Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation

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Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation Geraldine W.-L. Toh 1 , Aisling M. O’Shaughnessy 1 , Sonia Jimeno, Ian M. Dobbie, Muriel Grenon, Stefano Maffini, Anne O’Rorke, Noel F. Lowndes ∗ Genome Stability Laboratory, Department of Biochemistry and National Centre for Biomedical Engineering Science, National University of Ireland, University Road, Galway, Ireland

a r t i c l e

i n f o

a b s t r a c t

Article history:

In budding yeast, the Rad9 protein is an important player in the maintenance of genomic

Received 17 November 2005

integrity and has a well-characterised role in DNA damage checkpoint activation. Recently,

Received in revised form 6 March

roles for different post-translational histone modifications in the DNA damage response,

2006

including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been

Accepted 7 March 2006

demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs

Published on line 2 May 2006

specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophospho-

Keywords:

rylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated,

RAD9

checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase

Saccharomyces cerevisiae

and two independently regulated histone modifications, H2A phosphorylation and Dot1-

DNA repair

dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52

Histone modification

repair foci. These results, together with the observation that rad9
cells are defective in repair of IR breaks in G2 , strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Eukaryotic cells have evolved sophisticated ‘checkpoint’ or signal transduction pathways that respond to DNA damage by controlling cell cycle progression, transcriptional induction of damage response genes and DNA repair, thus helping to maintain genomic integrity [1–3]. Rad9 is an important player in the DNA damage response in Saccharomyces cerevisiae, being required for efficient DNA damage checkpoint signalling in all stages of the cell cycle. While checkpoint pathways are highly conserved in eukaryotes, there is no single obvious

∗

homologue of RAD9 in vertebrate cells. However, a number of proteins, including NFBD1/MDC1, 53BP1, BRCA1, have been identified as possible Rad9 homologues [4–6]. The Rad9 protein has a known biochemical role in DNA damage checkpoint signal transduction. Rad9 activates the downstream checkpoint kinase Rad53 by facilitating its Mec1-dependent phosphorylation and catalysing its in trans autophosphorylation [7,8]. A requirement for Rad9 in maintaining genome stability is reflected in the increased rates of spontaneous chromosome loss and rearrangement seen in rad9
cells [9–11]. However, the functions of Rad9 in other processes that contribute to

Corresponding author. Tel.: +353 91 492706/495504; fax: +353 91 512504. E-mail address: [email protected] (N.F. Lowndes). 1 Contributed equally to this work. 1568-7864/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2006.03.005

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genome stability, including DNA repair, remain poorly understood. There is increasing evidence that chromatin remodelling activities and post-translational modification of chromatin components, including histones, may influence damagedependent checkpoint signalling and DNA repair. Recent studies in yeast and mouse cells indicate a role for a DNA damage-induced histone modification termed ␥-H2AX (␥-H2A in yeast) in promoting sister chromatid-based repair of DSBs [12–14]. ␥-H2AX is generated by ATM/ATR-dependent phosphorylation of histone H2AX serine 139 (yeast H2A serine 129) and is rapidly induced in response to DNA DSBs. H2AX phosphorylation results in megabase regions of ␥-H2AX around each DSB (up to 100 kb in yeast), thus ‘marking’ a large domain of chromatin surrounding a lesion [13,15]. Another histone modification, methylation of histone H3 on lysine 79 (H3 K79Me), has been linked to DNA damage checkpoint signalling in yeast and human cells. In budding yeast, Dot1-dependent methylation of histone H3 on lysine 79 was reported to be required for efficient checkpoint signalling, although the role of this modification in checkpoint regulation in higher eukaryotic cells has not yet been determined [16–18]. In human cells, methylation of H3 K79 by the histone methyltransferase DOT1L is proposed to mediate recruitment of the checkpoint signalling protein 53BP1 to DSBs via its Tudor domain [17]. Rad9, the putative yeast orthologue of h53BP1, also contains a conserved Tudor motif that is required for efficient interaction of Rad9 with H3 K79Me in vitro [17]. In this study, we show that Rad9 is recruited to foci and bulk chromatin in response to ␥-irradiation. Strikingly, this stable Rad9 recruitment occurs only after down-regulation of checkpoint signalling, as measured by Rad53 phosphorylation. Consistent with a post checkpoint activation role, it is the hypophosphorylated form of Rad9 that is retained on chromatin. In addition, our results indicate that two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation, are non-redundantly required for Rad9 accumulation in foci after IR. Focal colocalisation of Rad9 and Rad52 in repair foci in G2 cells indicates that Rad9 is recruited to a subset of lesions undergoing repair by HR. Moreover, rad9
cells are defective in G2 phase repair of IR-induced DSBs. Taken together, our results support a novel role for Rad9 in DNA repair that is distinct from its function in checkpoint signalling and Rad53 activation.

