Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks
2007; Springer Nature; Volume: 26; Issue: 18 Linguagem: Inglês
10.1038/sj.emboj.7601835
ISSN1460-2075
AutoresHaico van Attikum, Olivier Fritsch, Susan M. Gasser,
Tópico(s)Acute Lymphoblastic Leukemia research
ResumoArticle30 August 2007free access Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks Haico van Attikum Haico van Attikum Division of Epigenetics, Friedrich Miescher Institute for Biomedical Research, Basel, SwitzerlandPresent address: Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, Leiden 2333, The Netherlands Search for more papers by this author Olivier Fritsch Olivier Fritsch Division of Epigenetics, Friedrich Miescher Institute for Biomedical Research, Basel, SwitzerlandPresent address: Department of Biological Clinical Sciences, Centre for Biomedicine, University of Basel, Basel-4058, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Division of Epigenetics, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Haico van Attikum Haico van Attikum Division of Epigenetics, Friedrich Miescher Institute for Biomedical Research, Basel, SwitzerlandPresent address: Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, Leiden 2333, The Netherlands Search for more papers by this author Olivier Fritsch Olivier Fritsch Division of Epigenetics, Friedrich Miescher Institute for Biomedical Research, Basel, SwitzerlandPresent address: Department of Biological Clinical Sciences, Centre for Biomedicine, University of Basel, Basel-4058, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Division of Epigenetics, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Author Information Haico van Attikum1, Olivier Fritsch1 and Susan M Gasser 1 1Division of Epigenetics, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland *Corresponding author. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel-4058, Switzerland. Tel.: +41 61 697 7255; Fax: +41 61 697 3976; E-mail: [email protected] The EMBO Journal (2007)26:4113-4125https://doi.org/10.1038/sj.emboj.7601835 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (γH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, γH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation. Introduction Cells constantly encounter potentially harmful DNA lesions. DNA double-strand breaks (DSBs) pose the most severe risks for cell viability, since their inefficient or inaccurate repair can result in deleterious mutations, chromosomal translocations, cancer or cell death. Cells respond to DNA breaks by rapidly deploying a host of proteins to the site of damage. Some of these factors engage in DNA repair, while others trigger signaling pathways known as DNA damage checkpoints, which delay cell cycle progression until repair is complete. Two evolutionary conserved pathways mediate DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR starts with the processing of the DNA ends into single-stranded DNA (ssDNA), which is bound by proteins that invade and copy information from a homologous DNA duplex to repair the break in an error-free manner. In contrast, the alternative pathway of repair, NHEJ, requires little or no processing and involves either an error-free or error-prone mechanism for re-ligation of the ends (Khanna and Jackson, 2001; van Gent et al, 2001). In eukaryotes, genomic DNA is organized into a nucleoprotein structure called chromatin, whose basic unit, the nucleosome, consists of ∼147 bp of DNA wrapped around a protein octamer of histones H2A, H2B, H3 and H4. This structure generally reduces accessibility for enzymes involved in DNA-based cellular processes. To overcome this nucleosome barrier, cells possess histone-modifying enzymes and chromatin remodeling complexes (Marmorstein, 2001). These latter complexes use the energy of ATP hydrolysis to disrupt contacts between DNA and histones in order to reposition or remove nucleosomes, or exchange histone variants (Lusser and Kadonaga, 2003). A number of reports have demonstrated that both histone modifications and chromatin remodeling