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Molecular and Cellular Biology, January 2006, p. 39-49, Vol. 26, No. 1
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.1.39-49.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences

Guochun Jiang and Aziz Sancar^*

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Received 6 September 2005/ Returned for modification 5 October 2005/ Accepted 6 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G₁ and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G₁ and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA damage checkpoints are regulatory pathways that slow or arrest the progression of the cell cycle in response to DNA damage (1, 14, 29, 37, 56). Conceptually, the checkpoint response encompasses damage sensors, mediators, transducers, and effector proteins. The checkpoint response is elicited by alkylating agents such as methyl methanesulfonate that generate nonbulky base lesions, UV, and cisplatin that produce bulky dinucleotide adducts and ionizing radiation that induce double-stranded breaks. An important question in the field of checkpoint response is the nature of the structure that activates the checkpoint. In particular, it remains to be determined whether the primary lesions generated by the various DNA-damaging agents are recognized by sensors specific for each class of damage or if all lesions are processed by replication/repair enzymes to a common intermediate that then acts as a signal for the checkpoint sensor proteins. A current view is that a member of the phosphoinositide 3-kinase related protein kinase (PIKK) family, such as ATM and ATR, with its cognate smaller molecular weight partner working in concert with the checkpoint-specific clamp loader/clamp complex (Rad17-RFC-9-1-1 complex) are necessary and sufficient to activate the DNA damage checkpoint in response to all types of DNA lesions (8). Thus, it is proposed that when the DNA damage is a double-stranded break, ATM is recruited to the damage site by Nbs1 and that the 9-1-1 complex is loaded onto DNA at the site of the double-strand break independent of ATM; the proximity of ATM and the 9-1-1 complex enables ATM to phosphorylate downstream targets and activate the checkpoint (9, 11, 18, 40). In case of base damage, it has been reported that checkpoint activation does not occur unless the cells enter or are in S phase regardless of when the damage took place (52) because ATR and the 9-1-1 complex can be loaded only on RPA-covered single-stranded DNA by ATRIP (57) and Rad17-RFC (10, 58), respectively. Moreover, in the case of checkpoint activation by the ATR-ATRIP-Rad17-RFC-9-1-1 ensemble, in addition to RPA-covered single-stranded DNA, a primer/template-like structure is needed because complete inhibition of DNA synthesis also abolishes checkpoint activation (7, 26) by ATR.

Studies with budding yeast have, in general, yielded results consistent with those obtained in mammalian cells. In yeast, a double-stranded break in the genome can be generated in a controlled manner by expressing the HO endonuclease placed under the regulation of the GAL4 promoter. With this system, it was shown that the yeast functional counterparts of ATM (Mec1) and the 9-1-1 complex (Rad17-Mec3-Ddc1) were recruited independently to the double-stranded break (15, 25). Similarly, it was found that, at moderate UV doses, yeast cells irradiated in G₁ or G₂ did not activate the checkpoint response until they entered S phase (27). However, further analysis of the checkpoint response revealed that the checkpoint response in G₁ and G₂ could be activated in wild-type cells but not in strains deficient in nucleotide excision repair (12, 13). It was proposed that the 30-nucleotide (nt)-long postexcision gaps containing RPA were the signal for checkpoint activation.

We are interested in the UV-activated DNA damage checkpoint in mammalian cells. Previously, we reported that human ATR, with and without its small partner ATRIP, can bind DNA directly without the intermediacy of RPA (45, 46) and that ATR exhibited higher affinity to UV-damaged DNA than undamaged DNA (45). Similarly, we found that in vitro the Rad17-RFC complex loaded the 9-1-1 ring onto a primer/template-like structure in the absence of RPA and that in fact RPA, under our experimental conditions, in contrast to other reports (10, 58), does not stimulate but rather inhibits 9-1-1 loading (5, 19). These findings, which suggest that, at least in the initial stages of sensing of DNA damage, the primary lesion is recognized by checkpoint proteins are consistent with some in vivo observations as well. It has been found that at moderate UV doses, p53 is efficiently phosphorylated and retained in the nucleus in excision repair-deficient XP-A cell lines (28, 53). These and related observations using different endpoints for testing checkpoint activation (21) raised some questions about the generality of the RPA-covered single-stranded DNA as the common intermediate for activating the ATR-initiated checkpoint response. In the current study, we have performed chromatin immunoprecipitation (ChIP) experiments to test the alternative model of direct damage sensing in UV-activated DNA damage checkpoint response in human cells. Our results, while not contradicting the model positing RPA-covered single-stranded DNA as a common intermediate for eliciting DNA damage checkpoint response by UV and UV-mimetic agents, strongly indicate that the primary UV lesions can be directly recognized by the DNA checkpoint sensor proteins RPA, ATR, and the 9-1-1 complex and activate the checkpoint response in the absence of replication or repair.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmids and antibodies. pcDNA3-Flag-RPA32, pcDNA4-Flag-Rad9, pcDNA3-Flag-Rad17, or pcDNA3-Flag-ATR were constructed in our lab and have been described previously (20, 45). Mouse monoclonal anti-Flag antibody was obtained from Sigma Chemical Company. Mouse monoclonal antibody against human RPA32 (hRPA32) was obtained from Oncogene Research Products. Rabbit polyclonal antibody against hATR was obtained from Affinity Bioreagents. Monoclonal hOrc2 antibody was purchased from BD Pharmingen.