2.

Materials and methods

2.1.

Yeast strains and plasmids

All strains used in this study are in the W303 background (MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3 can1-100 rad5535). See Supplementary Table 1 for strain genotypes. Rad9 was C-terminally tagged with GFP using plasmid pJK1 (a gift from D. Toczyski). A PCR-based integration approach [19] was used to C-terminally tag Ddc2 with GFP(S65T) or with 3xHA. To generate pML4-mRFP, an mRFP-encoding PacI–AscI fragment was PCR-amplified from plasmid pRSETB-mRFP1 (a gift from R. Tsien) and used to replace the PacI–AscI GFP-encoding fragment of pML4 (pFA6a-GFPS65T-kanMX6; a gift from M.

Longtine) [19]. pML4-mRFP was then used with appropriate primers to generate a PCR fragment for C-terminal mRFP tagging of Rad52. dotl
mutant strains were generated using either a PCR-based integration approach [20] or an adaptamermediated PCR method [21]. Integrating plasmids YIp-RAD97xA HA and YIp-RAD9-HA (a gift from K. Sugimoto) were used to generate rad9-7xA and isogenic WT strains.

2.2.

Western blotting

Immunoblot analysis was performed as previously described [22]. Rad9 and Rad53 were detected using polyclonal sera NLO5 and NLO16. Other proteins were detected using antibodies to Orc6 (a gift from J. Diffley), Rad52 (a gift from R. Rothstein) and alpha-tubulin (B512, Sigma). 3xHA-tagged Ddc2 was detected using 12CA5 monoclonal antibody.

2.3. Cell cycle arrests, checkpoint experiments and
-irradiation Cell cycle arrests and checkpoint experiments were performed as previously described [23–26]. ␥-irradiation was carried out using a 137 Cs source at a dose-rate of 23.5 Gy/min (Mainance Engineering, UK).

2.4.

Live cell imaging and fluorescence microscopy

Cells were harvested for microscopy and washed 5 times with saline pre-warmed to 30 ◦ C before mounting in low melting point agarose. Live cell images were captured using an OrcaER camera (Hamamatsu) mounted on a Zeiss Axiovert S100 microscope (Carl Zeiss) enclosed in a temperature-controlled box at 30 ◦ C, with a PlanApo 63× 1.4 NA objective, excitation and emission filter wheels (Sutter), transmission and fluorescence illumination shutters (Uniblitz) controlled by a PC running AQM software (Kinetic Imaging). For each field of cells, DIC and fluorescence images were taken in 15 Z-positions at 0.3 ␮m intervals. Fluorescence illumination was with an HBO 100 lamp, using either a single GFP filter set (excitation 470/40 nm, dichroic 440/80, emission 525/50, Omega Optical) or a dual GFP/RFP dichroic with separate excitation/emission filters (excitation 485/15 and 575/25, dichroic 490/575, emission 525/50 and 615/45, Omega Optical). Images were prepared for publication in Photoshop (Adobe). To minimise the effect of lamp variability on the measurements, experiments involving WT and mutant strains were paired. Colocalisation of Rad9 and Rad52 foci was confirmed by measuring the 3-D distance between the background-subtracted centres of gravity of the focal intensity (3-D distance <0.9 ␮m), using ImageJ software with plugin Measure Sync 3D (W.S. Rasband, US National Institutes of Health, Bethesda, Maryland, http://rsb.info.nih.gov/ij/).

2.5.

Chromatin fractionation

Experiments were performed as previously described [27], with minor modifications. Spheroplasts were prepared using oxalyticase (Enzogenetics), washed, resuspended in lysis buffer with protease and phosphatase inhibitors and lysed by addition of Triton X-100 to 0.5%. The chromatin-enriched

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fraction (Chr) was separated from the soluble fraction (Sol) by centrifugation for 15 min at 15,000 × g through 30% sucrose. Successful fractionation was confirmed by probing fractions with antibodies against alpha-tubulin and Orc6 as controls for soluble and chromatin-bound fractions, respectively.

2.6.

