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Previous Article | Next Article 
Molecular and Cellular Biology, March 2006, p. 1839-1849, Vol. 26, No. 5
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.5.1839-1849.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
and
Torsten Krude*
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom
Received 24 October 2005/ Returned for modification 20 November 2005/ Accepted 13 December 2005
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ABSTRACT |
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INTRODUCTION |
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For an efficient DNA damage response, not only must the DNA damage be repaired, but the surrounding chromatin must be accessed, remodeled, and restored as well (reviewed in references 13 and 36). Chromosomal DNA is compacted through association with histone proteins into arrays of nucleosomes, which further condense into high-order chromatin fibers. Histone modifications encode epigenetic information, which constitutes an essential property of the chromatin fiber. An efficient DNA damage response in eukaryotic cells therefore relies on DNA damage recognition, access, and repair within chromatin as well as on the successful reestablishment of chromatin structure and information following DNA repair. These requirements are contextualized in the access-repair-restore model (15).
NER is associated with temporary yet extensive rearrangements of chromatin structure (43), even though sites of DNA repair synthesis are confined to short patches of 
30 nucleotides (9, 16). The molecular mechanism by which nucleosome formation de novo is linked to NER was investigated in cell-free systems (11, 12). NER was initiated from a uniquely placed bulky cisplatin-DNA adduct on a circular plasmid, and the assembly of new nucleosomes was found to initiate at this site and to propagate bidirectionally from it, even when DNA synthesis was inhibited (12). This process created a regular nucleosomal array extending well beyond the initiation site. The propagation of nucleosomal arrays was shown to depend on the presence of DNA single-strand breaks (SSBs) and gaps formed as intermediates during DNA damage processing (31). These lesions recruited proliferating cell nuclear antigen (PCNA) and chromatin assembly factor 1 (CAF-1) in an ATP-dependent manner. CAF-1 is a histone chaperone that targets acetylated histones H3 and H4 to sites of DNA synthesis during DNA replication and NER as the first step of nucleosome assembly (45; reviewed in references 15 and 22). PCNA recruits the p150 subunit of CAF-1 to the DNA template through direct intermolecular interactions (31, 42). Nucleosome assembly on UV-damaged DNA depends on both PCNA and CAF-1, arguing that recruitment of these two proteins to sites of DNA damage repair is an essential step for chromatin assembly during NER. Furthermore, PCNA and CAF-1 are recruited locally to intranuclear sites of UV-induced DNA damage in vivo (14, 34).
In contrast, the factors that mediate chromatin dynamics during the NHEJ and HR pathways of repairing DNA double-strand breaks (DSBs) have not been identified. DNA DSBs can be generated globally by exposure to ionizing radiation or radiomimetic chemicals such as bleocin (a bleomycin compound). They can also arise as by-products of oxidative metabolism or by replication of damaged DNA. If misrepaired, DSBs have the potential to lead to chromosome translocations, genomic instability, and, in higher eukaryotes, cancer predisposition and cell death (18).
The NHEJ pathway predominates in quiescent cells, which represent most cells in an adult organism, and in the G1 phase of the proliferative cell division cycle. It is an inherently inaccurate process, often causing the loss of nucleotides from the site of the DNA break. The DNA repair process is mediated by a conserved set of cellular proteins that in vertebrates include DNA-PKcs, Ku, Artemis, DNA ligase IV, and XRCC4 (reviewed in references 18 and 26). A heterodimer of Ku70 and Ku80 subunits binds with a very high affinity to the ends of double-stranded DNA, independent of the sequence or precise structure of the DNA ends. It associates with the 470-kDa catalytic subunit, DNA-PKcs, which is involved in bringing two opposing DNA ends together into synapsis. This is followed by nucleolytic processing of DNA ends to remove 3' and 5' overhangs. Finally, resealing of the DNA ends is catalyzed by XRCC4/DNA ligase IV within the complex.
An early step in the response of vertebrate cells to the formation of DSBs is the local phosphorylation of histone variant H2AX (
-H2AX) by ATM/ATR protein kinases, which can extend megabases away from the break site (40, 41). -H2AX forms large intranuclear foci that can be detected by immunofluorescence microscopy. Subsequently, many proteins involved in NHEJ are recruited to these -H2AX foci (13, 36), including replication protein A (RPA) and PCNA (3, 4). A recent report (25) has shown that caf1 asf1 double mutants in yeast are 10-fold less competent in repairing DSBs via recombination and also exhibit 3- to 4-fold decreased repair proficiency and accuracy in NHEJ-mediated repair. These findings thus suggest the involvement of a chromatin assembly factor in the physiological response to DSBs.
