Chromatin modification and remodeling during repair of a double‐strand break

Abstract

A single double‐strand break (DSB) can be induced synchronously in a population of budding yeast, allowing us to monitor the kinetics of repair and the dynamics of DNA damage‐induced chromatin modification. When a DSB is induced at the MAT locus, it can be repaired by recombining with the heterochromatic donor loci, HML or HMR. The two donors are in competition with each other, but surprisingly the donor choice is not changed when one of the donors is unsilenced. Nevertheless, there are substantial chromatin changes when the Rad51‐coated single‐stranded DNA at the resected end of MAT invades the heterochromatic donor. These changes can be seen by analysis of the protection of MNase‐resistance of nucleosome‐bound sequences. Although strand invasion and synapsis occur in cells lacking the Swi2/Snf2 homologue, Rad54, we find important differences in the alterations of nucleosome protection relative to wild type cells.A single DSB provokes modification of about 50 kb of chromatin around the DSB by phosphorylation of histone H2AX (γ‐H2AX) by ATM or ATR. Heterochromatic regions are refractory to modification but do not prevent spreading of γ‐H2AX beyond such regions. γ‐H2AX can be removed by 5′ to 3′ resection of the DSB ends (moving about 4 kb/hr) but also in a more rapid and concerted fashion when the DSB is repaired. Once liberated from chromatin γ‐H2AX is dephosphorylated by Pph3. The roles of various chromatin remodeling complexes in the removal of γ‐H2AX will be discussed. A second modification, independent of ATM/ATR kinases, is the casein kinase II‐dependent phosphorylation of histone H4‐S1. Unlike γ‐H2AX, γ‐H4 is not constitutively found near telomeres. The kinetics and extent of spreading of “γ‐H4” are markedly different from γ‐H2AX. Strikingly, γ‐H4 is found very close to the DSB site, even when these regions appear to be resected and single‐stranded. The loss of γ‐H4 when the DSB is repaired also exhibits different kinetics. The role of phosphatases and chromatin remodelers in γ‐H4 dynamics are under investigation.