PFGE analysis of DSB repair

Cells were arrested in G2 and irradiated on ice (800 Gy; doserate 23.5 Gy/min). Samples were collected directly before and after irradiation and at intervals over the subsequent 3 h period, during which nocodazole arrest was maintained. Yeast chromosome plugs were prepared as previously described [28] and subjected to pulsed field gel electrophoresis in 1% SeaPlaque GTG agarose (BioWhittaker Molecular Applications) for 30 h at 110 V, switch time ramped from 50 to 150 s, in a Gene Navigator system (Amersham Biosciences). Gels were stained with ethidium bromide and visualised using a ChemiImager digital imaging system (Alpha Innotech). Intensity of intact and broken chromosomal DNA was quantitated using Multi Gauge software (Fuji) and used to determine the fraction of DNA in intact chromosomes recovered during repair. Fraction of intact DNA (intensity within chromosomal bands as a fraction of total DNA intensity below the well) was plotted against time post-irradiation. Signals were normalised to the total amount of DNA present and expressed as a fraction of that in the unirradiated sample (−0), which was set to 1.

3.

Results

3.1. Kinetics of Rad9 retention at DSBs undergoing repair by HR To directly examine Rad9 recruitment to sites of DNA damage in live cells, we used a carboxy-terminal Rad9-GFP fusion protein, expressed from the Rad9 chromosomal locus under its endogenous promoter. The fusion protein showed wild-type expression levels and functionality in DNA damage sensitivity and checkpoint assays, as well as damage-induced Rad9 and Rad53 phosphorylation (data not shown). In asynchronously growing cells, 60 min after 200 Gy of ␥-irradiation, a dose which causes 10–20 DNA double-strand breaks per yeast cell [29], we observed Rad9-GFP foci in live cells against a diffuse nuclear background signal (Fig. 1A). See Supplementary Fig. S1 for additional images of Rad9-GFP foci in wild-type cells after IR. Note that the presence of non-focal Rad9 nuclear intensity indicates that only a fraction of total cellular Rad9 becomes stably retained in foci. The Ddc2 checkpoint protein, similarly tagged with GFP, was included as a control. Unlike Rad9-GFP, Ddc2-GFP forms bright foci with little or no diffuse nuclear signal, suggesting that most of the Ddc2-GFP protein in the cell becomes recruited to these foci. See Supplementary Fig. S1 for representative images of IR-induced Ddc2-GFP foci. We then analysed the kinetics of IR-induced Rad9-GFP and Ddc2-GFP focus formation in G1 and G2 . In alpha-factor arrested cells treated with 200 Gy IR, Ddc2-GFP foci were observed by 30 min post-irradiation in 70–80% of cells. In contrast, no Rad9-GFP foci were observed in G1- arrested

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cells (Fig. 1B). In nocodazole-arrested cells, Rad9 foci were detectable from 30 min after DNA damage was induced by 200 Gy IR and reached a maximum (25% of cells) 90 min after irradiation (Fig. 1C). This was considerably later than for Ddc2 foci, which peaked at 30–60 min post-irradiation (70–80% of cells contained Ddc2 foci). For Ddc2, but not Rad9, appearance of foci correlated with checkpoint signalling, as indicated by Rad53 phosphorylation at the relevant time-points (Figs. 1C and 2B and data not shown). While the number of Ddc2 foci peaked at a time when Rad53 is clearly hyperphosphorylated, the number of Rad9 foci reached a maximum only after loss of Rad53 hyperphosphorylation, indicating downregulation of checkpoint signalling. In budding yeast, DSBs are primarily repaired by homologous recombination (HR), which is active during the G2 phase of the cell cycle [30,31] and requires members of the RAD52 epistasis group [32]. To determine if Rad9 localises to the vicinity of DSBs undergoing repair by HR, cells co-expressing wild-type levels of C-terminally tagged Rad52-RFP and Rad9GFP were G2 -arrested and Rad9/Rad52 focal co-localisation was assessed 90 min after 200 Gy IR (Fig. 1D). As the presence of diffuse Rad9 background nuclear intensity surrounding the Rad9 foci complicates analysis of Rad9/Rad52 colocalisation, we used numerical criteria for assessing co-localisation of the ‘centres of gravity’, or maximum intensity, of Rad9 and Rad52 foci in cells after background subtraction of nonfocal intensity, as detailed in Materials and Methods. Rad52 foci were observed in 72% of cells, whereas Rad9 foci were observed in 31% of cells. In all cases, cells that contained a Rad9 focus also contained a co-localised Rad52 focus. We observed no cells that contained Rad9 but not Rad52 foci. Similar results were obtained with asynchronously growing cultures (data not shown). Our data suggest that focal recruitment of Rad9 to a subset of Rad52-containing repair foci occurs only at late time points after irradiation. A specific role for Rad9 in repair of DSBs by HR is further supported by the observation that Rad9 and Rad52 foci are not formed in G1 cells after IR, nor in G2 cells after UV irradiation (data not shown), neither of which results in DSB repair by HR.