We therefore examined the role of CAF-1 in the response of human cells to DSBs, focusing on quiescent cells to exclude replication- and recombination-associated repair pathways. We found that the expression of CAF-1 and PCNA is induced in quiescent cells upon the generation of DSBs in a DNA-PK-dependent manner. These proteins are recruited to damaged chromatin undergoing DNA repair of single- and double-stranded DNA breaks by the BER and NHEJ pathways, respectively. These results support a novel role for CAF-1 in the response of quiescent human cells to DNA strand breaks.
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MATERIALS AND METHODS |
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DNA-damaging agents.
Asynchronously proliferating and quiescent cells cultured in low-glucose Dulbecco's modified Eagle's medium were treated with bleocin (Calbiochem, United Kingdom) without prior permeabilization. Irradiation was performed using a Faxitron cabinet X-ray system (model 43855D; Faxitron X-Ray Corporation) at a dose rate of 1.15 Gy/min for a total dose of 5 Gy.
Immunofluorescence staining and Western blot analysis.
Immunofluorescence microscopy was performed as detailed previously (46). Primary antibodies against the following epitopes were used for this study: XRCC1 (AHP832; Serotec), XRCC4 (AB145; Abcam), Rad51 (rabbit polyclonal PC130; Oncogene), -H2AX (mouse monoclonal; Upstate Technologies), PCNA (mouse monoclonal PC-10; Cancer Research UK), p60 (pAb1) (29), RPA (pAb-RPA1) (46), and p150 (SS-1) (44). Immunofluorescence detection of PCNA was carried out as specified previously (30). For Western blotting, equal amounts of protein samples were loaded in denaturing sodium dodecyl sulfate-polyacrylamide gels, blotted, and identified using primary antibodies as detailed above.
Silencing of CAF-1 and cell viability assays. The protocol for small interfering RNA (siRNA)-mediated silencing of p60 and the colorimetric assessment of cell viability were performed exactly as described previously (33). For trypan blue staining, cells were trypsinized and mixed with an equal volume of 0.8 mM trypan blue solution diluted in phosphate-buffered saline (Sigma). Two microliters of this mixture was then transferred to a hemocytometer, and 200 cells per sample were counted and scored for viability.
Pulsed-field gel electrophoresis analysis. Isolated nuclei (300,000) were encapsulated in a 0.5% low-melting-point agarose block, incubated overnight at 55°C in 500 µl lysis buffer (50 mM Tris, pH 8.0, 50 mM EDTA, pH 8.0, 2% sarcosyl, 2 mg of proteinase K per ml of buffer), and washed twice with 1 ml of Tris-EDTA at room temperature for 10 min. Electrophoresis of the prepared samples was performed using the Gene Navigator system (Amersham Biosciences). The electrophoretic conditions were as follows: 195 V; north-south switch time, 40 s; east-west switch time, 40 s; run time, 19 h; angle, 120°; and temperature, 9°C. After electrophoresis, gels were stained with SYBR gold (Molecular Probes).
Partial purification of CAF-1 and nucleosome assembly reactions. cDNA strand synthesis and nucleosome assembly reactions were performed as described elsewhere (23). Nuclear extracts were prepared as detailed previously (33). For partial purification of CAF-1 from quiescent EJ30 cells, nuclear extracts were diluted to 0.2 M KCl and loaded on a Mono S cation-exchange column using the ÄKTA fast-performance liquid chromatography system (Amersham Biosciences). The column was then equilibrated with buffer A (20 mM HEPES-KOH, pH 7.8, 1 mM EGTA, 1 mM dithiothreitol) containing 0.2 M KCl before a linear gradient elution was performed using buffer A containing between 0.2 and 0.6 M KCl. Twelve 0.5-ml fractions were collected, and equivalent volumes of all samples were subjected to Western blot analysis. The peak fractions containing p60 were pooled, concentrated fivefold using Ultrafree-CL centrifugal filters (Millipore), dialyzed against buffer A containing 0.1 M KCl, and then immediately used for replication reactions.
