yeast Saccharomyces cerevisiae to monitor nucleosome dynamics during repair of an HO endonuclease-induced DSB. Nucleosome occupancy around the break is lost following DSB formation, by 5’ to 3’ resection of the DSB end. Soon after repair is complete, nucleosome occupancy is partially restored in a repair-dependent but cell cycle-independent manner. Full re-establishment of nucleosome protection back to the level prior to DSB induction is achieved when the cell cycle resumes following repair. These findings may have implications to the mechanisms by which cells sense the completion of repair.

Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication

“…HR involves the generation of single-stranded DNA (ssDNA) around the DSB via end-resection machinery and subsequently requires the presence of a homologous sequence of DNA (most commonly a sister chromatid) to serve as a template that can be used to resynthesize DNA sequences. However, except in special circumstances, such as MAT switching where the donor is heterochromatic, with highly positioned nucleosomes that are not found at the recipient locus (Weiss and Simpson 1998;Ravindra et al 1999) , it is difficult to distinguish the chromatin structures of homologous regions undergoing recombination (Hicks et al 2011;Tsabar et al 2016) . NHEJ, however, is a simpler process and requires no template for repair of the DNA strand.…”

“…These immediate changes in chromatin structure at the break were followed by the MRE11 -dependent broader eviction of histone octamers. The early and local changes to chromatin were reversible through repair of the genetic lesion by NHEJ, and occurred in a replication-independent manner.Previous investigations of chromatin structure surrounding a DSB have relied on ChIP or PCR/probe based methods to query the presence of a specific sequences/loci(Tsukuda et al 2005;Shim et al 2007;Goldstein et al 2013;Tsabar et al 2016;Li and Tyler 2016) . These methods have established a low-resolution model of broad nucleosome eviction dependent on MRX activity and facilitated by Sgs1, Exo1, Dna2, Fun30, and Ino80(Dubrana et al 2007;Mimitou and Symington 2008;Eapen et al 2012;Westmoreland and Resnick 2015) .…”

Processing of DNA Double-Strand Breaks by the MRX Complex in a Chromatin Context

“…Chromatin immunoprecipitation experiments support nucleosome disassembly near DSBs in both yeast and human cells (Li and Tyler, 2016; Tsabar et al, 2016), suggesting that nucleosome eviction occurs during resection. A key question is whether nucleosomes are evicted prior to the onset of resection or whether chromatin remodelers help the resection machinery to navigate through chromatin, with nucleosome loss occurring as a consequence of nucleolytic degradation.…”

Quantitative mechanisms of DNA damage sensing and signaling

“…H2A is an integral component of chromatin and as such immediately available as substrate for phosphorylation. Therefore, and despite the fact that histones were shown to display plasticity at DSBs (Hauer et al 2017;Hauer and Gasser 2017;Adam et al 2016;Tsabar et al 2016), encounters between Mec1 and H2A over the broad γH2A domain will rather be defined by chromatin architecture and mobility than recruitment (Caron et al 2015;Aymard and Legube 2016;Lee et al 2013;Renkawitz et al 2013;Zimmer and Fabre 2019). Taken together, we therefore propose that Mec1 targets are phosphorylated dependent on the frequency of kinase-substrate encounters, which in the global signaling circuit is determined by recruitment and in the local signaling circuit depends on chromatin architecture (Fig.…”