3.2. Kinetics of chromatin retention of hypophosphorylated Rad9 after ionising radiation We have also studied the recruitment of different checkpoint and DNA repair proteins to damaged chromatin using a biochemical fractionation assay [27]. After irradiation in G2 , cells were spheroplasted, lysed and separated into soluble and chromatin-enriched fractions by centrifugation through a 30% sucrose cushion (Fig. 2A, left panel). Analysis of total protein (Fig. 2A, middle panel) and DNA (Fig. 2A, right panel), as well as controls for the soluble and chromatin-enriched fractions, using antibodies to alpha tubulin and Orc6, respectively, indicated effective fractionation. Damage-induced Rad9 hyperphosphorylation is Mec1/ Tel1-dependent [22,33]. In WT cells, Rad9 was retained on chromatin from 30 to 90 min after irradiation, reaching its maximum at 60 min post-IR (Fig. 2B). Although hyperphosphorylated Rad9 could be detected in the total spheroplast extract and in the soluble fraction at these time points,

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Fig. 1 – Rad9-GFP focus formation after IR. (A) Representative images of Rad9 foci in live RAD9-GFP cells 60 min after irradiation with 200 Gy. This IR dose is predicted to result in 10–20 double strand breaks (DSBs) per yeast cell, equivalent to a dose of 0.3 Gy to human cells [29]. Scale bar, 5 ␮m. (B) Analysis of Rad9-GFP and Ddc2-GFP focus formation in alpha-factor arrested RAD9-GFP and DDC2-GFP cells, respectively, after 200 Gy IR. Ddc2-GFP – solid circles. ≥100 cells were analysed per time point; n = 2. Note that no Rad9-GFP foci were observed in G1 -arrested cells. Arrest was maintained throughout the experiment. (C) Analysis of Rad9-GFP and Ddc2-GFP focus formation in nocodazole-arrested RAD9-GFP and DDC2-GFP cells, respectively, after 200 Gy. Rad9 – solid squares (mean ± S.E.M., n = 3); Ddc2 – solid circles (n = 2). ≥100 cells were analysed per time point. Nocodazole arrest was maintained throughout the experiment. (D) Colocalisation of Rad9 and Rad52 foci in nocodazole-arrested cells 90 min after irradiation (200 Gy). Colocalisation was confirmed by measuring the 3-D distance between the background-subtracted centres of gravity of foci intensity, as described in Materials and Methods, with a cut-off distance for co-localisation of <0.9 ␮m.

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Fig. 2 – Chromatin retention of hypophosphorylated Rad9 after IR requires Rad9 SQ/TQ motifs. (A) Left panel, schematic of the fractionation of total yeast spheroplast extract (Tot) into soluble (Sol) and chromatin-enriched (Chr) fractions. Middle panel, electrophoretic analysis of fractionated proteins. Right panel, electrophoretic analysis of fractionated DNA. The low molecular weight band visible in the total and soluble fractions is a small endogenous yeast plasmid that does not cofractionate with large chromosomal DNA. (B) Western blot analysis of total, soluble and chromatin-enriched fractions from nocodazole-arrested WT cells before (−0) and at 30, 60, 90 and 180 min after irradiation (200 Gy). Fractions were probed using antibodies directed against the indicated proteins. 5× volume equivalents of ‘Chr’ (relative to ‘Sol’ and ‘Tot’) fractions were analysed. (C) Western blot analysis of Rad9 in ‘Chr’ fractions from G2 -arrested WT and rad9-7xA cells after irradiation (200 Gy).

