While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.

Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.

Budding yeast cells suffering a single unrepaired double-strand break (DSB) trigger the Mec1 (ATR)-dependent DNA damage response that causes them to arrest before anaphase for 12-15 h. Here we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both Esp1 and Pds1. Transient relocalization of Pds1 is also seen in wild-type cells lacking vacuolar protease activity after induction of a DSB. Arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes. Permanent arrest can be overcome by blocking autophagy, by deleting the vacuolar protease Prb1, or by driving Esp1 into the nucleus with a SV40 nuclear localization signal. Autophagy in response to DNA damage can be induced in three different ways: by deleting the Golgi-associated retrograde protein complex (GARP), by adding rapamycin, or by overexpression of a dominant ATG13-8SA mutation.adaptation | separase | securin | cell cycle arrest I n the presence of a single unrepairable DNA double-strand break (DSB), Saccharomyces cerevisiae arrest is due to Mec1 checkpoint kinase-dependent cell cycle arrest, but cells eventually escape from the arrest and reenter the cell cycle in a process termed adaptation (1-6). Arrest before adaptation typically lasts 12-15 h, a time equivalent to five to six normal cell cycles. Adaptation is accompanied by the loss of checkpoint-induced hyperphosphorylation of checkpoint kinases Rad53 and Chk1 and the loss of association with damaged DNA of the Mec1 (ATR) kinaseassociated Ddc2 (ATRIP), despite the persistence of DNA damage (7,8). Several proteins are required for adaptation, including those with known roles in DNA repair (Yku70, Yku80, Rdh54/ Tid1, Rad51, Srs2, Sae2, Fun30, and Sgs1), as well as proteins that are required to turn the checkpoint off (the PP2C phosphatases Ptc2 and Ptc3 and casein kinase II) and the Polo kinase Cdc5, which plays several roles in regulating mitosis (5,6,(9)(10)(11)(12)(13)(14).When DSB repair is relatively rapid, as during HO endonuclease-induced mating-type (MAT) switching, there is no cell cycle arrest or Rad53 phosphorylation (7), although Mec1 and Tel1 kinases rapidly phosphorylate histone H2A (15, 16). However, when DSB repair is slower, e.g., during ectopic homologous recombination or long-distance single-strand annealing, the DNA damage checkpoint is fully activated and G2/M arrest is maintained for several hours until repair is effected and cells reenter the cell cycle in a process termed recovery (12, 17). Recovery is an active process that requires the functions of the Srs2 helicase and also the casein kinase II and PP2C phosphatases to dephosphorylate checkpoint kinases (11-13). None of the other adaptation mutants tested (yku70Δ, rdh54Δ, rad51Δ, sae2Δ, cdc5-ad, and fun30Δ) is deficient in recovery (12, 14) although a number of double mutants are recovery defective (3). Analysis of checkpoint recovery in human cells has shown a requirement ...