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. 2006 Sep 1;20(17):2437-49.
doi: 10.1101/gad.1440206.

Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptation in response to DNA damage

Manolis Papamichos-Chronakis  1 , Jocelyn E Krebs, Craig L Peterson
Affiliations

Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptation in response to DNA damage

Manolis Papamichos-Chronakis et al. Genes Dev. .

Abstract

Ino80 and Swr1 are ATP-dependent chromatin remodeling enzymes that have been implicated in DNA repair. Here we show that Ino80 is required for cell cycle checkpoint adaptation in response to a persistent DNA double-strand break (DSB). The failure of cells lacking Ino80 to escape checkpoint arrest correlates with an inability to maintain high levels of histone H2AX phosphorylation and an increased incorporation of the Htz1p histone variant into chromatin surrounding the DSB. Inactivation of Swr1 eliminates this DNA damage-induced Htz1p incorporation and restores H2AX phosphorylation and checkpoint adaptation. We propose that Ino80 and Swr1 function antagonistically at chromatin surrounding a DSB, and that they regulate the incorporation of different histone H2A variants that can either promote or block cell cycle checkpoint adaptation.

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Figures

Figure 1.
Figure 1.
Ino80 does not play a major role in recombinational repair. The indicated yeast strains were grown in raffinose media, galactose was added for 45 min to induce HO expression, glucose was then added to repress HO expression, and genomic DNA was isolated at the indicated time points and analyzed either by real-time PCR (B,C) or by Southern blotting (D). (A) Schematic representation of chromosome III in yeast strain JKM154 (switching strain) bearing a GAL–HO gene and containing the HMLα homologous donor site. The arrow indicates the location of the HO cut site. (B) DSB occurs normally in the ino80 strain. Percent cut (%Cut) was measured as loss of the PCR product spanning the DSB (primers: MAT unique and DSB) and normalized to thePHO5 ORF region. (C) Kinetics of strand invasion and branch extension during switching. (Left) Strand invasion in the indicated strains was detected by PCR using the strand invasion primer that is 30 bp upstream of the HO recognition site atHMLα, and the MAT unique primer that is 428 bp downstream from the HO site atMAT. (Right) Branch extension was detected by PCR using the HMLα upstream primer that is 470 bp upstream of the HO recognition site at HMLα and the MAT unique primer. Values of the strand invasion and branch extension PCR products were normalized to the percentage of DSB formation and the PHO5 ORF region. (D) Kinetics of gene conversion. Southern blot analysis for the switched product was conducted for wild-type (WT) and ino80 strains. Genomic DNA was isolated at the indicated time points, digested with StyI, and the values of the MATα final product were quantified by PhosphorImager analysis and normalized to the initial DSB product and the MAT distal region.
Figure 2.
Figure 2.
Ino80 does not play a major role in nonhomologous end joining. (A) Schematic representation of chromosome III in yeast strain JKM179 (CY915, donorless) bearing a GAL–HO gene and deleted for the HMLα and HMRa homologous donor sites. Arrows indicate the location of the HO cut site and of the two primers used to detect it. (B) DSB occurs normally in the ino80 “donorless” strain. GAL–HO was induced by addition of galactose in the indicated strains, and genomic DNA was isolated at the indicated time points and analyzed by real-time PCR. Percent cut (% Cut) was measured as loss of the PCR product spanning the site of the break and normalized to the PHO5 ORF region. (C) Analysis of DSB formation and 5′ → 3′ resection in wild-type (WT), ino80, and mre11 strains. (Top) StyI restriction map at the MATα locus. (Bottom) GAL–HO was induced by addition of galactose in the indicated strains, and genomic DNA was isolated at the indicated time points, digested with StyI and analyzed by Southern blot using the DNA probe illustrated at the top. The ssDNA generated by 5′ → 3′ resection cannot be digested by StyI, leading to a gradual loss of