interestingly, it was the hypophosphorylated form of Rad9 that was found in the chromatin-enriched fraction. Rad53, detected only in the total spheroplast extract and in the soluble fraction, had largely returned to the hypophosphorylated state at 60 min, indicating down-regulation of checkpoint signalling. Ddc2 was also detectable in the chromatin fraction between 30–90 min, but in this case the hyperphosphorylated form was retained on chromatin (Fig. 2B). Thus, protein accumulation in damage-induced foci correlates with increased chromatin association. We also examined the fractionation of Rad52; this protein was significantly depleted from the soluble fraction at 30 and 60 min

and correspondingly appeared in the chromatin fraction at these time points. At 90 min Rad52 had largely returned to the soluble fraction and was no longer detectable in the chromatin fraction. Significant Rad52 chromatin retention at 30 and 60 min suggests that the bulk of DSB repair in these cells occurs between 30 and 60 min post-irradiation (Fig. 2B). Thus, hypophosphorylated Rad9 is associated with damaged chromatin at time points when DSB repair by HR is ongoing, with maximal Rad9 association correlating with late stages of Rad52 association, as well as with downregulation of checkpoint signalling, as judged by loss of Rad53 hyperphosphorylation.

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3.3. Chromatin retention of hypophosphorylated Rad9 requires a central cluster of Rad9 SQ/TQ motifs Rad9 undergoes Mec1/Tel1-dependent hyperphosphorylation after DNA damage [22,33]. To determine whether Mec1/Tel1dependent hyperphosphorylation of Rad9 is required for its recruitment, we examined Rad9 chromatin retention after IR in a rad9-7xA strain. This strain expresses a mutant Rad9 protein in which seven SQE/TQE motifs redundantly required for

damage-induced Rad9 hyperphosphorylation are mutated to AQE motifs [34]. rad9-7xA cells are defective in Rad9 and Rad53 hyperphosphorylation after UV irradiation and MMS treatment and to a single DSB induced by HO endonuclease cleavage [34,35], as well as to IR (data not shown). In G2 -arrested rad9-7xA cells, the mutant Rad9 protein could not be detected in the chromatin fraction after irradiation (Fig. 2C). It should be noted that rad9-7xA cells display significantly less sensitivity to a range of DNA damaging agents than rad9
cells ([34];

Fig. 3 – Rad9 focal recruitment requires H2A phosphorylation and Dot1 histone methyltransferase. (A) Asynchronously growing cultures of the indicated strains were arrested with nocodazole and irradiated (200 Gy). Cells containing one or more foci were counted after 1 h and plotted as a percentage of the total population. (B) Representative images of Rad9-GFP localisation in WT and dot1
and h2a-S129A mutant cells before and after irradiation (200 Gy). No IR-induced Rad9-GFP foci were observed in dot1
and h2a-S129A mutant cells.

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A.O’S. & G.T., unpublished observations), indicating that most RAD9 functions are unaffected by the seven alanine substitutions. Thus, although the form of Rad9 retained on chromatin after IR is non-hyperphosphorylated, prior Rad9 hyperphosphorylation may be required for its subsequent chromatin retention.

3.4. Rad9 recruitment to foci requires H2A phosphorylation and the Dot1 histone methyltransferase We next examined the genetic dependency of Rad9 focus formation in G2 cells 60 min after irradiation with 200 Gy in a number of mutant backgrounds. Rad9-GFP foci were detected in tel1
, rad24
, mec3
and rad53
mutants identically to wild-type, whereas mecl
and mec1-81 mutant cells did not form Rad9-GFP foci (Fig. 3A). Rad9-GFP focus formation was also abolished in h2a-S129A cells, which carry a serine 129 to alanine (S129A) mutation in the C-terminus of histone H2A [29], and in cells lacking the histone methyltransferase Dot1, whose only known target is H3 lysine 79 (Fig. 3A and B). Thus, the formation of Rad9-GFP foci specifically required functional Mec1 kinase activity but, intriguingly, not the related PI3K-like kinase Tel1, although this kinase is also able to hyperphosphorylate Rad9 [22]. Confirming the previous observations, the PCNA-like and RFC-like checkpoint complexes and the checkpoint signalling kinase Rad53 were also not required for Rad9-GFP focus formation [36]. However, Rad9-GFP focus formation specifically required H2A serine 129, as well as the histone methyltransferase Dot1, which

