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.

Fig. 1.

H2A S129 phosphorylation is required for G1 and S phase checkpoint delays. (A) Arrest defect in H2A-S129A after DNA damage in G1. αf-arrested cells were irradiated with 300 Gy, released into fresh media, removed at 5- min intervals, mixed with αf/noc trapping media, and incubated for an additional 90 min. The percentage of G1 cells was assayed by counting buds under phase microscopy. (B) tel1Δ, H2A-S129A and tel1Δ H2A-S129A share a G1 checkpoint defect. After αf arrest and 300 Gy IR, cells were released into fresh media, removed at 15-min intervals, and analyzed by flow cytometry. (C) Intra-S phase checkpoint defect in H2A-S129A cells. αf-arrested cells were treated with 0.03% methyl methane sulfonate for 30 min before release into fresh media and analyzed as in B.

Fig. 2.

G1-specific loss of Rad9 phosphorylation and recruitment and Rad53 activity in H2A-S129A cells. (A) WT and H2A-S129A strains expressing Rad9–13Myc were synchronized in either G1 with αf (Upper) or G2/M with noc (Lower) and irradiated and arrest maintained for the duration of the experiment. Aliquots were subjected to Western blot analysis to detect the mobility shift of Rad9. (B) WT and H2A-S129A strains expressing Rad53–13Myc were synchronized in G1 with αf and irradiated and arrest maintained for the duration of the experiment. Immunoprecipitates (Upper) were analyzed by Western blotting, and kinase assays (Lower) were analyzed by PhosphorImager analysis. (C) Loss of Rad9-HA recruitment to an HO-induced DSB in G1 in H2A S129 mutant. WT, dot1Δ, H2A-S129*, and H2A-S129* dot1Δ cells carrying Rad9-HA were synchronized in G1 with αf and incubated in galactose for 45 min to induce HO. Chromatin immunoprecipitation was used to determine Rad9-HA occupancy 60 bp (HO site), 1.5 kb (HO + 1.5 kb), and 10 kb (HO + 10 kb) from the HO-cutting site and at a large intergenic region on chromosome V (control). Occupancy (IP/input) is shown as ratio of IP to input PCR signals after galactose induction, with background subtraction using an anti-Myc control antibody. Glucose controls showed no change over background levels (data not shown). IP, immunoprecipitate.

Fig. 3.

NuA4, but not Swr or Ino80 complexes, affect G1 checkpoint persistence. (A) G1 checkpoint persistence in NuA4-deficient cells. WT, yng2, and yng2 rad9Δ cells were αf-arrested, irradiated with 300 Gy, and released into fresh media. Aliquots were removed at 5-min intervals, mixed with αf/noc trapping media, and incubated for an additional 90 min. The percentage of G1 cells was assayed by counting buds under phase microscopy. (B) Intact G1 checkpoint in the absence of SWR1 or INO80 activity. swr1 and arp8 mutants were analyzed as in A. (C) Prolonged G1 delay in ARP4 mutants cells after IR. WT, arp4–12, and arp4–12 rad9Δ cells were analyzed as in A.