Cell culture, cell synchronization, and transfection. The simian virus 40-transformed human embryonic kidney 293 cells (HEK293T), XP-A cells (XP20SV), XP-C cells (XP4PA.SV), and HeLa S3 cells were obtained from the Lineberger Comprehensive Cancer Center. The cell lines were maintained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum containing 100 µg/ml streptomycin and 100 units/ml of penicillin. Synchronized cells were obtained by subjecting 2.5 x 10⁶ cells in R150 dishes to a double block with 3 mM thymidine. Following release from the second thymidine block, fluorescence-activated cell sorter (FACS) analysis revealed that cells were in S and G₁ phases 3 h and 14 h after release, respectively. For the transfection of HEK293T cells, 2.5 x 10⁶ cells were plated on R150 dishes 24 h before transfection. Then, 10 µg of appropriate expression plasmid or control vector was transfected using the calcium-phosphate precipitation method. After 16 h of incubation at 37°C in 5% CO₂, the medium was replaced with fresh DMEM plus 10% fetal bovine serum, and incubation was continued for another 48 h. For transfecting XP-C cells, 2.5 x 10⁶ cells were incubated with expression plasmid or vector DNA and 40 µl FuGene (Roche) for 48 h.

ChIP assay. ChIP assays were carried out as described elsewhere (16) with the following modifications. Cells in R150 dishes were either left untreated or treated with UV or N-acetoxyacetylaminofluorene (N-AAAF) before formaldehyde cross-linking. For UV irradiation, the growth medium was removed, and the cells were washed once with phosphate-buffered saline and then exposed to UV light (254 nm) from a germicidal lamp at a fluence rate of 0.5 J/m²sec. Then the growth medium was added back and cells were incubated for the indicated time before processing. For N-AAAF treatment, the growth medium was removed and cells were washed once with buffer A (137 mM NaC1, 5.4 mM KCl, 4.2 mM NaHCO₃, pH 7.2, and 0.1% glucose) and incubated with 20 µM of N-AAAF (dissolved in dimethyl sulfoxide) in buffer A at 37°C for 30 min. Then the buffer was replaced with growth medium, and incubation was continued for an additional 20 min (42). Following the DNA-damaging treatments, cells in 20 ml DMEM were transferred into 50-ml conical polypropylene tubes, fixing solution (11% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Tris-Cl, pH 8.0) was added to the cell suspension to a final concentration of 1% formaldehyde, and the mixture was incubated for 10 min at 22°C. Then glycine was added to 125 mM and incubation was continued for another 5 min. Chromatin was pelleted by centrifugation and washed once with wash solution 1 (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0) and once with wash solution 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0). Then, the cross-linked chromatin was dissolved in 0.5 ml TEE (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0). To obtain 0.5- to 2.0-kbp DNA fragments, the chromatin suspension was sonicated with 3 pulses of 30-s each at a setting of 6.5 and 1-min intervals on ice, using a Fisher Model 60 sonic dismembranator. Then the suspension was centrifuged, and the supernatant was transferred into 1.5-ml polypropylene tubes. The protein concentration was determined by the Bradford assay, and the samples were stored at –80°C until further use.

ChIPs were performed on precleared chromatin from 400 µg cross-linked and sonicated chromatin in 700 µl immunoprecipitation buffer (16). For the Flag-tagged proteins, 10 µl of anti-Flag immobilized beads (Sigma) was added to the suspension and incubated at 4°C overnight. The beads were collected by centrifugation and were washed two times in low-salt buffer (10 mM Tris-HCl, pH 8.0, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.1% Na deoxycholate, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), two times in high-salt buffer (10 mM Tris-HCl, pH 8.0, 1% Triton X-100, 0.1% SDS, 0.1% Na deoxycholate, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), twice in LiCl buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1% NP-40, 1% Na deoxycholate, 1 mM EDTA), and two times in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The DNA protein complex was eluted with 70 µl Flag peptide (0.2 mg/ml) in Tris-buffered saline buffer at 4°C for 15 min. For ChIP with antibodies against endogenous untagged proteins, 1.5 µg hRPA32, 2.5 µg hOrc2, and 2 µg ATR antibodies were used to precipitate the cross-linked chromatin. The antibody-chromatin mixture was incubated at 4°C overnight. Then, 8 µl protein A/G agarose was added, and the mixture was incubated at 4°C for another 45 min on a rotator. The immune complexes were washed as described above for the Flag immunoprecipitates, and the DNA-protein complexes were eluted in 100 µl 0.2% SDS-0.1 M NaHCO₃. To the immunoprecipitates isolated by either method, 1 µg of RNase A was added and the mixtures were incubated at 37°C for 30 min. The samples were adjusted to 0.5% SDS and 0.5 mg/ml proteinase K and incubated at 55°C for 1 h, followed by incubation for 6 h at 65°C. The DNA was extracted with phenol-chloroform. To 200 µl of the extracted DNA in TE buffer, 20 µg of glycogen was added, and after addition of Na acetate (pH 5.2), the DNA was precipitated with 2.5 volumes of 100% ethanol, washed with 70% ethanol, briefly air dried, resuspended in 25 µl distilled water, and stored at 4°C.