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methylates histone H3 lysine 79. In contrast to Rad9, Ddc2-GFP focus formation occurs in both G1 and G2 (Fig. 1B and C; [36]), is not specific to IR and does not require H2A serine 129 nor Dot1dependent methylation. We observed no qualitative or kinetic differences in Ddc2-GFP focus formation between wild-type, h2a-S129
or dot1
mutant cells (M.G. et al., manuscript in preparation). To clarify the nature of the Rad9 function associated with its stable accumulation in foci and chromatin after irradiation, we investigated DNA damage sensitivity and checkpoint signalling in strains lacking either or both H2A S129 and Dot1 (Fig. 4). Confirming previous observations, we observed only mild IR sensitivity in h2a-S129A cells [29], and slightly greater IR sensitivity in dot1
mutant cells [16,18], as compared to rad9
, mecl
sm11
, or rad52
cells (Fig. 4A). Combining the dot1
and h2a-S129A mutations did not result in significantly increased IR sensitivity, suggesting that Dot1 and H2A S129 operate in at least one common pathway for promoting IR survival (Fig. 4A). Rad9 hyperphosphorylation after irradiation was wild-type in the h2a-S129A, dot1
and h2a-S129A dot1
mutants. A slight but reproducible reduction in Rad53 hyperphosphorylation after IR treatment was observed in dot1
and dot1
h2a-S129A cells, relative to WT cells. This could indicate that Dot1 is partially required for checkpoint activation in G2 . In contrast, the h2a-S129A mutant shows wild-type Rad53 phosphorylation after IR treatment (Fig. 4B), consistent with H2A phosphorylation having little or no role in G2 checkpoint activation in budding yeast. Thus, lack of Rad9 focal accumulation does not appear to affect

Fig. 4 – DNA damage sensitivity and checkpoint signalling in h2a-S129A and dot1
mutant strains. (A) Survival of WT, dot1
, h2a-S129A and dot1
h2a-S129A strains after ␥-irradiation. 10-fold serial dilutions from exponentially growing cultures of the indicated strains were plated onto YEPD and either mock-treated (Control) or treated with the indicated doses of IR. (B) Western blot analysis of Rad9 and Rad53 in nocodazole-arrested cultures of WT, dot1
, h2a-S129A and dot1
h2a-S129A strains before (0) and at indicated times after irradiation (400 Gy).

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its phosphorylation status, since Rad9 hyperphosphorylation is wild-type in dot1
, h2a-S129A and dot1
h2a-S129A cells, nor does it necessarily cause a defect in Rad53 phosphorylation, since this occurs in h2a-S129A cells identically to wild-type.

3.5. Rad9 is required for efficient genome repair of IR damage in G2 The observed kinetics and specificity (IR but not UV, and G2 but not G1 cells) of Rad9 accumulation in foci and chromatin, as well as the co-localisation with Rad52 at sites of repair, suggest a repair-related role for Rad9 in responding to DSBs. Indeed, radiation sensitivity and genome instability phenotypes have been observed in rad9
cells [11]. These defects have been attributed primarily to defective RAD9-dependent G2 /M cell cycle arrest [9,10]. To determine whether Rad9 has roles in G2 phase repair that are distinct from its function in checkpointdependent cell cycle arrest, we directly monitored repair of chromosomal DSBs induced by IR in nocodazole-arrested cells

by pulsed field gel electrophoresis (PFGE). Yeast chromosomes from nocodazole-arrested WT, rad9
and rad52
cells were analysed by PFGE before and at intervals after treatment with a dose of IR expected to produce 40–80 DSBs per cell. Intact chromosomes from unirradiated cells show a characteristic banding pattern, whereas after irradiation a low molecular weight smear reflects extensive chromosomal fragmentation (Fig. 5A). We found that in WT cells, intact chromosome bands reappeared by 30 min and by 60–90 min cells had substantially repaired their chromosomes, as indicated by the reappearance of the highest molecular weight DNA bands (Fig. 5A). The rad9
and rad52
mutants showed persistence of fragmented chromosomes during the remainder of the experiment, indicating a significant DSB repair defect in both strains (Fig. 5B). We also observed a modest repair defect, less than that observed in rad9
cells, in cells lacking Dot1 and H2A S129 (Fig. 5C and D). This defect is consistent with the slight IR sensitivity observed in the dot1
, h2a-S129A and dot1
h2a-S129A strains and supports the conclusion that Rad9 focal recruitment, which requires H2A phosphoryla-