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RESULTS |
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-H2AX in both proliferating and quiescent EJ30 cells during a 24-h treatment (Fig. 1A). In order to visualize the extent of DSBs induced by bleocin, we analyzed the integrity of genomic DNA in these cells directly by pulsed-field gel electrophoresis (Fig. 1B). In both proliferating and quiescent EJ30 cells, the genomic DNA became increasingly fragmented with increasing concentrations of bleocin. For both cell populations, the amount of fragmented DNA correlated with the level of -H2AX induced in response to bleocin treatment.
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Induction of chromatin assembly factor CAF-1 by formation of DSBs. We next investigated the expression levels of the chromatin assembly factor CAF-1 (via the p150 and p60 subunits) and its interacting protein, PCNA, in response to the induction of DSBs by bleocin. In proliferating cells, both CAF-1 and PCNA were expressed, and no significant change in their expression levels was observed upon the addition of bleocin (Fig. 2A). In contrast, CAF-1 and PCNA were detected only in trace amounts in whole-cell extracts prepared from untreated quiescent human cells (Fig. 2A), consistent with a recent report (37). Importantly, upon induction of DSBs by bleocin, the levels of both CAF-1 and PCNA strongly increased (Fig. 2A). For comparison, the canonical DNA replication and repair protein RPA was expressed in proliferating and quiescent cells, and its levels did not change upon bleocin treatment (Fig. 2A). However, the 32-kDa subunit of RPA became increasingly phosphorylated upon treatment with increasing doses of bleocin, indicating that DNA damage signaling involving RPA was activated in proliferating and quiescent cells.
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We conclude that the induction of DSBs in quiescent cells leads to the induction of CAF-1 and PCNA expression, resulting in enhanced competence to assemble new nucleosomes during DNA synthesis.
CAF-1 is recruited to chromatin in damaged cells.
To further characterize the physiological significance of CAF-1 and PCNA induction in response to the generation of DSBs, we investigated their intracellular localization by immunofluorescence microscopy. We used bleocin as a chemical means to induce DSBs, which has been demonstrated to generate DSBs with a higher efficiency and selectivity than ionizing radiation (IR) (38). However, we confirmed these results by treating cells in parallel with IR at a dosage of 5 Gy, followed by a recovery period of 2 h. This treatment was chosen because previous studies had shown a peak of transient colocalization of repair proteins such as RPA, BRCA1, and Rad51 with -H2AX by 2 h after irradiation (4, 35). As an initial control, we observed that the p150 and p60 subunits of CAF-1 colocalized at discrete sites in the detergent-resistant fractions of untreated proliferating cell nuclei (19, 29) and of damaged proliferating and quiescent EJ30 cell nuclei (see Fig. S1 in the supplemental material).
We first visualized the localization of the p60 subunit of CAF-1 in relation to sites of DSBs, as detected by -H2AX staining (Fig. 3A). In 40% of the untreated proliferating nuclei, p60 exhibited an S-phase-specific staining pattern (19; data not shown). No -H2AX staining was detected in these S-phase nuclei (Fig. 3A). After bleocin treatment or application of IR, p60 was distributed in over 85% of the cells in a punctate pattern throughout the nucleus (Fig. 3A). These nuclei also showed discrete sites of -H2AX staining. However, despite some localized overlap, the majority of the p60 foci did not specifically colocalize with sites of -H2AX staining. In untreated quiescent cells, p60 showed a very low level of staining within the nucleus (Fig. 3A). After treatment with bleocin or IR, p60 levels increased significantly in over 85% of the nuclei to levels and distribution patterns comparable to those in bleocin-treated proliferating cells. Again, the majority of the newly formed p60 foci did not specifically colocalize with sites of -H2AX. The p150 subunit of CAF-1 showed identical behavior to that of p60 in these experiments (data not shown).
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We asked next whether significant DNA synthesis was occurring at these sites. Cells were pulse labeled for 1.5 h with bromodeoxyuridine (BrdU), and sites of BrdU incorporation were detected by immunofluorescence microscopy (Fig. 3D). In asynchronously proliferating EJ30 cells, sites of BrdU incorporation colocalized with PCNA in S-phase-specific patterns in 40% of nuclei. However, in the presence of bleocin, no BrdU incorporation was detected, although PCNA was recruited to damaged chromatin (Fig. 3D). This observation is consistent with our flow cytometry data showing an accumulation of bleocin-treated proliferating cells in the G2/M phases of the cell cycle (Fig. 1D). For comparison, no BrdU incorporation was detectable in quiescent cells, whether or not bleocin was added (Fig. 3D). These data suggest that no extensive DNA synthesis occurred around DSB sites in bleocin-treated cell nuclei.
