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 Tripuraneni December 6, 2019 2 observed the re-deposition and precise re-positioning of nucleosomes at the originally-occupied positions. This re-establishment of the pre-lesion chromatin landscape suggests that a DNA replication-independent mechanism exists in G1 cells to preserve epigenome organization following DSB repair.

Genome-wide sequencing analysis of Sgs1, Exo1, Rad51, and Srs2 in DNA repair by homologous recombination

“…However, mre11D cells still generate an asymmetric short-range resection profile, indicating that this event is independent of MRX activity. In agreement with this observation, loss of nucleosome occupancy and incorporation of repair factors is faster at the left side of the HO break in an Mre11-independent manner (Tripuraneni et al, 2021). Contrary to this initial stage, long-range resection proceeds symmetrically and at a constant rate (Figure 4E), a feature that can be attributed to the canonical DNA damage-dependent exonucleases Sgs1/Dna2 and Exo1.…”

“…Accordingly, cell viability in response to galactose was reduced to 35% (Figure S1D). In line with recent findings on nucleosome occupancy at an induced HO break (Tripuraneni et al, 2021), a mre11D mutant displayed a similar asymmetric short-range resection profile to control cells (Figure S11B), indicating that short-range resection is MRX independent. However, long-range resection was significantly affected, as denoted by the symmetrical reduction of the long-range coverage profiles (Figure S11C), with averaged values of 1.4 kb/h and 2.2 kb/h to the left and right sides from the HO site, respectively (Figure S11D).…”

Linking the dynamics of chromatin occupancy and transcription with predictive models

“…Some chromatin statistics require more spatial precision than occupancy provides, for example when determining a factor’s position or organization. In these cases, we used cross-correlation scores in a similar manner to that described in Tripuraneni et al (2019). Around each gene’s TSS, a per-bp cross-correlation score was computed to smooth positional variation and filter out non-relevant fragments.…”

“…A cross-correlation was computed in a similar manner described in (Tripuraneni et al 2019). Around each gene's TSS, a per bp cross-correlation score was computed to smooth the positional variation and filter out non-relevant fragments.…”

“…Digestion by a nuclease, such as micrococcal nuclease (MNase), provides a complementary perspective to understand chromatin occupancy as it can probe accessibility at base-pair precision. Recent genome-wide mapping studies have used nucleosome-sized MNase-seq fragments to characterize the dynamics of nucleosomes under various conditions, including the cell cycle (Nocetti and Whitehouse 2016), DNA damage (Tripuraneni et al 2019), and heat shock (Teves and Henikoff 2011). Additionally, studies have attempted to understand the roles of the smaller DNA-bound factors that correspond to subnucleosomal MNase-seq fragments (Belsky et al 2015; Brahma and Henikoff 2019; Chereji et al 2017; Henikoff et al 2011; Kubik et al 2017; Ramachandran et al 2017; Teves and Henikoff 2011).…”