Interrogating Chromatin Dynamics Surrounding a DNA Double-Strand Break and Ensuing Non-Homologous End-Joining Mediated Repair

Abstract

The DNA double-strand break (DSB) is one of the most toxic genomic lesions that can occur in any living cell. Failure to repair DSBs results in cell cycle arrest and ultimately programmed cell death, while improper repair can lead to profound alterations or loss of genomic information through translocations, inversions, deletions and other genomic aberrations. 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. I tested several site-specific DSB induction systems and found that that the HO endonuclease was able to rapidly and synchronously induce a site-specific DSB in Saccharomyces cerevisiae upstream of the PHO5 locus. This region of the genome is recognized for its chromatin organization, which is comprised of well-positioned nucleosomes. Utilizing MNase digestion of chromatin followed by paired-end fragment sequencing I was able to interrogate 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 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. I also examined the temporal dynamics of non-homologous end joining (NHEJ) mediated repair in a G1-arrested population. Concomitant with DSB repair, I 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.