play important roles in controlling transcription. While histone modifications have long been recognized to be part of the cellular response to DNA damage, the importance of chromatin remodeling in DNA repair has only recently been addressed (Peterson and Cote, 2004; van Attikum and Gasser, 2005). One of the most rapid events that occurs in yeast in response to a DSB is the phosphorylation of a conserved serine residue (S129) near the C-terminus of histone H2A by ATM and ATR checkpoint kinases (scTel1 and scMec1, respectively; (Downs et al, 2000; Burma et al, 2001; Shroff et al, 2004)). In mammalian cells, their target is a variant histone, H2AX, producing γH2AX. While γH2AX occurs over megabases of chromatin surrounding DSBs (Rogakou et al, 1999), in yeast, H2A phosphorylation spreads over ∼50 kb (Shroff et al, 2004). For simplicity we will use the mammalian nomenclature γH2AX to describe the analogously modified yeast histone H2A. In budding yeast, mutation of the H2A S129 acceptor site leads to mildly elevated sensitivity to DNA damaging agents, and defects in DSB repair by NHEJ (Downs et al, 2000; Moore et al, 2006). Moreover, yeast γH2AX is required for the recruitment and loading of two types of protein complexes: Cohesin, which aids repair of DSBs through sister chromatid recombination (Strom et al, 2004; Unal et al, 2004), and chromatin remodeling complexes from the Snf2 superfamily (Downs et al, 2004; Morrison et al, 2004; van Attikum et al, 2004; Chai et al, 2005; Shim et al, 2005). Mutations in four members of the Snf2 superfamily of ATPases, Snf2, Sth1, Ino80 and Swr1, which are present in large multi-protein complexes called SWI/SNF, RSC, INO80 and SWR1, respectively, have been shown to result in hypersensitivity to a wide range of DNA damaging agents (Shen et al, 2000; Kobor et al, 2004; Mizuguchi et al, 2004; van Attikum et al, 2004; Chai et al, 2005; Shim et al, 2005). Although all four complexes also alter nucleosome structure and control transcription of large sets of genes (Ebbert et al, 1999; Shen et al, 2000; Saha et al, 2006), it is unlikely that their involvement in the DNA damage response stems solely from their role in transcription. Chromatin immunoprecipitation (ChIP) experiments revealed the direct binding of Snf2, Sth1 and Ino80 ATPases, along with other subunits, at an HO endonuclease-induced DSB at the yeast mating-type locus (Downs et al, 2004; Morrison et al, 2004; van Attikum et al, 2004; Chai et al, 2005; Shim et al, 2005). Amongst the proteins recruited to the DSB were unique subunits of the SWI/SNF2 (Snf5) and INO80 (Arp5 and Arp8) complexes, as well as shared subunits such as Arp4 and Rvb1 (Bird et al, 2002; Downs et al, 2004; Chai et al, 2005). Given that the yeast SWR1 complex shares Act1, Arp4, Rvb1 and Rvb2 subunits with the INO80 complex, it was unclear whether the SWR1 complex was recruited. Several laboratories have examined how chromatin remodeling alters nucleosomal structure at the site of damage. Mutations in RSC2 or STH1 of the RSC complex, or ARP8 of the INO80 complex, impair core histone loss near the HO-induced DSB (Tsukuda et al, 2005; Shim et al, 2007). Consistently, some authors detected a delayed conversion of double-stranded (ds) DNA into ssDNA (van Attikum et al, 2004; Shim et al, 2005, 2007). This suggested that chromatin remodeling by RSC and/or INO80 facilitates access to DNA ends for enzymes involved in end-processing and DSB repair. Indeed, downregulation of Sth1 was then shown to impair loading of yKu70, Mre11 and RPA and to delay the loading of Rad51 at DSBs (Shim et al, 2007). Arp8, on the other hand, was reported to facilitate Rad51, but not RPA loading (Tsukuda et al, 2005). It was not addressed whether INO80 regulates the binding of other repair proteins, such as yKu70 or Mre11. Despite strong similarities in the catalytic subunits, and the presence of shared components, the two yeast remodeling complexes, INO80 and SWR1, show distinct affinities for histone H2A variants: INO80 binds γH2AX uniquely, while SWR1 binds both