Fig. 5 – Rad9 is required for efficient genome repair in G2 . (A) PFGE analysis of DSB repair during G2 /M in WT, rad9
and rad52
cells. Exponentially growing cultures of the indicated strains were nocodazole-arrested and irradiated on ice (800 Gy at a dose-rate of 23.5 Gy/min). This dose of IR is estimated to result in approximately 40–80 DSBs per cell. Samples were collected immediately before and after irradiation at indicated intervals over the subsequent 3 h period, during which nocodazole arrest was maintained. Yeast DNA plugs were then prepared and analysed by PFGE. (B) Time course for recovery of intact chromosomal DNA during repair in WT, rad9
and rad52
cells. Fraction of intact DNA (intensity within chromosomal bands as a fraction of total DNA intensity below the well) was plotted against time post-irradiation. Signals were normalised to the total amount of DNA present and expressed as a fraction of that in the unirradiated sample (−0), which was set to 1. (C) PFGE analysis of DSB repair during G2 /M in WT, rad9
and dot1
h2a-S129A cells, as in (A). (D) Time course for recovery of intact chromosomal DNA during repair in WT, rad9
and dot1
h2a-S129A cells, as in (B).

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tion and Dot1-dependent H3 methylation, is involved in DSB repair.

4.

Discussion

The Rad9 protein is required for an effective cellular response to diverse types of DNA damage in yeast. IR treatment of cells in G2 , but not G1 , induced the relocalisation of Rad9-GFP to nuclear foci, whereas Ddc2-GFP formed foci after irradiation in both G1 and G2 (Fig. 1B and C). Surprisingly, Rad9-GFP focal accumulation in G2 occurred significantly later than Ddc2-GFP (Fig. 1C). The peak of Ddc2-GFP focal accumulation occurred 0.5 h after irradiation, correlating with active checkpoint signalling, while that of Rad9 was significantly shifted to later time points, peaking at approximately 1.5 h (Fig. 1C). Retention of Rad9 on damaged chromatin was also observed after IR treatment and its kinetic broadly resembled that of Rad9 focus formation (Fig. 2B). Similar to the nuclear foci assay, Rad9 chromatin retention also occurred later than for Ddc2. Moreover, both Rad9 foci and chromatin retention correlate with down-regulation of checkpoint signalling, as measured by loss of Rad53 hyperphosphorylation (Figs. 1C and 2B). Note that the kinetics of Chk1 dephosphorylation under these conditions was similar to that of Rad53 (data not shown). Rad9 chromatin retention also corresponded to later time points of Rad52 retention (Fig. 2B) and recovery of intact chromosomes after ␥-irradiation (Fig. 5A). Thus, both live-cell microscopy and biochemical fractionation strongly suggest a role for Rad9 after its involvement in the initial phase of checkpoint activation, that correlates with later stages of bulk genome repair after IR in G2 . Although not detectable by live-cell microscopy, initial Rad9 recruitment leading to checkpoint activation must occur at least transiently in both G1 and G2 . It is possible that stable Rad9 retention at lesions may occur through a similar mechanism to this initial recruitment, although this later accumulation of Rad9 in foci is observed only in G2 . Strikingly, it is the hypophosphorylated form of Rad9 that is specifically associated with damaged chromatin, even though both hyperphosphorylated and hypophosphorylated Rad9 are present in cells when this association occurs (Fig. 2B). In rad9-7xA cells, Rad9 hyperphosphorylation after IR was abolished and chromatin retention of Rad9 could not be detected (Fig. 2C). This result is consistent with a previous report that Rad9 recruitment to a HO-induced DSB requires these phosphorylation motifs [35]. However, a more trivial possibility, that the 7xA mutant Rad9 protein, although retaining much of the function of the wild-type protein ([34]; A. O’S. and G. T., unpublished observations) may have specifically lost its DSB-binding ability, irrespective of its phosphorylation status, cannot be excluded. In addition, although Mec1 and Tel1 are redundant for hyperphosphorylation of Rad9 after IR in G2 -arrested cells ([22] and M.G., unpublished results), Rad9 accumulation in foci specifically requires Mec1 kinase activity (Fig. 3 A). Thus, some aspect of Mec1 regulation or activity that is distinct from Tel1, perhaps its recruitment to ssDNA or other structures not recognised by Tel1, is specifically required for this Rad9 function. A role for Rad9 in repair of IR-induced lesions is consistent with the observation that Rad9 foci always co-localise with a

