Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling

Abstract

Recent studies of yeast G1 DNA damage response have identified characteristic changes in chromatin adjacent to double-strand breaks (DSBs). Histone H2A (yeast H2AX) is rapidly phosphorylated on S129 by the kinase Tel1 (ATM) over a domain extending kilobases from the DSB. The adaptor protein Rad9 (53BP1) is recruited to this chromatin domain through binding of its tudor domains to histone H3 diMe-K79. Multisite phosphorylation of Rad9 by Mec1 (ATR) then activates the signaling kinase Rad53 (CHK2) to induce a delay in G1. Here, we report a previously undescribed role for Tel1 in G1 checkpoint response and show that H2A is the likely phosphorylation target, in a much as S129 mutation to Ala confers defects in G1 checkpoint arrest, Rad9 phosphorylation, and Rad53 activation. Importantly, Rad9 fails to bind chromatin adjacent to DSBs in H2A-S129A mutants. Previous work showed that H2A phosphorylation allows binding of NuA4, SWR, and INO80 chromatin remodeling complexes, perhaps exposing H3 diMe-K79. Yet, mutants lacking SWR or INO80 remain checkpoint competent, whereas loss of NuA4-dependent histone acetylation leads to G1 checkpoint persistence, suggesting that H2A phosphorylation promotes two independent events, rapid Rad9 recruitment to DSBs and subsequent remodeling by NuA4, SWR, and INO80.H2AX ͉ histone ͉ Rad9 F ailure to repair even one DNA double-strand break (DSB) can be lethal. Surviving cells may display aneuploidy or other chromosome defects predisposing to cancer (1-3). Eukaryotic cells remain poised to respond to DSBs at any point in the cell cycle. DNA damage checkpoint and repair factors are rapidly recruited to the break site to form characteristic DNA damage foci, leading to significant reorganization of chromatin structure (4, 5). In metazoans, phosphorylation of the histone variant H2AX at a conserved carboxyl-terminal SQEL motif by the PIKK-related kinases ATM, ATR, and͞or DNA-PK (6, 7) is required for normal recruitment and͞or retention of signaling and repair factors such as MDC1, 53BP1, and NBS1 to DNA damage foci (8-10). H2AX deficiency confers DNA damage sensitivity and genomic instability (11)(12)(13)(14).When yeast HO endonuclease is induced in G1, the Mre11͞ Rad50͞Xrs2 complex rapidly accumulates at the HO break site, followed by the ATM kinase paralog Tel1, which can then phosphorylate the yeast core histone H2A at S129 to form a chromatin domain extending up to 20 kb on either side of the break site (15). S129 phosphorylation is necessary for binding of chromatin remodeling complexes and cohesins in asynchronous cells (16)(17)(18)(19)(20). However, in as much as cohesins and partners for homologous exchange are absent before the onset of DNA replication, the function of H2A S129 phosphorylation in G1 remains obscure.Activation of the DNA damage checkpoint in G1 results in rapid phosphorylation of the yeast 53BP1 ortholog and checkpoint adaptor protein Rad9 and subsequent binding and recruitment of the checkpoint effector kinase Rad53, which then transautophosphorylates and becomes active...

The Dot1 Histone Methyltransferase and the Rad9 Checkpoint Adaptor Contribute to Cohesin-Dependent Double-Strand Break Repair by Sister Chromatid Recombination in Saccharomyces cerevisiae

“…We show that gH2A and H3K79 methylation contribute to SCR through the same mechanism, consistent with previous studies reporting overlapping functions for these histone modifications in the DNA damage response ( Javaheri et al 2006;Toh et al 2006). We also demostrate that, like gH2A, Dot1 and Rad9 promote DSB-induced loading of cohesin onto chromatin.…”

“…However, a ''late'' role for Rad9 in DSB repair by HR, distinct from its ''early'' checkpoint function on Rad53 activation, has been proposed because Rad9 chromatin retention and colocalization with a subset of Rad52 foci correlate with checkpoint signaling downregulation and reconstitution of intact chromosomes after IR treatment (Toh et al 2006). Moreover, the impaired G2/M checkpoint arrest of the rad9 mutant cannot solely explain the defect in SCR, because the dot1 and H2A-S129 mutants are quite proficient at DNA-damageinduced G2/M arrest (Wysocki et al 2005;Javaheri et al 2006), but they display similar SCR defects.…”

“…gH2A contributes to SCR: gH2A is necessary for accumulation of Rad9 at IR-induced foci in G2 (Toh et al 2006) and, together with Dot1, promotes chromatin association of Rad9 at DSB sites in G1 cells ( Javaheri et al 2006;Hammet et al 2007). gH2A also mediates recruitment of cohesin to DSB sites (Unal et al 2004), which is involved in repair by SCR (Cortes-Ledesma and Aguilera 2006).…”

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

“…The time required to progress from G 1 to S was also highly variable among mutant strains in our hands, leading to inconsistencies in the fraction of cells in S at the time of UV irradiation. Furthermore, in most strains, UV irradiation caused G 1 arrest in a proportion of the cell population, as reported previously (44,45). To circumvent these confounding problems associated specifically with UV treatment, we decided to induce replicative stress using 4-nitroquinoline 1-oxide (4NQO), a UV-mimetic agent that generates helix-distorting DNA adducts removed by NER.…”

Histone modifications in DNA damage response

“…et al, 2002;Xie et al, 2004). Additionally, although H2AX is dispensable for the constitution of the primary DNA damage signal and the initial recruitment of repair factors, it is critical for the accumulation and retention of Rad50, Rad51, BRCA1 (breast cancer 1), MDC1 (mediator of DNA damage checkpoint 1) in human cells and 53BP1 homolog in yeast in response to DNA damage (Celeste et al, 2003b;Javaheri et al, 2006;Nakamura et al, 2004;Paull et al, 2000;Stucki et al, 2005). Moreover, H2AX does not affect chromatin organization in the initial stage of DNA damage recognition and signaling (Fink et al, 2007).…”