γH2AX and H2A.Z, with a preference for H2A.Z (encoded by HTZ1 in budding yeast) (Morrison et al, 2004). SWR1 catalyzes the incorporation of H2A.Z into chromatin at promoters, centromeres and subtelomeric regions, with resulting effects on gene expression (Krogan et al, 2003; Kobor et al, 2004; Mizuguchi et al, 2004; Guillemette et al, 2005; Raisner et al, 2005). A recent report suggested that Swr1 could incorporate H2A.Z into chromatin proximal to an HO-induced DSB at MAT, yet only in the absence of Ino80 and when γH2AX levels were reduced (Papamichos-Chronakis et al, 2006). Here we have carefully examined the roles of INO80 and SWR1 with respect to H2A.Z deposition, nucleosome removal and downstream events at a DSB. We have established that both the Ino80 and Swr1 ATPases are recruited to DSBs at two different loci (MAT and PDR10). Surprisingly, Swr1 is not recruited to incorporate or remove H2A.Z. Rather, at DSBs H2A.Z, γH2AX and histone H3 are removed in an INO80-dependent, but SWR1-independent, manner. Efficient binding of the Mre11 nuclease, processing of the ends into ssDNA and recruitment of Mec1 kinase are all INO80-dependent. Loss of Swr1, on the other hand, affects yKu80 binding, but not Mre11 association or end-resection. Consistent with these phenotypes, we find that swr1 deletion selectively impairs error-free NHEJ events, while mutations in the INO80 complex reduce Mec1-dependent checkpoint activation. Results Ino80 and Swr1 are recruited to chromosomal DSBs Mating-type switching in budding yeast provides a well-controlled system to study events that occur at a specific DSB. During mating-type switching the HO endonuclease cleaves uniquely at the MAT locus, an event followed by efficient gene conversion requiring donor sequences at HML or HMR. In the absence of these donors, cells must repair the break by NHEJ to survive (Figure 1A). As mentioned above, ChIP studies have shown that the INO80 chromatin remodeling complex is recruited to the DSB at MAT, while the situation for SWR1 remained unclear (Downs et al, 2004; Morrison et al, 2004; van Attikum et al, 2004; Tsukuda et al, 2005). Equally unclear was whether or not the recruitment of nucleosome remodelers to breaks is locus-specific. Figure 1.Ino80 and Swr1 are recruited to chromosomal DNA DSBs. (A, B) Schematic representations of genes and rt–PCR primer sets on chromosome III (A) and XV (B) in the haploid yeast strain JKM179 and its derivative GA3632. Potential donor loci HML and HMR are deleted. The cleavage efficiency at MAT and PDR10 upon induction of HO is shown over time. (C) Absolute fold enrichment of Ino80-Myc and Swr1-HA ChIPs at the indicated distances from the HO site at MAT and PDR10 for strains without a DSB. Cells were grown in the presence of glucose to repress HO. (D) Relative fold enrichment of Ino80-Myc and Swr1-HA ChIPs at the indicated distances from the HO cut site at MAT, and the indicated times on galactose. The value at 0 h is arbitrarily set as 1. Cleavage efficiencies were 96, 98 and 99% (Ino80-Myc ChIP), and 89, 98, 99% (Swr1-HA ChIP) at 1, 2 and 4 h after HO induction, respectively. (E) As panel D, except that this shows Ino80-Myc and Swr1-HA ChIPs at the DSB in the PDR10 locus. Cleavage efficiencies were 93, 97 and 99% (Ino80-Myc ChIP), and 89, 98, 99% (Swr1-Myc ChIP). Download figure Download PowerPoint To test this, we generated a second yeast strain lacking the HO consensus at MAT, but with an HO cleavage site at the 3′ end of a non-essential gene, PDR10, located on chromosome XV. Real-time (rt)PCR probes were designed to monitor protein binding up to 23 kb from the cleavage site at MAT, and up to 4.4 kb from the cut site at PDR10 (Figure 1A and B). Importantly, ChIP experiments for Swr1-Myc and Ino80-Myc detected no significant binding of either remodeler at MAT or PDR10 when the galactose-inducible HO endonuclease in these strains was repressed by growth on glucose (Figure 1C), in contrast to an earlier report (Tsukuda et al, 2005). Following HO induction by the addition of galactose, cleavage