PCR analysis was carried out with primers for 250-bp regions in intron 7 of p53 and the middle of the ß-globin pseudogene (Locus AF 339400). The PCR primers for p53 amplified a fragment of 248 bp, and those for ß-globin amplified a fragment of 251 bp. The sequences for the p53 amplicon were as follows: forward primer, 5'-CCTCTTACCGATTTCTTCCA; reverse primer, 5'-GCAAGAGGCAGTAAGGAA-3'. Primers for ß-globin were as follows: forward primer, 5'-CCTTTGCTACACTGAGT; reverse primer, 5'-CATAGTCCTTGCTCTACC. Amplification was carried out with the Promega GoTaq DNA polymerase system, and the products were analyzed on 1.5% agarose gels containing ethidium bromide. Quantitative analysis was carried out by the Kodak Gel Logic100 image system.

Western blotting. We used Chk1-S345P antibodies (Cell Signaling Technology, Inc.) for Western blotting with cell lysates that were used in the ChIP experiments. The phosphoprotein was visualized by nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate system (Promega).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Method for detecting protein binding to DNA lesions at defined genomic sites. ChIP is most commonly used to analyze the binding of transcription factors to defined DNA sequences in the genome. To apply this method for binding of repair and checkpoint proteins to DNA damage, it would be desirable to have cells containing a single lesion at a defined genomic position. This has been achieved for recruitment of checkpoint proteins to double-strand breaks by employing inducible sequence-specific endonuclease, such as the HO endonuclease in yeast (15, 25) and the SceI endonuclease in a human cell line (2), constructed for this purpose. There is no comparable method for introducing base damage at a unique site to investigate binding of repair and checkpoint proteins to such a site in vivo. Because of our interest in the activation of the DNA damage checkpoint response by UV and UV-mimetic agents, we designed an alternative strategy to analyze the recruitment of checkpoint proteins to the lesions in specific regions of the genome (Fig. 1A). We used the p53 gene in this and subsequent experiments because the damage and repair of this gene has been extensively studied (44). Cells are treated with a DNA-damaging agent such as UV or N-AAAF that produces a relatively precise number of lesions in the genome for a given dose of damaging agent. Moreover, while both DNA-damaging agents exhibit some sequence specificity at nucleotide resolution (42, 43, 44, 55), damage distribution when integrated over 200-bp to 2,000-bp segments is essentially random throughout the genome and, importantly, in transcriptionally active and inactive sequences (17, 32, 47, 51, 55). Therefore, provided that a fragment of 200 bp or longer is probed, binding of checkpoint proteins to various regions of the genome can be analyzed by this method. The method consists of treating cells with a DNA-damaging agent to produce 0.1 to 0.5 lesion per kbp and then to carry out ChIP experiments with chromatin fragments in the range of 0.5 to 2.0 kbp using antibodies to specific checkpoint proteins and amplifying specific regions that would be immunoprecipitated if they are within a 0.5- to 2.0-kbp distance from a lesion.

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FIG. 1. (A) Method for detecting protein binding to UV photoproducts in a given genomic region. Cells are irradiated with UV (or treated with a UV-mimetic agent), incubated for an appropriate period of time, and then treated with formaldehyde, and ChIP is carried out by standard procedures. Binding of repair/checkpoint proteins to UV photoproducts (or chemical lesions) within 1 kbp on either side of the target sequence leads to enhancement of the target sequence in the immunoprecipitate. In most of our studies, a p53 fragment spanning the region of nt 13251 to 13498 (in intron 7) from the initiation codon was amplified, and thus, bindings of checkpoint proteins to the area extending from exon 4 to intron 9 are detected. (B) Binding of RPA to UV and N-AAAF lesions in the p53 of HEK293T cells. The cells were transfected with 10 µg pcDNA3-Flag-RPA32, then 72 h later, cells were either irradiated with 50 J/m2 or treated with 20 µg/ml N-AAAF for 30 min and then incubated with formaldehyde, ChIP was carried out by standard procedures. Top panel: ChIP data. UV and – indicate irradiated and unirradiated cells, respectively, and AAF and DMSO indicate cells treated with the carcinogen or with solvent, as indicated. C indicates the control reaction using cells that were transfected with pcDNA3 vector and used in ChIP with anti-Flag antibodies. Bottom panel: quantitative analysis of ChIP data. The averages of the results from three experiments, including the one shown in the top panel, are plotted. The values are relative to the vector-transfected cell control. Error bars indicate standard errors of the means.