The recruitment of CAF-1 and PCNA to damaged chromatin was partly reversible after the removal of bleocin from proliferating and quiescent cells for 24 h (Fig. 3E). Immunofluorescence microscopy revealed that in proliferating cells, both proteins colocalized at fewer sites on damaged chromatin following removal of bleocin. This finding correlated with a reduced extent of H2AX phosphorylation, as detected by Western blotting. However, the expression levels of CAF-1 did not change significantly, suggesting that CAF-1 may concentrate at sites of unrepaired DNA damage under these conditions and/or that it relocates to DNA replication foci in repaired cell nuclei. In contrast, in quiescent cell nuclei, CAF-1 and PCNA colocalized at few residual sites following the removal of bleocin, and the expression levels of CAF-1 were significantly reduced (Fig. 3E). The level of this residual CAF-1 correlated with a greatly reduced amount of -H2AX, strongly suggesting that CAF-1 is displaced from chromatin after the repair of DNA strand breaks. These data indicate that the induction of CAF-1 and PCNA and their recruitment to damaged chromatin are dynamic and part of a regulated response of the cell to DNA damage.
Taken together, these data establish that the expression of CAF-1 and PCNA is induced in quiescent human cells upon induction of DSBs and that both proteins are recruited to the damaged chromatin. However, despite a partial overlap, the majority of CAF-1 and PCNA did not directly overlap with sites of DSBs, as visualized by -H2AX staining. In the next set of experiments, we therefore investigated by which pathway(s) the damaged DNA was repaired in these cells and whether CAF-1 colocalized with the respective repair marker proteins in these nuclei.
Recruitment of CAF-1 to damaged chromatin in NER-deficient human cells. Repair intermediates arising during NER recruit proteins such as PCNA and RPA (2). This raised the question of whether the recruitment of PCNA and CAF-1 to damaged chromatin in our experiments might be triggered through the activation of the NER pathway, as seen after UV irradiation (14). Therefore, we used transformed fibroblasts derived from patients with xeroderma pigmentosum (XP), which are defective at the initial incision stage of the NER pathway (XP-A mutant). After localized UV irradiation, it has been shown that XP-A cells do not recruit CAF-1 to chromatin at sites of photodamage (14).
First, we induced quiescence in XP-A cells by serum deprivation. The percentage of nuclei actively incorporating BrdU was reduced from 44% for proliferating cells to 4% for quiescent cells, and CAF-1 expression was significantly down-regulated during quiescence (see Fig. S2A in the supplemental material). We treated proliferating and quiescent XP-A cells with bleocin and, in both cases, observed a formation of -H2AX, indicating that these NER-deficient cells detect DNA double-strand breaks. As seen in NER-proficient EJ30 cells, CAF-1 was recruited to damaged chromatin, partially overlapping with sites of -H2AX staining (see Fig. S2B in the supplemental material). These results show that the induction and recruitment of CAF-1 to damaged chromatin upon bleocin treatment do not occur through activation of the NER pathway.
Induction of CAF-1 and PCNA in quiescent cells upon damage induction depends on DNA-PK. To determine whether the induction of CAF-1 and PCNA in response to bleocin treatment depends on a specific response of the cell to the formation of DSBs, we compared the human glioblastoma sister cell lines MO59J and MO59K. Both cell lines were derived from the same tumor, but MO59J cells lack DNA-PK activity, are deficient in the repair of DSBs, and display a 30-fold increased sensitivity to ionizing radiation and bleocin compared to their nonmutated, DNA-PK-proficient MO59K counterparts (1, 24).
We induced quiescence in MO59J and MO59K cells by serum deprivation for 7 days. The percentage of S-phase cells was reduced from 36% in proliferating cells to 1 to 3% in quiescent cells (data not shown). Compared to the case in proliferating cells, the expression of CAF-1 and PCNA was significantly down-regulated during quiescence in both MO59J and MO59K cells (Fig. 4). Treatment with bleocin led to the induction of CAF-1, PCNA, and -H2AX in DNA-PK-proficient quiescent MO59K cells (Fig. 4), as similarly observed in EJ30 cells (compare with Fig. 2). In contrast, CAF-1 and PCNA were not induced in response to bleocin treatment in DNA-PK-deficient quiescent MO59J cells (Fig. 4). As a control, H2AX was still phosphorylated upon bleocin treatment in these cells, confirming that ATM/ATR-dependent DNA damage signaling pathways were active.