the 0.7- and 2.2-kb bands. (D) Quantification of the 5′ → 3′ resection rate in the indicated strains. The values of the 0.7-kb product from C were quantified by PhosphorImager analysis and normalized to the URA3 locus and the 1.8-kb MATα product at 0 h. The normalized 1.8-kb MATα product value was arbitrarily set as 100. (E) Ino80 is not necessary for error-free NHEJ. Mid-log cells from wild-type (WT), ino80, and ku70 donorless strains were grown in raffinose media and plated in 10-fold dilutions in YPD plates with or without prior addition of galactose for3hto induce HO expression. Growth after galactose treatment requires NHEJ. Note that after 3 h, >90% of cells harbor a DSB (B; data not shown). (F) Analysis of error-prone NHEJ. Mid-log cells from the indicated strains were grown in raffinose media and plated in 10-fold dilutions on YPD or YP-Gal plates in order to induce constant HO expression. Under these conditions the cells can repair the break and form colonies only by ligating the DSB and mutating the HO cut site.
Figure 3.
Figure 3.
Ino80 is required for cell cycle checkpoint adaptation in response to a persistent DNA DSB. (A) Mid-log cells from wild-type (WT) and ino80 MATα donorless strains were spread on galactose plates to induceGAL–HO. G1-phase (unbudded) cells were congregated on the plates by micromanipulation. Morphology and growth of the cells were observed and pictures were taken at 20 h. (B)The Rad53p kinase is persistently active in the absence of INO80. In situ autophosphorylation assay was performed for TCA-extracted proteins from wild-type (WT) and ino80 donorless strains at the indicated times following DSB formation. Similar results were obtained from two independent experiments. (C–F) G1 cells (0 h) of the indicated donorless strains were micromanipulated onto galactose plates and their division was monitored at 8 and 24 h. Microcolonies with a number of cells/buds that equals 2 represent the percentage of cells that have arrested at G2/M, whereas colonies with more than two cells/ buds indicate cells that adapt and resume division. (C) Wild type (WT). (D) ino80. (E) rad17. (F) rad17 ino80. (G) The ATPase domain of INO80 is required for adaptation. G1 cells (0 h) of the indicated donorless strains carrying either the empty vector, a plasmid expressing wild-type INO80 (pINO80), or anATPase-defective allele of INO80 (pINO80-K737A) were micromanipulated onto galactose plates and monitored at 24 h. Analysis of cell number was performed as in B. (H) Ino80 is dispensable for SSA. (Top) Schematic representation of the SSA strain bearing a GAL–HO gene and deleted for the MAT, HMLα, and HMRa loci. The cells contain an HO cut site within the LEU2 gene (leu2::cs), and a fragment proximal to the cut site of the LEU2 gene has been inserted 30 kb distal. The galactose-induced DSB is repaired by SSA of the complementary LEU2 sequences, which are revealed as a result of the 5′ → 3′ resection of the DSB ends. (Bottom) Mid-log cells from the indicated SSA strains were grown in raffinose media and plated in 10-fold dilutions ontoYEPD and YEP-galactose plates. DSB occurred at equal levels in all strains as measured by quantitative PCR (data not shown; see also Fig. 4D, right panel). (I,J) G1 cells (0 h) of the indicated SSA strains were micromanipulated onto galactose plates and monitored at 8 and 24 h as in B. (I) rad52. (J) ino80 rad52.
Figure 4.
Figure 4.
Ino80 is required for high levels of DNA damage-induced H2AX phosphorylation. (A) Immunoblot analysis of H2AX-phos in wild-type (WT) and ino80 mutant cells exposed to the indicated amounts ofMMS, Phleo, and CPT for the indicated time. Acid-extracted proteins were separated by SDS page and analyzed with antiserum against the H2AX C-terminal phospho-peptide. In all Western blots, equal loading of the samples was confirmed by Ponceau-S staining of the membranes and verified byCoomassie staining of a parallel gel. The 10- to 20-kDa region of the respective Coomassie-stained gels is shown. We note that the levels of H2AX-phos are nearly equivalent in wild-type and ino80Δ cells if higher concentrations of MMS (0.2%–0.3%) are used, consistent with a previous study (Morrison et al. 2004). Wild-type and ino80 samples treated with the same DNA damaging agent were electrophoresed in the same gel and processed together. (B) Phosphorylation state of H2AX near the HO DSB is dependent on Ino80. ChIP analysis of H2AX-phos was conducted from asynchronous cultures of wild-type and ino80 donorless strains grown in galactose for the indicated times. Occupancy at 5 and 8 kb next to the HO break was measured by quantitative PCR. Values reflect the fold enrichment of the tested DNA relative to the H2AX-phos levels before HO induction (time 0) after correction for the ratios of amplification achieved using input DNA.