is equally efficient at PDR10 and MAT (Figure 1B), and both remodelers are efficiently recruited to the induced DSBs (Figure 1D and E). Their binding increases over 4 h, yet the kinetics and efficiency of recruitment were locus-dependent. At MAT Ino80 peaked very close to the cut site, spreading weakly by 2–4 h after HO induction, whereas at PDR10 Ino80 was bound equally over the 5-kb region analyzed. Swr1, on the other hand, rapidly accumulated close to the cut site at both MAT and PDR10 (Figure 1D and E). We conclude that both INO80 and SWR1 are recruited to DSBs. INO80 and SWR1 recruitment to DSBs requires γH2AX Several studies examined the prerequisites for recruiting different chromatin remodeling complexes to DSBs. In the case of RSC, two rapidly recruited repair proteins, yKu70 and Mre11, were found necessary for the association of RSC with the break (Shim et al, 2005). Recruitment of Ino80, on the other hand, was reduced in the absence of H2A phosphorylation (Morrison et al, 2004; van Attikum et al, 2004). We next checked whether there was a similar effect on the recruitment of Swr1 in an H2A phospho-acceptor mutant. Using a double S129-to-stop mutation of the H2A phospho-acceptor residue, the efficiency of both Ino80 and Swr1 recruitment near the MAT DSB was reduced by 75–80% (hta1/2S129* mutant; Figure 2). Our results argue that these chromatin remodeling complexes are recruited following modification of γH2AX by either Tel1 or Mec1 kinase (Morrison et al, 2004; Shroff et al, 2004). Arp4, a subunit shared by INO80, SWR1 and NuA4 complexes, was shown to interact with yeast γH2AX, and is likely to be involved in the recruitment of these complexes (Downs et al, 2004), although Nhp10 was also implicated in a stable INO80–γH2AX interaction (Morrison et al, 2004). The kinetics of accumulation and distribution of SWR1 and INO80 near the DSBs at MAT and PDR10 differ, suggesting that chromatin context may modulate both binding and distribution. Nonetheless, γH2AX seems to function as a common recognition signal that triggers the recruitment of both SWR1 and INO80 to DSBs. Figure 2.H2AX phosphorylation at sites near a DSB at MAT is required for recruitment of INO80 and SWR1. (A) Relative fold enrichment of Ino80-Myc ChIP for WT and hta1/2S129* strains at the indicated distances from the HO cut site, and the indicated times on galactose. The enrichment is corrected for cleavage efficiencies, which were 96, 98 and 99% (WT), and 89, 98, 99% (hta1/2S129*). (B) As panel A, except that this shows ChIP for Swr1-HA. Cleavage efficiencies were 89, 98 and 99% (WT), and 78, 92, 95% (hta1/2S129*). Download figure Download PowerPoint The SWR1 complex does not incorporate H2A.Z. near DSBs The SWR1 complex catalyzes the incorporation of the histone variant H2A.Z into chromatin at promoters, centromeres and telomeres (Krogan et al, 2003; Kobor et al, 2004; Mizuguchi et al, 2004; Guillemette et al, 2005; Raisner et al, 2005). We therefore examined whether SWR1 was recruited to a chromosomal DSB to insert H2A.Z. We performed ChIP using an antibody against H2A.Z and probed for its presence near the HO site at MAT under conditions that either repress or induce the HO endonuclease. In the absence of cleavage, we detected a significant enrichment for H2A.Z at the MATalpha2 and BUD5 promoter regions (+0.6 and +1.6 kb, respectively), whereas little or no H2A.Z was detected in coding regions on the centromere proximal side of the HO consensus (+4.5, +9.6 and +23 kb, respectively; Figures 1A and 3A). Similarly, and consistent with earlier reports showing that promoters are generally enriched for H2A.Z (Krogan et al, 2003; Kobor et al, 2004; Mizuguchi et al, 2004; Guillemette et al, 2005; Raisner et al, 2005), we detected a significant enrichment for H2A.Z at the telomere-proximal PER1 promoter (+7.2 kb), but not in the TAF2 coding region (+0.9 and +1.9 kb) (Supplementary Figure 1). The presence of H2A.Z at these sites is SWR1-dependent: we found no detectable H2A.Z at MAT in the swr1 mutant (Figure 3A, and data not