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NHEJ, but not HR, is activated in quiescent cells upon bleocin treatment.
DSBs can be repaired by either the HR or NHEJ pathway. To determine which of these pathways is activated in human quiescent EJ30 cells upon bleocin treatment, we first visualized the HR-specific protein Rad51 under these conditions (Fig. 5A). Rad51 is required for strand invasion and exchange during HR (5). Insoluble pools of Rad51 protein became enriched in the nuclei of proliferating EJ30 cells after bleocin treatment, mostly colocalizing with sites of -H2AX (Fig. 5A). In contrast, no induction of Rad51 protein or recruitment to -H2AX sites was detectable in quiescent cells after bleocin treatment. This finding suggests that HR pathways of DNA damage repair are not activated in quiescent cells after treatment with bleocin.
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-H2AX (Fig. 5B). Importantly, bleocin treatment resulted in a recruitment of XRCC4 to chromatin and its colocalization with sites of -H2AX in both proliferating and quiescent cells (Fig. 5B). These data indicate that DSBs in bleocin-treated quiescent human cells are repaired by the NHEJ pathway. We observed by confocal immunofluorescence microscopy that a small proportion of these bleocin-induced XRCC4 sites did not contain CAF-1 (Fig. 5C, red foci in the merged image) and that approximately half of the CAF-1 sites did not contain XRCC4 (Fig. 5C, green foci in the merged image). However, a subpopulation of the bleocin-induced sites of XRCC4 perfectly colocalized with detergent-resistant sites of the p150 subunit of CAF-1 in treated proliferating and quiescent cells (Fig. 5C, yellow foci in the merged image). These observations suggest that only a subpopulation of CAF-1 is recruited to sites on damaged chromatin undergoing the NHEJ pathway of DSB repair, whereas a second subpopulation of CAF-1 is recruited to other sites on damaged chromatin. We next investigated whether these extra sites could be sites of DNA SSB repair.
Activation of BER pathway upon bleocin treatment.
DNA-PK can also be activated by SSBs (8, 32). SSBs represent 90% of all DNA breaks generated by bleocin (39) and can be repaired by short- and long-patch BER pathways (6). Therefore, we investigated the pattern and distribution of CAF-1 in relation to the SSB repair protein XRCC1. XRCC1 is a molecular scaffold protein that appears to stimulate SSB repair through direct physical interaction with essential components such as PARP, APE1, DNA polymerase ß, and DNA ligase III
, forming dynamic complexes at SSB sites (reviewed in reference 6). These sites can recruit PCNA, DNA ligase I, FEN1, and DNA polymerases 
and 
to execute long-patch base excision repair. The involvement of PCNA in this pathway could allow for a possible recruitment of CAF-1 through direct protein-protein interactions (31, 42).
We first probed for XRCC1 and -H2AX in order to determine the extent of overlap between SSB and DSB repair sites (Fig. 5D). In untreated proliferating and quiescent EJ30 cell nuclei, XRCC1 was detected only as a diffuse insoluble pool in the absence of -H2AX (Fig. 5D). However, bleocin treatment of proliferating and quiescent cells resulted in a recruitment of XRCC1 to chromatin and the formation of -H2AX foci. We obtained no direct evidence for a specific overlap of XRCC1 and -H2AX foci (Fig. 5D; note the absence of entirely yellow foci: only partial overlaps of sections of some -H2AX foci with neighboring XRCC1 sites are apparent). These data indicate that bleocin-induced DNA damage also activates BER pathways at sites that are mostly distinct from sites of activated NHEJ.
We observed by confocal immunofluorescence microscopy that a small proportion of the XRCC1 sites did not contain CAF-1 (Fig. 5E, red foci in the merged image) and that approximately half of the CAF-1 sites did not contain XRCC1 (Fig. 5E, green foci in the merged image). However, a subfraction of the bleocin-induced sites of XRCC1 perfectly colocalized with detergent-resistant sites of the p150 subunit of CAF-1 in bleocin-treated proliferating and quiescent cells (Fig. 5E, yellow foci in the merged image). This finding suggests that a subpopulation of CAF-1, induced in response to the DNA damage generated by bleocin, is recruited to sites of activated BER pathways.
Taking these data together, we conclude that the expression of CAF-1 is induced in quiescent cells upon treatment with bleocin and that small subpopulations of CAF-1 are found at chromatin at sites of BER and NHEJ repair of single- and double-strand DNA breaks, respectively.