An application of the method is shown in Fig. 1B. HEK293T cells expressing Flag-RPA32 were treated with 50 J/m² of UV or 20 µM of N-AAAF, both of which introduced on the average 1 base lesion per 2 kbp (42, 49, 51). Then ChIP was performed, and a 248-bp fragment from intron 7 of p53 was amplified to detect binding of RPA to lesions within 2 kbp on either side of the amplified fragment. As seen in Fig. 1B, RPA does bind to lesions of UV and N-AAAF in the vicinity of intron 7 of p53. While these experiments show that the method can be used to detect specific binding of a protein to lesions in a defined region of the genome, they do not necessarily mean that the binding constitutes an initial step in checkpoint response because RPA functions as a damage sensor in both excision repair and checkpoint response reactions (8, 36). Therefore, to test if proteins that are considered to function as damage sensors strictly in the DNA damage checkpoint response are recruited to the damage site, we performed ChIP with such proteins. We will present data obtained only with UV damage for the sake of simplicity, but essentially the same results were obtained with N-AAAF in all key experiments that were performed.

Binding of the ATR and the 9-1-1 complex to UV-damaged DNA. To assess the recruitment of checkpoint-specific damage sensors to the site of UV photoproducts, we tested the binding of Rad17 (presumably in the form of Rad17-RFC), Rad9 (presumably in the form of 9-1-1 complex), and ATR to a 2-kbp segment of p53 around intron 7 in UV-irradiated cells. As a positive control for ChIP, we used Orc2, which is known to be bound at Ori sites at all times (4). The results are shown in Fig. 2. In addition to RPA, Rad9 and ATR are also recruited to the damage site. Interestingly, Rad17 is not bound to the target region with and without damage. The data on Rad9 and Rad17 are in concert with a subcellular fractionation study that found that ionizing radiation led to the association of members of the 9-1-1 complex but not of Rad17 to a high-salt-resistant association with chromatin (33). As expected, UV irradiation did not augment the binding of Orc2 to p53. In fact, we reproducibly observed a small decrease in the amount of Orc2-associated p53 after UV irradiation. While the significance of this finding remains to be determined, Orc2 does nevertheless provide a valuable negative control as a protein that associates with chromatin, but its association does not increase by UV irradiation, suggesting that increased association of any protein with DNA after UV damage is most likely due to the higher affinity of that protein to either the primary DNA lesions induced by UV or the processed forms of these lesions. This point is further elaborated below.

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FIG. 2. Binding of checkpoint proteins to UV lesions in p53 in HEK293T cells. The cells were transfected with vectors expressing the indicated proteins containing Flag tag or with control (C) vector pcDNA3, irradiated where indicated with 50 J/m2, and 20 min after irradiation, treated with formaldehyde, and ChIP was carried out with anti-Flag antibodies. Top panel: representative ChIP data. +, irradiation; –, no irradiation. Bottom panel: average of the results from three experiments, including the one for which results are shown in the top panel. Error bars indicate standard errors of the means.

Dose response and time course of checkpoint protein binding to damaged DNA. A widely accepted model for DNA damage checkpoint activation is that RPA-covered single-stranded DNA that forms at replication forks stalled at DNA lesions in S phase or at the 30-nt single-stranded DNA gaps that are generated by nucleotide excision repair in any phase of the cell cycle is the signal for checkpoint activation (8, 30, 37, 54). To test this model, we wished to analyze the recruitment of checkpoint sensor proteins to DNA under a variety of conditions, including in the absence of replication, in the absence of transcription, and in the absence of repair. We chose to use the XP-C cells for most of these analyses because these cells are defective in general genomic repair (50) and the particular XP-C strain we are using, XP4PA.SV, can be readily synchronized. First, we determined the binding of two DNA damage sensor proteins, RPA and ATR, to UV lesions in the vicinity of p53 intron 7 as a function of UV dose and as a function of time following irradiation to determine optimal time-dose coordinates in subsequent experiments aimed at more specific questions. Figure 3A shows the binding of RPA and ATR to the p53 gene as a function of UV dose and time after irradiation. Some binding can be detected by a dose as low as 10 J/m² that produces about 1 photoproduct per 10 kbp. However, the amount of binding at this dose over the background is too low for this dose to be used for quantitative comparison of binding of checkpoint proteins under a variety of experimental conditions. The level of binding of both RPA and ATR increases linearly with dose, reaching five- to sevenfold of the background value at 50 J/m². Therefore, this dose was used in the majority of our ChIP experiments. Figure 3B shows the kinetics of RPA and ATR binding to UV lesions (primary or processed) in the p53 gene. For ATR, because of higher damage density with 50 J/m², the maximum binding, as expected, occurs faster, 20 min after irradiation, than binding after 25 J/m², which reaches its maximum at about 60 min. Then the level of bound proteins decreases gradually for both UV doses over the course of the experiment (4 h), likely as a consequence of damage removal by transcription-coupled repair that is operational in XP-C cells. In the case of RPA, interestingly, the kinetics of association with and dissociation from DNA follow the same kinetics at both doses. We do not have an explanation for the different kinetics for the two proteins at present. Nevertheless, these experiments do show that a UV dose of 50 J/m² followed by DNA-protein cross-linking 20 min after irradiation are appropriate for detecting a robust checkpoint protein-DNA complex signal that can be used to monitor checkpoint protein recruitment under a variety of experimental conditions aimed at addressing specific questions regarding the initiation of checkpoint response by DNA damage.