shown). Importantly, however, after 4 h of HO cleavage, there was no increase in H2A.Z levels at the DSB. Instead, the H2A.Z signal decreased to background level seen in the swr1 strain, suggesting that histone removal occurred (Figure 3A and Supplementary Figure 1). Thus, rather than H2A.Z recruitment, we scored an eviction of H2A.Z on both centromere- and telomere-proximal sides of the HO cut. Figure 3.H2A.Z is evicted from sites near a DSB at MAT and PDR10. (A) Absolute fold enrichment of H2A.Z ChIP at the indicated distances from the HO site at MAT for a WT strain with and without DSB, and a swr1 mutant without DSB is shown. Cells were grown in the presence of glucose to repress HO, or in the presence of galactose for 4 h to induce HO. Cleavage efficiency was 99% at 4 h after HO induction. (B) As panel A, except that this shows H2A.Z ChIP at the DSB in the PDR10 locus for a WT strain. Cleavage efficiency was 97%. (C) As panel A, except that this shows H2A.Z ChIP at MATinc for a WT strain. Cells were grown in the presence of glucose to repress HO or in the presence of galactose for 4 h to induce HO. On galactose no DSB induction at MATinc occurs. (D) As panel C, except that this shows histone H3 ChIP. Download figure Download PowerPoint In order to examine whether the break-induced eviction of H2A.Z was MAT-specific, we probed for H2A.Z near the HO consensus at the PDR10 locus. Again under conditions that repress HO cleavage, we scored a significant enrichment for H2A.Z at the SNC2 promoter (+4.5 kb), but not in the coding regions (+0.9 and +1.9 kb; Figure 3B). However, upon induction of the DSB, the H2A.Z signal at PDR10 dropped to near background levels (Figure 3B). We conclude that H2A.Z eviction is a general phenomenon at a DSB. To confirm that H2A.Z eviction is a direct consequence of HO cleavage, and not a reflection of transcriptional changes provoked by galactose, we monitored H2A.Z and histone H3 levels in a strain carrying an uncleavable HO site (MATinc). Indeed, when cells with MATinc were placed on galactose, H2A.Z levels increased slightly at +0.6 kb, whereas histone H3 levels remain unchanged (Figure 3C and D). The H2A.Z increase is found at a promoter, and may reflect transcriptional potentiation by galactose. We conclude that the observed loss of H2A.Z at MAT and PDR10 occurs exclusively in response to a DSB. Thus, Swr1 is not recruited to incorporate H2A.Z at promoters near a break. Instead, when present, H2A.Z disappears from chromatin with kinetics that mirror the recruitment of both SWR1 and INO80 remodelers. INO80, but not SWR1, is required for variant histone eviction at a DSB If SWR1 does not insert H2A.Z. at a DSB, then why is it recruited? The Swr1 complex is often considered equivalent to the mammalian and fly TIP60 complexes, which are implicated in the repair of DNA damage largely due to their ability to acetylate histone tails. However, unlike SWR1, TIP60 possesses both acetylation and ATPase activities. It is thought that the Drosophila TIP60 complex first binds to and acetylates phosphorylated histone H2Av, and then exchanges it for the unmodified form. Consequently, cells lacking a functional TIP60 complex accumulate phospho-H2Av following ionizing radiation (Kusch et al, 2004). We therefore examined whether either the INO80 or the SWR1 complex exchanges yeast γH2AX for unmodified H2A at sites of DNA damage. To do this, we tested the presence of γH2AX near the HO-induced DSB at MAT, comparing the wild-type (WT) situation with mutants lacking either Swr1 or the INO80 subunits Arp8 or Nhp10. By 1 h of HO induction in WT and swr1 strains, γH2AX has accumulated and spread from the DSB at MAT, peaking at +9.6 kb from the DSB (Figure 4A). This accumulation is slower in nhp10 and arp8 mutants, where it peaks at 2 h (Figure 4A and Supplementary Figure 2), suggesting that INO80 actually facilitates H2A phosphorylation (Papamichos-Chronakis et al, 2006). However, by 2–4 h of HO induction, the levels of γH2AX decreased significantly in WT and swr1 cells, but not in the