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

Abstract

Although the molecular events required for the repair of double-strand breaks (DSB) have been well characterized, the role of epigenetic processes in the recognition and repair of DSBs has only been investigated at low resolution. We rapidly and synchronously induced a site-specific DSB in Saccharomyces cerevisiae upstream of the PHO5 locus, which has well-positioned nucleosomes. Utilizing MNase-seq epigenome mapping we interrogated the order of chromatin changes that occur immediately following a DSB by generating a base-pair resolution map of the chromatin landscape.In wild type cells, the first nucleosome left of the break was rapidly evicted. The eviction of this flanking nucleosome was dynamic and proceeded through an early intermediate chromatin structure where the nucleosome was repositioned in the adjacent linker DNA.Other nucleosomes bordering both sides of the break were also shifted away from the break; however, their loss was more gradual. These local changes preceded a broader loss of chromatin organization and nucleosome eviction that was marked by increased MNase sensitivity in the regions ~8 kb on each side of the break. While the broad loss of chromatin organization was dependent on the end-processing complex, Mre11-Rad50-Xrs2 (MRX), the early remodeling and repositioning of the nucleosome adjacent to the break was independent of the MRX and YKU70/80 complexes. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population, where 5' to 3' end-resection of DSB ends is blocked. Concomitant with DSB repair, we

Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites

“…Assuming nucleosome spacing length of around 200 bp, this implies that the up to 2 kb accessible region from Fig. 5c lost up to ten nucleosomes 47 , 48 . We further uncovered a subpopulation of ATAC–seq reads spanning the target sites that significantly increased after Cas9 delivery ( P = 1.44 × 10 −18 , Student’s t -test) (Fig.…”

SMC protein RecN drives RecA filament translocation and remodelling for in vivo homology search

“…In vitro studies have shown that RecA molecules in the nucleoprotein filament possess ATPase activity that can influence (a) formation of a continuous filament during RecA loading on ssDNA 39 , (b) growth and shrinkage of the filament [40][41][42] , (c) release of the filament from heterologous pairing 35,43 , and (d) RecA turnover during strand invasion [42][43][44] . In vivo studies on double-strand break repair have made use of engineered endonuclease-based DSB-inducing systems (such as the I-SceI system) to make targeted breaks on the chromosome and track the associated dynamics [4][5][6]11,12,[45][46][47] . Using such a system in E. coli to study dynamics of long-distance homology search, imaging of RecA has revealed the presence of large assemblies of RecA, described as RecA filaments (or 'bundles'), that can even span the length of the cell 5,6 .…”

The histone demethylase JMJ27 acts during the UV-induced modulation of H3K9me2 landscape and facilitates photodamage repair

“…Indeed, the dense constitutive heterochromatin structure hinders an efficient DNA repair process and thus requires decompaction (Schieferstein and Thomä, 1998; Liu, 2015). Alteration of chromatin structure occurs during several DNA repair processes and often results from a change in nucleosome density (Waters et al ., 2015; Tripuraneni et al ., 2021; Chakraborty et al ., 2021). Few emerging models proposed that different chromatin remodeling events facilitate DNA repair, including nucleosome PTM erasure (Palomera-Sanchez et al ., 2010; Jeon et al ., 2020), nucleosome sliding and eviction (Dinant et al ., 2012; Matsumoto et al ., 2019; Nodelman and Bowman, 2021; Chakraborty et al ., 2021).…”