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FIG. 3. Binding of RPA and ATR to the p53 gene in XP-C cells as a function of UV dose and incubation time following UV irradiation. (A) Dose response. Cells were irradiated with the indicated doses of UV, and following a 20 min incubation, ATR- or RPA-bound DNA was immunoprecipitated with anti-ATR and anti-RPA antibodies using standard ChIP procedures. The C lane in the top panel contained DNA nonspecifically bound to agarose beads. The bottom panel shows quantitative analysis of the data from three experiments, including the one for which results are shown in the top panel. The values are relative to the irradiated control. (B) Time course. Cells were irradiated with either 25 J/m2 or 50 J/m2 and incubated for the indicated times before ChIP assays were performed with either anti-RPA or anti-ATR antibodies. The top panel shows representative ChIP assays, and the bottom panel shows quantitative analysis of data from three experiments. The values are relative to ChIP signals at 25 J/m2 and 50 J/m2 at time zero after irradiation. Note that, at both UV doses, RPA binding reaches a maximum at 20 min, whereas ATR binding reaches a maximum at 20 min at 50 J/m2 and gradually decreases thereafter, but the binding of ATR after 25 J/m2 proceeds at a slower rate and decays at a slower rate as well. Error bars indicate standard errors of the means.

Effect of cell cycle phase on recruitment of checkpoint proteins to UV photo damage. The experiments discussed so far were done with asynchronous cell cultures. Hence, it is conceivable that in fact the recruitment to UV damage we detect with our ChIP assay measures the recruitment of checkpoint proteins to replication forks stalled at the target sequence and not to the primary damage. To address this issue, we synchronized cells by double thymidine block and tested the recruitment of checkpoint proteins to the vicinity of p53 intron 7 both in G₁ and S phases. As seen in Fig. 4, the three key damage sensors in checkpoint response, RPA, ATR, and Rad9, are recruited to the damage site with comparable efficiencies in G₁ and S phases. Note that, in these experiments, ChIP was performed with antibodies against endogenous proteins to avoid potential artifacts that may arise from immunoprecipitation of proteins overexpressed by transient transfection. The recruitment of checkpoint proteins to the p53 gene clearly indicates that replication is not necessary for binding of checkpoint sensors RPA, ATR, and Rad9 to damaged DNA. In the case of RPA and ATR, where an XP-C cell line was used for ChIP, the possibility exists that DNA repair-generated postexcision gaps resulting from transcription-coupled repair covered with RPA recruited the ATR protein (50). This issue is addressed in the following sections. In the case of Rad9, anti-Rad9 antibodies of suitable quality for ChIP were not available nor could we generate an XP-C cell line stably expressing a tagged Rad9 protein. Therefore, for Rad9 ChIP, we used a HeLa cell line expressing Flag-Rad9. This cell line enabled us to demonstrate that Rad9 is recruited to the damage site in the absence of replication. However, the data in Fig. 4 does not allow us to discriminate among the possibilities of binding of RPA, ATR, and Rad9 to (i) a transcription bubble immobilized by DNA damage in the path of RNA polymerase, (ii) postexcision gaps generated by transcription-coupled repair, or (iii) direct binding of these checkpoint proteins to UV photoproducts. These issues are addressed in the following set of experiments.

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FIG. 4. Binding of checkpoint proteins to the p53 gene in G1 and S phases of the cell cycle. XP-C cells or HeLa cells expressing Flag-tagged Rad9 protein were synchronized by double thymidine block and then irradiated in either G1 or S phase, and 20 min after irradiation, ChIP was carried out with antibodies against Orc2, RPA, or ATR in XP-C cells and with anti-Flag antibodies in HeLa cells stably transfected with a Flag-Rad9 vector. The top panel shows the FACS analysis of XP-C cells before synchronization (Asyn) and of XP-C cells in G1 and S phases that were used for the ChIP experiments. The FACS profiles of G1- and S-phase HeLa cells were similar to that of XP-C cells and are not shown for clarity. The bottom panel shows the results of the ChIP experiments. The C columns contained ChIP material from control beads with no antibodies that were mixed with chromatin from irradiated cells. +, irradiation; –, no irradiation. Note the decreased binding of UV-irradiated DNA to all probed checkpoint proteins in S phase compared to the level of binding in G1 phase. Error bars indicate standard errors of the means.