nhp10 and arp8 mutants, indicating that γH2AX is removed from chromatin surrounding the DSB in an INO80-dependent, but SWR1-independent manner (Figure 4A and Supplementary Figure 2). Figure 4.INO80 is involved in γH2AX, H2A.Z and histone H3 eviction from sites near a DSB at MAT. (A) Relative fold enrichment of γH2AX ChIP at the indicated distances from the HO cut site at MAT, and the indicated times on galactose for WT, nhp10, and swr1 strains is shown as in Figure 1. Cleavage efficiencies were 90, 98 and 99% (WT), 44, 75 and 95% (nhp10), and 76, 91 and 96% (swr1). (B) As panel A, except that this shows H2A.Z ChIP for WT, nhp10 and hta1/2S129* strains. Cleavage efficiencies were 92, 97 and 99% (WT), 62, 83 and 94% (nhp10), and 88, 96 and 97% (hta1/2S129*). (C) As panel A, except that this shows histone H3 ChIP for WT, nhp10 and swr1 strains. Cleavage efficiencies were 89, 96 and 98% (WT), 46, 79 and 94% (nhp10), and 89, 96 and 98% (swr1). Download figure Download PowerPoint We showed in Figure 2A and Supplementary Figure 1 that H2A.Z is also evicted around the HO-induced cut at MAT in a WT strain. We next examined whether the INO80 complex is required for H2A.Z eviction, even though no association between INO80 and H2A.Z had been reported. Indeed, by 2–4 h after cleavage, H2A.Z was lost within a 5-kb region of MAT in WT, but not in nhp10 cells. Moreover, the hta1/2S129* phospho-acceptor mutant, which recruits INO80 inefficiently (Figure 2A), is similarly impaired for H2A.Z eviction (Figure 4B). While there is detectable H2A.Z at MAT in the nhp10 and hta1/2S129* phospho-acceptor mutants, there is none in a swr1 mutant (Figure 3A and Supplementary Figure 2), and HO-mediated cleavage does not alter this in swr1 cells (Supplementary Figure 2). We conclude that SWR1 neither inserts nor removes H2A.Z at a DSB, and that H2A.Z and γH2AX are both evicted near a DSB in an INO80-dependent manner. Loss of H2A.Z. and γH2AX reflects general nucleosome eviction To test whether the loss of H2A.Z and γH2AX near a DSB reflects general nucleosome eviction, we performed ChIP under similar conditions using an antibody that recognizes both modified and unmodified forms of histone H3. Upon HO induction, histone H3 levels decreased significantly by 2–4 h in both WT and swr1 cells, but not in the nhp10 mutant (Figure 4C). This mirrors results reported earlier for an arp8 mutant (Tsukuda et al, 2005). A significant decrease in histone H3 levels was also detected on the other side of the break in the TAF2 coding region (+0.9 and +1.6 kb), but not at the PER1 promoter region (+7.2 kb; Supplementary Figure 1). Despite the relative stability of histone H3 at the PER1 promoter, we scored a significant drop in H2A.Z levels, which may indicate that H2A.Z is selectively removed from this promoter to regulate a transcriptional response to the DSB (Supplementary Figure 1). In contrast, H2A.Z and histone H3 loss at TAF2 and MAT is cleavage-specific (Figure 3D). To examine whether the mechanisms and kinetics of histone eviction occur similarly at other cleavage sites, we induced the HO DSB at PDR10 and monitored H2A.Z and histone H3 levels by ChIP. H2A.Z levels significantly decreased within 4.5 kb of the DSB at PDR10, as early as 1 h after HO induction, and maximal displacement occurred by 4 h (Figure 5A). Histone H3 levels also decreased within a 4.5-kb region near this DSB, although with slightly slower kinetics (Figure 5B). Surprisingly, in both the nhp10 and swr1 mutants the H3 levels first significantly increased after 1 h on galactose, and only later decreased to reach WT levels of loss (Figure 5C). This increase was not seen in WT cells. Importantly, the absolute levels of histone H3 at PDR10, as at MAT, are not reduced in the nhp10 or swr1 mutant when compared with WT on glucose (Figure 5D), ruling out the possibility that the increase at 1 h in these mutants reflects a return to WT levels. We conclude that INO80, but not SWR1, is needed to evict both core histones and histone variants at MAT. In contrast, at PDR10 the