Figure 5.
Figure 5.
Ino80 regulates the Swr1-dependent deposition of Htz1p near a DSB. (A–D) ChIP analysis of Htz1 using polyclonal α-Htz1 antiserum was conducted in the wild-type (WT) and ino80 donorless strains grown in galactose for the indicated times. Occupancy over the 20-kb region next to the HO break was measured by quantitative PCR with primers corresponding to regions 0.75, 2.5, 5.0, 8.0, and 20 kb distal to the DSB. In C, primers were used corresponding to regions 30, 35, 45, and 50 kb distal to the DSB. Values reflect the fold enrichment of the tested DNA relative to an rDNA region (A–C) or to a region of the ASL1 ORF (D) after correction for the ratios of amplification achieved using input DNA and normalized to the percentage of DSB formation in each strain. (E) Recruitment of Htz1 requires H2AX S129. ChIP analysis of Htz1p was conducted in wild-type, ino80Δ, hta1-S129A hta2-S129A, and ino80Δ hta1-S129A hta2-S129A donorless strains grown in galactose for 2 h. Occupancy was measured as in D. (F) H2AX-phos and Htz1 occupy the region near the HO break in a reciprocal manner. ChIP analyses of H2AX-phos (left panel) and Htz1 (right panel) were conducted in the wild-type (WT) and ino80 donorless strains grown in galactose for the indicated time. Occupancy over the 8-kb region next to the HO break was measured by quantitative PCR with primers corresponding to regions 1.5, 2.5, 5.0, and 8.0 kb distal to the DSB. Analysis was performed as in D.
Figure 6.
Figure 6.
Inactivation of Swr1 restores H2AX-phos and checkpoint adaptation in the absence of Ino80. (A) Recruitment of Htz1 near the HO break is dependent on Swr1. ChIP analysis of Htz1p was conducted in ino80 and ino80 swr1 donorless strains grown in galactose for 2.0 h. Occupancy was measured by quantitative PCR using the same primers as in Figure 5D. Values are normalized to the percentage of DSB formation of each strain and reflect the ratio of the precipitated over the input DNA of each region. (B) Deletion of SWR1 restores the H2AX-phos levels near the HO DSB. ChIP analysis of H2AX-phos was conducted in the indicated donorless strains grown in galactose for 1.30 h. Occupancy was measured by quantitative PCR as in Figure 5D. (C,D) H2AX-phos is recovered in the ino80 htz1and ino80 swr1 double-mutant strains. (C) Immunoblot analysis of H2AX-phos in wild-type (WT), ino80, htz1, and ino80 htz1 cells exposed to 25 μM CPT for the indicated times. (D) Immunoblot analysis of H2AX-phos in wild-type (WT), ino80, swr1, and ino80 swr1 cells exposed to0.1% MMS for the indicated time. Analysis was performed as in Figure 4A. (E–G) Cell cycle checkpoint adaptation defect of the ino80 strain is alleviated by either a swr1 or htz1 deletion. G1 cells (0 h) of ino80 (E), swr1 ino80 (F), and htz1 ino80 (G) donorless strains were micromanipulated onto galactose plates and monitored at 8 and 24 h. Analysis of cell number was performed as in Figure 3B.
Figure 7.
Figure 7.
(A–F) Ino80 defines a novel checkpoint adaptation pathway. G1 cells (0 h) of the indicated donorless strains were micromanipulated onto galactose plates and their division was monitored at 8 and 24 h. Microcolonies with a number of cells/buds that equals 2 represent the percentage of cells that have arrested atG2/M, whereas colonies with more than two cells/buds indicate cells that adapt and resume division. (A) ino80. (B) mre11 ino80. (C) ku70. (D) ino80 ku70. (E) rad51. (F) rad51 swr1. (G) Proposed model for the role of Ino80 and Swr1 chromatin remodeling complexes during DNA damage. See the text for details.
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