Effect of transcription on recruitment of checkpoint proteins to UV-induced DNA damage. To determine whether checkpoint sensor proteins bind to primary UV photoproducts or the intermediate structures arising from processing of the damage by replication, repair, or transcription, we analyzed the binding of checkpoint proteins RPA, ATR, and Rad9 to transcribed and nontranscribed sequences in XP-C cells or HeLa cells in G₁ phase. We chose the p53 gene and the ß-globin gene for this purpose. It has been determined that UV produces DNA lesions in the range of 0.8 to 1.0 lesions per kbp per 100 J/m² in both sequences (44, 48). Hence, any difference between bindings of checkpoint proteins to these two genomic sequences is expected to be due to the fact that p53 is transcribed while ß-globin is not. The results of semiquantitative ChIP experiments with RPA, ATR, and Flag-Rad9 are shown in Fig. 5. All 3 proteins bind more efficiently to p53 than ß-globin, indicating that either the act of transcription itself or transcription-coupled repair facilitates the binding of checkpoint proteins to UV-damaged DNA. Importantly, however, we also observe that both RPA and ATR bind to UV-damaged ß-globin with moderate but significantly higher affinity than undamaged DNA in the absence of replication, transcription, or repair. Similarly, after UV damage, Rad9 binds with higher affinity to the p53 gene than undamaged DNA, again consistent with an order of binding preference of transcribed UV-DNA > nontranscribed UV-DNA > nontranscribed undamaged DNA. However, in the case of Rad9, the ChIP, by necessity, was carried out in an excision-proficient cell line; hence, whether Rad9 was recruited to the damage directly or to an excision repair intermediate cannot be ascertained from these experiments. This qualification notwithstanding, our results show that at least one, ATR, of the two proteins (ATR and the 9-1-1 complex) considered to be checkpoint-specific UV damage sensors can be recruited to damaged DNA in the absence of replication, transcription, or repair, and hence, checkpoint signal transduction can be activated in the absence of these chromosome activities.

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FIG. 5. Binding of checkpoint proteins to UV damage in transcribed and nontranscribed genes in G1 phase. For ChIP with RPA and ATR, XP-C cells were used. ChIP with Rad9 was carried out with HeLa cells expressing Flag-tagged Rad9. (A) ChIP assays performed with serial dilutions (4 µl of undiluted ChIP DNA and two- or fourfold dilutions, respectively) of the immunoprecipitated DNA to ensure the PCR amplification were within the linear range. The "input" gel in the RPA and ATR assays contains DNA from UV irradiated cells (lane 1), nonirradiated cells that were used for ChIP with anti-RPA and anti-ATR antibodies (lane 2), and DNA from irradiated cells that was used for the control no-antibody reaction (lane 3). In the Rad9 ChIP, the input DNAs were from cells that were transfected with Rad9 and UV irradiated (lane 1) or nonirradiated (lane 2) and cells that were transfected with control vector (lane 3). (B) Quantitative analysis of the ChIP data. Averages of the results from three experiments are plotted. The values were expressed relative to that of RPA, ATR, or Rad9 binding to UV-damaged p53 DNA at the highest DNA concentration used in the PCR. Significance is indicated by asterisks: **, P < 0.01; *, P < 0.05. +, irradiation; –, no irradiation.

Transcription or transcription-coupled repair as a signal for checkpoint activation. The preferential binding of ATR and RPA to nontranscribed DNA suggest that unprocessed DNA base lesions can be a signal for checkpoint activation. However, the preferential binding to damaged DNA compared to undamaged DNA in XP-C cells is much more pronounced in transcribed sequences such as p53 and dihydrofolate reductase (data not shown) than in nontranscribed sequences such as ß-globin (Fig. 5) and immunoglobulin E (data not shown). Because these cells are defective in general genomic repair but carry out transcription-coupled repair, it is unclear from the data presented so far whether the binding to the UV-damaged p53 gene is due to postexcision gaps generated by transcription-coupled repair or to stalled elongation complexes.

We addressed this question by performing ChIP experiments in XP-A cells that are totally defective in excision repair. The results are shown in Fig. 6. Even in XP-A cells, binding of RPA and ATR to the p53 gene is stimulated by UV to a level comparable to that seen in XP-C cells in G₁ phase. Although this result does not necessarily eliminate postexcision gaps as a signal for checkpoint activation, it does show that such repair intermediates are not necessary and that stalled transcription complexes are high-affinity targets for checkpoint damage sensors, in agreement with reports indicating that stalled elongation complexes activate the DNA damage checkpoint (21, 23, 31).

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FIG. 6. Binding of checkpoint damage sensors, RPA and ATR, to DNA damage in transcribed DNA in the absence of transcription-coupled repair or replication. XP-A cells were synchronized by double thymidine block and were irradiated in G1 or S phase with 50 J/m2 of 254-nm light. Following irradiation, ChIP was performed at 20 min with either anti-RPA or anti-ATR antibodies. (A) FACS profile of asynchronous (Asyn) and synchronized XP-A cells. (B) ChIP of p53 intron 7. Top panels show ChIP data, and bottom panels show quantitative analysis of data from three experiments with standard errors of the means (error bars). +, irradiation; –, no irradiation.

Activation of DNA damage checkpoint in G₁ phase by UV. It is generally assumed that the ATM-initiated checkpoint response to double-strand break induced by ionizing radiation can be activated in any phase of the cell cycle but that ATR-initiated checkpoint response to UV can be activated only in S phase (8). Because the results presented above indicate that ATR can be recruited to DNA damage in G₁ phase, we wished to determine if this recruitment resulted in checkpoint activation. We analyzed the phosphorylation of Chk1 at Ser345 as a readout for checkpoint activation. As seen in Fig. 7, UV is equally efficient in checkpoint activation in asynchronous cells and in cells in G₁ or S phases, even in the total absence of excision repair (XP-A cells), as well as in the absence of general genomic repair only (XP-C cells). These results suggest that ATR recruitment to DNA lesions, to stalled transcription complexes, or to intermediates of transcription-coupled repair in the absence of DNA replication can activate the checkpoint response.