absence of either complex results in a transient increase in histones near the break, although eviction eventually succeeds. Figure 5.H2A.Z and histone H3 are evicted from sites near a DSB at PDR10. (A) Relative fold enrichment of H2A.Z ChIP at the indicated distances from the HO cut site at PDR10, and the indicated times on galactose for a WT strain is shown as in Figure 1. Cleavage efficiencies were 84, 95 and 97%. (B) As panel A, except that this shows histone H3 ChIP. Cleavage efficiencies were 87, 92 and 94%. (C) As panel B, except that this shows histone H3 ChIP for nhp10 and swr1 strains. Cleavage efficiencies were 72, 88 and 91% (nhp10), and 89, 96 and 98% (swr1). (D) Absolute fold enrichment of histone H3 ChIP at the indicated distances from the HO site at MAT and PDR10 for WT, nhp10 and swr1 strains in the absence of a DSB. Cells were grown in the presence of glucose to repress HO. Download figure Download PowerPoint INO80 and SWR1 differently affect the binding of Mre11 and yKu80 to DSBs What is the outcome of nucleosome eviction on other events at DSBs? It has been proposed that the removal of histones may facilitate the binding of repair proteins, such as Mre11 and yKu80, to broken ends (Shim et al, 2007). To test the impact of histone removal on their binding, we monitored their presence at the HO-induced DSB at MAT in mutants deficient for either INO80 or SWR1 function. Consistent with published ChIP experiments, we monitored significant recruitment of Myc-tagged Mre11 and yKu80 by 1 h of HO induction, with signals peaking at 0.6 kb from the cleavage site (Figure 6A and B). If a similar ChIP was performed on a strain with no Myc epitope tag, we saw no precipitation of the induced DSB at MAT. If we monitored this in the arp8 mutant, we scored a 65% reduction in the efficiency of Mre11 recruitment, whereas it retained WT levels in the swr1 background (Figure 6A). In contrast, the efficiency of yKu80 recruitment was significantly reduced in both the arp8 and swr1 strains (by 70–85%; Figure 6B). Thus, both INO80 and SWR1 facilitate the binding of yKu80, yet only INO80 affects the binding of Mre11. Since INO80, but not SWR1, removes nucleosomes at the DSB, we propose that INO80-mediated histone eviction specifically facilitates the binding of Mre11. Figure 6.Effect of ARP8, NHP10 and SWR1 deletion on Mre11 and yKu80 recruitment, end-resection and non-homologous end-joining. (A) Absolute fold enrichment of Mre11–Myc ChIP at the indicated distances from the HO cut site at MAT at 0 and 1 h on galactose. The enrichment is corrected for cleavage efficiencies, which were 86% (WT), 84% (arp8), 86% (swr1) and 96% (WT no tag). (B) As panel A, except that this shows ChIP for yKu80–Myc. Cleavage efficiencies were 98% (WT), 85% (arp8), 90% (swr1), and 96% (WT no tag). (C) Schematic representation of the QAOS assay (see text). DNA template molecule, generated using primer HO1-ss, is quantified by rt–PCR using primers HO1-f and Tag. (D) QAOS assay for ssDNA formed at the HO1 site at the indicated times on galactose in WT, arp8, nhp10 and swr1 strains. The amount of ssDNA is corrected for cleavage efficiencies, which were 92, 97, 99% (WT), 56, 78, 85% (arp8), 48, 79, 93% (nhp10) and 77, 90, 96% (swr1). (E) Error-prone NHEJ repair of a DSB at MAT is slightly reduced in mutants in INO80, but not SWR1. Repair of the DSB at MAT can only occur by NHEJ. Error-prone NHEJ was monitored by scoring survival of the indicated strains after plating serial 10-fold dilutions on medium with either 2% glucose or galactose. (F) Error-free NHEJ repair of a DSB at MAT is reduced in swr1, but not in arp8 or nhp10 mutants. NHEJ proficiency was determined by rt–PCR, using primers that span the DSB at MAT or anneal to the SMC2 control locus. HO was repressed (t=0) and induced for 1 h (t=1). After 1 h, cells expressing HO were shifted to a glucose medium and incubated for 2 h (t=3) to allow error-free NHEJ. Error-free end-joining efficiency was calculated from PCR cyc
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