CAF-1 becomes essential for the survival of quiescent cells in the presence of DNA strand breaks. We finally asked whether CAF-1 plays an essential role in the cellular response to the formation of DSBs and SSBs in vivo. Previously, we had demonstrated that the silencing of CAF-1 by RNA interference (RNAi) in untreated quiescent EJ30 cells had no effect on cell viability, whereas CAF-1 was essential for the viability of proliferating cells (33).
Building on this finding, we treated quiescent EJ30 cells with bleocin and simultaneously performed RNAi to silence the p60 subunit. Within 48 h of transfection with two different p60-specific siRNAs, the p60 protein levels were reduced >95% in whole-cell extracts compared to those in control samples from cells transfected with a nontarget scrambled control siRNA (Fig. 6A). We observed a significant loss of viability in bleocin-treated cells after silencing of p60 compared to control samples transfected with the nontarget siRNA 48 h after transfection (Fig. 6B). This loss of cell viability was visually corroborated by phase-contrast microscopy of cells depleted of p60 in the presence of bleocin and quantified by determining the proportion of apoptotic cells under these conditions by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays (Fig. 6C). We therefore conclude that CAF-1 becomes essential for the viability of quiescent cells under conditions of genotoxic stress resulting from the formation of DNA strand breaks.
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DISCUSSION |
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Recruitment of CAF-1 to damaged chromatin following induction. CAF-1 is a key player for de novo nucleosome assembly during DNA replication (reviewed in reference 22) and NER DNA synthesis through its interaction with PCNA (reviewed in reference 15). CAF-1 is thereby recruited to sites of replication- and UV damage repair-associated DNA synthesis, apparently via the same mechanism. In this paper, we have shown that CAF-1 is recruited to damaged chromatin undergoing different DNA damage repair pathways which do not depend on extensive DNA synthesis, namely, NHEJ of DNA double-strand breaks and BER of DNA single-strand breaks.
Proteins required for DNA synthesis have also been reported to participate in NHEJ. Upon exposure of quiescent human fibroblasts to ionizing radiation or treatment with bleocin, PCNA was recruited into an insoluble pool bound to chromatin that largely colocalized with sites of DNA damage repair (3). PCNA further colocalizes with the Ku70/80 heterodimer under these conditions, suggesting that PCNA may assist Ku70/80 in the repair of broken DNA (3). PCNA may also serve as a docking platform to recruit the DNA synthesis machinery to carry out very limited gap filling of degraded or partially annealed DNA strands following irradiation or bleocin treatment (3). In support of this hypothesis, the replication protein RPA, which binds to single-stranded DNA during replication and NER, is also recruited to radiation-induced foci (4). Synthetic DNA substrates containing mimics of radiation-induced breaks are repaired by Ku70/80-dependent end joining in the presence of overhangs with partial complementarity of as little as 2 bases (7). In such a context, PCNA may facilitate limited gap-filling DNA synthesis after the pairing of one or more bases in the aligned termini. Our data are only partially consistent with these observations. Under our experimental conditions, the majority of PCNA and CAF-1 is present at intranuclear sites in damaged cells that are not labeled by the DSB marker -H2AX. This raises the possibility that the recruitment of PCNA and CAF-1 to damaged chromatin may be independent of the actual sites of DSBs. A signal may be generated at the DSB site, for instance, by phosphorylation of a diffusible protein, which could indirectly lead to the induction of CAF-1 and PCNA and their recruitment to the damaged chromatin at different sites. Alternatively, but not mutually exclusively, CAF-1 and PCNA may also be recruited to additional sites such as single-strand DNA breaks (see below). In fact, NHEJ has recently been reconstituted in vitro with purified Ku, DNA-PK, Artemis, XRCC4, and DNA ligase IV proteins on defined DNA substrates with incompatible ends in the absence of PCNA and RPA (28), which would be fully consistent with such a scenario.