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FIG. 7. Phosphorylation of Chk1-S345 residue in XP-A and XP-C cells that were irradiated with 50 J/m2 of 254-nm light while growing asynchronously (Asyn), in G1 phase, or in S phase. (A)The top panel shows the FACS analysis of XP-A cells used in the checkpoint activation experiment, and the bottom panel shows Western blot analysis of cell lysates that were used in ChIP assays probed with anti-S345P antibodies. The input gel represents a protein in the lysate that cross-reacts with anti-S345P antibody nonspecifically. (B) The top panel shows the FACS analysis of XP-C cells used in the checkpoint activation experiment, and the bottom panel shows Western blot analysis of cell lysates that were used in ChIP assays probed with anti-S345P antibodies. +, irradiation; –, no irradiation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Checkpoint signals and signal sensors. It is generally accepted that two classes of proteins recognize DNA damage and initiate checkpoint signaling. One class comprises the PIKK family kinases ATM and ATR and possibly DNA-PK. The other class consists of the checkpoint-specific clamp loader/clamp proteins, Rad17-RFC (clamp loader) and the 9-1-1 complex (checkpoint sliding clamp). The ATM pathway is activated by ionizing radiation, radiomimetic agents, and other conditions that produce double-strand breaks, whereas the ATR pathway is activated by UV and UV-mimetic agents, such as cisplatin, that produce base damages. It has been proposed that ATM is recruited to the site of double-strand breaks with the aid of the Nbs1 component of the MNR complex (9, 11, 18, 39). Similarly, it is thought that ATR is recruited to RPA-covered single-stranded DNA that is generated by stalled replication forks or by excision repair of base damage through its small subunit called ATRIP (8, 30, 57). In addition to the PIKK family proteins, the checkpoint-specific clamp loader/DNA clamp is needed for both ATM- and ATR-mediated checkpoint response (8, 35). Some evidence has been obtained that the Rad17-RFC-9-1-1 complexes are targeted to RPA-covered single-stranded DNA as well (10, 58).

Checkpoint activation by UV damage. There are a number of observations, however, that are not consistent with this unified model for the checkpoint response. In particular, it has been found that in XP-A cells, which are completely deficient in excision repair, UV does elicit a checkpoint response, as measured by p53 accumulation (22, 28, 53). Interestingly, in one of these studies, XP-A cells in G₁ phase required an about 10-fold lower UV dose to induce p53 to the same level as wild-type cells in G₁ phase (22), suggesting that the signal for the UV-activated checkpoint response is either the primary UV damage or a stalled RNA polymerase elongation complex. In our study, we demonstrate that RPA and ATR are recruited to damage in G₁ phase in XP-A cells. Second, the report that the ATR-ATRIP complex can bind to single-stranded DNA only when the DNA is covered by RPA has not been confirmed in a follow-up study (46) that found the ATR-ATRIP complex or ATR alone can bind to single- and double-stranded DNA with affinities comparable to that of RPA-covered DNA. In addition, proteins other than RPA have been proposed to mediate ATR-ATRIP-DNA binding (3, 6). Third, it was found that ATR binds to UV-induced (6-4) photoproducts with moderately higher affinity than undamaged DNA (45). The reports that RPA-covered template/primer type substrates are required for loading of the 9-1-1 checkpoint ring by Rad17/RFC are contradictory with respect to the type of primer required for binding, with one study reporting preferential dependence on the 5' overhang (58) and the other reporting absolute dependence on the 3' overhang (10). Moreover, in other studies with either the human Rad17-RFC-9-1-1 pair (5) or its functional equivalent in budding yeast, Rad24-RFC-Ddc1-Rad17-Mec3 (24), it was found that RPA either had no effect or mildly inhibited the loading of the 9-1-1 ring or its yeast equivalent onto primed DNA. While the cause of discrepancy between these reports remains to be investigated, clearly, at this point it is premature to consider a template-primer structure with RPA-covered single-stranded DNA as the optimal structure for loading of the 9-1-1 ring and activating the checkpoint response. Finally, several studies have provided strong evidence that, in mammalian cells, RNA polymerase stalling because of damage in the template strand or by specific RNA polymerase inhibitors activates the checkpoint response (21). Thus, an alternative view of checkpoint activation by UV is that ATR, RPA, and Rad17-RFC-9-1-1 may detect UV-induced base damage directly and they may function as sensors of stalled transcription and replication complexes.