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subset of Rad52 foci; Rad9 foci are not observed in cells that do not also contain a Rad52 focus (Fig. 1D). In addition, Rad9 focus formation does not occur in G1 , when homologous recombination is not active [30,31], although Rad9-dependent checkpoint functions are intact in this cell cycle phase [22,37]. Furthermore, Rad9 foci are specific to IR, which causes DSBs, and are not detected after UV irradiation of cells in G2 , a treatment that does not directly induce DSBs. It may be that Rad9 foci represent ‘complex’ or difficult to repair lesions that require stable Rad9 retention, whereas the majority of IR-induced breaks are more rapidly repaired. Intriguingly, genetic experiments in fission yeast indicate a role for Crb2, the S. pombe orthologue of Rad9, at late stages of recombinational repair of DSBs in G2 cells. Crb2 was reported to control a late stage of recombination by regulating the activity of topoisomerase III (Top3) and its associated RecQlikeDNA helicase, Rqh1 [38], which may be required for proper processing of a subset of lesions undergoing HR repair. The possibility that Rad9 might directly participate in the processing of recombination intermediates is also consistent with the observations that Rad9 has both checkpoint-dependent and checkpoint-independent roles in the formation of ssDNA following DNA damage near telomeres [39,40]. In this work, we demonstrate that Rad9 focal recruitment requires both Mec1-dependent H2A phosphorylation and Dot 1-dependent H3 methylation (Fig. 3). Histone H3 Lys 79 is the only known target of the Dot1 histone methyltransferase and appears to be evolutionarily conserved [41,42]. With respect to IR sensitivity, there is an epistatic relationship between DOT1 and the methylation status of H3 lysine 79 [43]. Thus, it is likely that Rad9 focus formation specifically requires H3 K79 methylation, in addition to H2A S129 phosphorylation. These two histone modifications are independently regulated; H3 K79 methylation is wild-type in h2a-S129A cells and damage-induced ␥-H2A formation is wild-type in dot1
cells (S.J., unpublished observations). The cell cycle phase and DSB specificity of Rad9 focal accumulation, and its dependency on multiple events, including Mec1-dependent phosphorylation and both H2A phosphorylation and Dot 1-dependent H3 methylation, is striking. In particular, the requirement for these independent chromatin modifications suggests that the chromatin environment around a DNA lesion may be particularly important in determining Rad9 retention. As one of the modifications appears to be constitutively present throughout the entire genome within the core nucleosome [44], this further suggests that remodelling of chromatin structure might be required to allow stable recruitment of Rad9. Indeed, damage-induced recruitment of ATP-dependent chromatin remodelling complexes was recently shown to be dependent on ␥-H2A and to be involved in promoting repair of DNA DSBs in yeast [45–48]. This is consistent with a scenario in which the formation of ␥-H2A at DSBs results in the recruitment of chromatin remodelling complexes as well as other activities. Chromatin remodelling could unmask the normally hidden H3-K79Me mark, thereby permitting stable accumulation of Rad9. Once stably retained in repair foci, Rad9 could mediate the recruitment of other activities required for the efficient and accurate repair of ‘complex’ DNA lesions. It is also worth noting that stable focal recruitment of 53BP1, the proposed vertebrate orthologue of Rad9, requires both H3 methylation

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and H2AX phosphorylation, although 53BP1 does not appear to interact directly with ␥-H2AX [17,49–51]. Interestingly, recent data suggests that the role of 53BP1 in DSB repair may be linked to NHEJ rather than HR ([52]; S. Takeda, pers. commun.). It is possible that during evolution, not only have the functions of RAD9 become distributed amongst the ‘RAD9-like’ genes of vertebrate cells, but that additional functions have been acquired, reflecting the greater complexity of these cells. Our results indicate a novel function for Rad9 in responding to DSBs that is distinct from its role in checkpoint signalling. This function involves stable recruitment of Rad9 late in the DSB response, after initial checkpoint activation, and may be important for efficient repair of a subset of IR-induced DNA lesions.

Acknowledgements We thank W. M. Bonner, J. Diffley, M. Longtine, R. Rothstein, K. Sugimoto, D. Toczyski and R. Tsien for generous gifts of yeast strains, plasmids and antibodies; S. Takeda for communicating results prior to publication. We thank C. Morrison and members of the Genome Stability Laboratory, in particular R. Bree, for comments and suggestions. This material is based upon work supported by the Irish Higher Education Authority (HEA) Programme for Research in Third Level Institutions (PRTLI3), by a Health Research Board (HRB) Programme grant, and by Cancer Research Ireland under grant CR105GRE. Support from an EMBO Long-Term Fellowship (to S.J.), from EU contract number Meif-ct-2003-501230 (to I. M. D.) and from the EU-IP6 “RISC-RAD” and “DNA Repair” projects is also gratefully acknowledged.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.dnarep.2006.03.005.

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