The expression of CAF-1 and PCNA is induced in direct response to the formation of DSBs and SSBs in quiescent human cells and depends on the activity of the protein kinase DNA-PK. Following induction, CAF-1 and PCNA are recruited to damaged chromatin. However, only a small subpopulation of the -H2AX sites actually colocalize with CAF-1. The partial overlap can be accounted for by the observation that bleocin produces a nearly equal mixture of staggered and blunt-ended cuts (38) and that only a fraction of the double-stranded repair sites would therefore require processing and DNA synthesis after they are annealed together. The DNA synthesis step would necessitate the activity of DNA polymerases and PCNA. The recruitment of PCNA could in turn target CAF-1 to those repair sites. This may explain the perfect colocalization of CAF-1 with PCNA (and RPA) after damage induction and also why only a fraction of -H2AX actually colocalizes with these proteins. Sites of CAF-1 and PCNA could therefore potentially represent sites where PCNA is recruited for DNA synthesis in the repair of double-stranded lesions that require strand processing in the postannealing stage. Alternatively, they could represent sites where single-stranded lesions, which are simultaneously induced by bleocin, are repaired by BER. These sites can recruit PCNA and DNA polymerases /, among other repair proteins, to execute long-patch base excision repair by synthesizing 2 to 15 nucleotides of DNA (reviewed in reference 6). Our data showing colocalization of subfractions of CAF-1 and XRCC1 sites would entirely support this possibility. Moreover, since we chose to induce low but chronic levels of damage in cells over a 24-h time course, it is possible that PCNA remains bound to the repaired DNA template after successful repair, perhaps serving as a marker for sites of CAF-1-mediated histone deposition and chromatin assembly.
Possible roles for CAF-1 in response to DNA breaks. CAF-1 purified from damaged cells supported nucleosome assembly during DNA synthesis, suggesting that it plays a similar role during or following repair of DSBs by NHEJ and of SSBs by BER. However, we were not able to detect extensive DNA synthesis at DSB and SSB sites by BrdU incorporation. This suggests that if limited DNA synthesis occurs at these sites, as discussed above, its extent is below the threshold required for detection by BrdU incorporation.
Green and Almouzni have suggested that, in principle, a histone chaperone like CAF-1 may also act as a local histone sink during chromatin disassembly at sites preparing for DSB repair in order to allow access of the repair machinery to the damaged sites (15). Following repair, chromatin could be reestablished through nucleosome assembly by CAF-1 using these histones, thereby maintaining the epigenetic information carried by histone variants or posttranslational modifications at the break site. This model is consistent with our data. However, CAF-1 exhibits preferential specificity for histone H3 variant H3.1, whereas variant H3.3 is specifically recruited to an assembly pathway that does not depend on DNA synthesis (47). This variant specificity would not entirely support a role for CAF-1 as a nondiscriminatory histone sink during DSB and/or SSB repair. Therefore, additional experimentation will be required to resolve the issue of conserving epigenetic information during DNA strand break repair.
In a previous study, we found that CAF-1 was not essential for the survival of quiescent human cells (33). In contrast, CAF-1 is essential in proliferating cells, and its functional or physical depletion leads to phenotypes ranging from DNA damage to S-phase arrest, checkpoint activation, and programmed cell death (17, 33, 48). These effects can be attributed to the failure of efficient chromatin assembly during chromosomal DNA replication.
When proliferating cells withdraw from the cell cycle and become quiescent, the expression of CAF-1 is down-regulated at the mRNA and protein levels, reflecting the diminished need for nucleosome assembly during DNA replication (37). Upon stimulation of quiescent cells to reenter the cell division cycle and proliferate, CAF-1 becomes induced again (37). Our data entirely confirm these observations. In this paper, however, we have identified a new essential role for CAF-1 in quiescent cells. Upon the formation of DSBs and SSBs by bleocin or ionizing radiation, CAF-1 expression is induced and contributes to the survival of the damaged quiescent cell. Furthermore, we observed that more DSBs accumulated in bleocin-treated quiescent cells in the absence of CAF-1 than in its presence. Additionally, the induction of CAF-1 and PCNA in response to damage was detected in a mutant cell line defective in NER, but not in a mutant cell line defective in NHEJ, suggesting that the induction is specific to DSB and/or SSB lesions and depends on signaling through DNA-PK. Taken together, our data suggest that CAF-1 forms an important component of the cell's machinery for coping with DNA breaks. This essential role for CAF-1 is most likely mediated by its activity of restoring and/or maintaining chromatin structure in the context of repairing broken DNA strands, which becomes essential for the survival of the quiescent cell.
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ACKNOWLEDGMENTS |
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This work was funded by research grants from the Human Frontier Science Program and Cancer Research UK. A.N. is supported by a fellowship from the Boehringer Ingelheim Fonds, and D.S. is supported by the Isaac Newton Trust.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
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