Model for ATR-dependent UV checkpoint response. In this study, we have presented further evidence for the model described above for the ATR-mediated UV-induced DNA damage checkpoint response. First, we detect binding of the three damage sensors, RPA, ATR, and Rad9 (presumably in the form of the 9-1-1 complex) to damage sites in nontranscribed DNA and in the absence of replication. This result indicates that the primary lesion itself can be detected by damage sensors. It should be noted, however, that the binding of RPA to the sites of damage cannot be exclusively ascribed to damage detection for the purpose of checkpoint response. RPA is one of the three factors involved in damage recognition and initiation of nucleotide excision repair (34). Therefore, it could be argued that the binding of RPA to the UV damage site is a function of its role in excision repair. A more realistic view is that the binding of RPA to damage serves both purposes, that is the assembly of repair excision nuclease and the assembly of the DNA damage checkpoint sensor complex. Incidentally, the demonstration of binding of RPA to UV damage by ChIP in the absence of XPC helps address some of the uncertainty that has existed about the role of RPA in damage recognition during nucleotide excision repair and the order of assembly of human excision nuclease. In one model the three damage recognition factors, RPA, XPA, and XPC, bind to the damage site in random order and cooperatively (34), whereas in an alternative model (41), the binding follows a rigid order in which the binding of XPC must precede the assembly of the other factors. The fact that RPA binds with higher affinity to UV-damaged DNA than to undamaged DNA in the absence of XPC both in vitro (34) and as shown here in vivo constitutes strong evidence for the random order-cooperative assembly model. In addition to RPA, we also detect specific binding of ATR and Rad9 to UV damage in untranscribed DNA and in the absence of replication, indicating that the two independent checkpoint damage sensors can assemble at nonprocessed UV damage sites. It must be noted, however, that the discrimination between undamaged and damaged DNA for all three factors as revealed by ChIP is modest in the absence of transcription. Nevertheless, considering that in a given cell type the vast excess of genome is not transcribed (32, 53), it is likely that, in cells irradiated with UV in the G₁ phase, a significant fraction of the checkpoint damage sensors are assembled at lesions in untranscribed sequences.

Effect of transcription on checkpoint activation. A second noteworthy finding of our study is that RPA, ATR, and Rad9 are recruited more efficiently to transcribed sequences than nontranscribed sequences after UV irradiation. In addition to p53 and ß-globin we have tested the DHFR and IgE genes as representatives of transcriptionally active and inactive sequences, respectively, and have obtained similar preference for transcribed DNA (data not shown). Thus, we believe that, as a general rule, after DNA damage by UV, checkpoint proteins are recruited to both transcribed and nontranscribed DNA but with higher affinity to transcribed DNA. Since in both wild-type and the XP-C cell lines used in our study transcribed DNA is repaired by excision repair, it could be argued that the higher binding affinity of checkpoint proteins to transcribed sequences may be due to binding to the RPA-covered postexcision gaps generated by transcription-coupled repair. While our data do not eliminate this possibility, we find that checkpoint proteins are recruited with equal efficiency to transcribed DNA after UV irradiation of XP-A cells that do not carry out transcription-coupled repair. Thus, it is likely that both stalled RNA polymerase ternary complexes and postexcision gaps contribute to the recruitment of checkpoint proteins in agreement with data indicating checkpoint activation by stalled RNA polymerase, independent of DNA damage (21). This issue is further discussed below. It is noteworthy that recently Derheimer and colleagues found that specific blockage of elongating RNA polymerase II with antibody microinjection resulted in phosphorylation of p53 on Ser-15 in an RPA- and ATR-dependent manner (F. A. Derheimer, H. M. O'Hagan, and M. Ljungman, personal communication). This study provides independent evidence that stalled RNA polymerase II can activate DNA damage checkpoint directly, independent of postexcision gaps.

We find that checkpoint proteins RPA, ATR, and Rad9 are recruited to transcribed DNA even in the absence of DNA damage (Fig. 5B). It is not possible to ascertain whether this recruitment occurs only by transcription elongation complexes at natural pause sites or if recruitment of checkpoint proteins to the elongation complex is a physiological feature of transcription because of the presence of single-stranded DNA in the transcription bubble. In fact, the difference between the two models might be only a matter of semantics because elongation by RNA polymerase proceeds at variable rates dictated by template sequence and other factors (38), and it is reasonable to assume that there is a basal level of checkpoint activation by transcription under physiological conditions.

In summary, our data, in combination with previous studies on activation of DNA damage checkpoint by UV and UV-mimetic agents, indicate that (i) the primary DNA lesion itself, (ii) RNA polymerase ternary complex stalled at a UV photoproduct, and (iii) a replication fork stalled at a UV lesion or collapsed at the damage site may be recognized by checkpoint sensors and initiate the DNA damage checkpoint response.

 
This work was supported by NIH grant GM32833.

We thank Tadayoshi Bessho, Keziban Unsal-Kacmaz, and Laura Lindsey-Boltz for useful discussions and critical comments on the manuscript. We are grateful to Mats Ljungman for sharing unpublished data.

 
* Corresponding author. Mailing address: Department of Biochemistry and Biophysics, Mary Ellen Jones Building CB 7260, University of North Carolina School of Medicine, Chapel Hill, NC 27599. Phone: (919) 962-0115. Fax: (919) 843-8627. E-mail: Aziz_Sancar{at}med.unc.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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