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Molecular Cancer Research 2:362-369 (2004)
© 2004 American Association for Cancer Research

DNA Damage and Cellular Stress Responses

Chromatin Association of Rad17 Is Required for an Ataxia Telangiectasia and Rad-Related Kinase-Mediated S-Phase Checkpoint in Response to Low-Dose Ultraviolet Radiation1

Renu Garg1, Shannon Callens1, Dae-Sik Lim2, Christine E. Canman2, Michael B. Kastan2 and Bo Xu1

1 Department of Genetics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana and 2 Department of Hematology and Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee

Requests for reprints: Bo Xu, Department of Genetics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, Room 406, CSRB Building, 533 Bolivar Street, New Orleans, LA 70112. Phone: 504-568-2228; Fax: 504-568-8500. E-mail: bxu{at}lsuhsc.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Activation of the S-phase checkpoint results in an inhibition of DNA synthesis in response to DNA damage. This is an active cellular response that may enhance cell survival and limit heritable genetic abnormalities. While much attention has been paid to elucidating signal transduction pathways regulating the ionizing radiation–induced S-phase checkpoint, less is known about whether UV radiation initiates the process and the mechanism controlling it. Here, we demonstrate that low-dose UV radiation activates an S-phase checkpoint that requires the ataxia telangiectasia and Rad-related kinase (ATR). ATR regulates the S-phase checkpoint through phosphorylation of the downstream target structural maintenance of chromosomal protein 1. Furthermore, the ATPase activity of Rad17 is crucial for its chromatin association and for the functional effects of ATR activation in response to low-dose UV radiation. These results suggest that low-dose UV radiation activates an S-phase checkpoint requiring ATR-mediated signal transduction pathway.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
UV light radiation is considered the most important environmental DNA damage agent and it contributes to the development of over 1 million skin cancer cases every year in the United States. UV-induced DNA bulky adducts and strand breaks can cause point mutations and chromosomal aberrations, each of which has been directly linked to the development of cancer. However, the mechanisms by which mammalian cells respond to UV radiation are poorly understood.

Activation of cell cycle checkpoints in response to DNA damage is an important step in maintaining genomic stability and limiting tumorigenesis (1). The cell cycle can potentially arrest at the G1, S, and G2 phases of the cycle after UV radiation. The G1 and G2 phase responses represent checkpoint activation to prevent the cell progression through DNA replication or aberrant segregation of damaged chromosomes in the presence of DNA damage (2). However, UV-induced S-phase arrest, measured as a transient decrease of DNA synthesis, represents two distinct mechanisms: the result of physical obstruction of the DNA replication apparatus (because of cyclobutane pyrimidine dimers and 6-4 photoproducts) at the sites of DNA damage (3, 4) and/or the activation of checkpoint signals (5). Physical obstruction of the DNA replication apparatus at sites of DNA damage may cause passive inhibition of replication, while activation of the S-phase checkpoint will result in active inhibition of DNA replicon initiation (5). While the ionizing radiation (IR)–induced S-phase checkpoint has been extensively studied, less is known about the S-phase cell cycle checkpoint pathway in response to UV radiation.

A pair of large protein kinases with homology to phosphatidylinositol-3-OH kinases—the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad-related (ATR) kinases (6)—is believed to play critical roles in cellular response to IR- and UV-induced DNA damage. Loss of ATM in humans causes ataxia telangiectasia, an autosomal recessive disease characterized by progressive neurodegeneration, immunodeficiency, hypersensitivity to radiotherapy, and enhanced risk of lymphoblastoid malignancies (7). The ATM kinase is remarkable for its role in cellular response to IR. ATM is activated through autophosphorylation at a particular amino acid (Ser1981; ref. 8) and then phosphorylates a series of targets, including p53 (9, 10), Brca1 (11-13), Chk2 (14, 15), FancD2 (16), Mdm2 (17), Nbs1 (18-21), Rad17 (22), and structural maintenance of chromosomal protein 1 (SMC1; refs. 23, 24), to execute cellular responses to IR. In contrast to ATM, ATR appears to be an essential gene, as genetically knocking out of the gene causes embryo lethality (6). The similarities of ATM and ATR include, but are not limited to, sharing an identical consensus target recognition motif (S/TQ motif; ref. 25) and functioning as signal transducers in DNA damage pathways (6). However, the major difference between ATM and ATR is the type of damage to which each responds. For example, although cells without ATM function are hypersensitive to IR, they have normal sensitivity to UV. Although no direct evidence has been reported, ATM and ATR play distinct roles in IR and UV response. Experiments on Xenopus model have revealed that XATR is required for cellular response to replication blockage and UV radiation (26). Recent studies have revealed that disruption of DNA replication activates the ATR kinase (27, 28). In response to UV radiation, ATR may phosphorylate several proteins, including p53 (29), Brca1 (30), Chk1 (31), and Rad17 (22), to facilitate certain cellular responses. Recently, Heffernan et al. (5) demonstrated that ATR participates in regulation of UVC-induced S-phase checkpoint by activating Chk1. However, it is still not fully understood how ATR participates in the cell cycle checkpoint in response to UV.

Among the proteins required for DNA damage–induced cell cycle checkpoints, Rad17 shares homology with all five subunits of replication factor C (32, 33), which recognizes the primer template junction and loads proliferating cell nuclear antigen onto DNA during replication. Recent reports have revealed that Rad17 recruits Rad9 complex, the essential DNA damage recognizing protein complex, onto chromatin after UV radiation (34, 35). Depletion of Rad17 results in defective ATR-mediated cell cycle checkpoints (36). Meanwhile, Rad17 is also phosphorylated by ATR at two serine sites (Ser635 and Ser645) after DNA damage (22), and the phosphorylation events are required for DNA damage–induced G2 arrest. Therefore, Rad17 appears to be a component of ATR-mediated pathway.

In addition to ATR (5), Rad9 (37) and claspin (38) have also been implicated in controlling UV-induced S-phase response. However, these studies used relatively high UV doses (above 10 J/m2) to induce S-phase arrest. To study the effects of UV-induced signaling pathways on DNA replication, we chose to minimize the structural limitations on DNA replication imposed by bulky DNA adducts by studying effects following very low dose UV radiation. In the present study, we report that low-dose UV radiation activates an S-phase cell cycle checkpoint that requires functional ATR and the chromatin association of Rad17. In response to low-dose UV, ATR phosphorylates SMC1 to regulate the S-phase response. Furthermore, we find that the ATPase activity of Rad17 is required for its chromatin binding and for the ATR-mediated S-phase checkpoint.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Low-Dose UV Radiation Activates a Transient S-Phase Cell Cycle Checkpoint That Is Sensitive to Caffeine
To more carefully characterize the UV-induced S-phase checkpoint pathway, we used a dose range from 0 to 0.12 J/m2 to determine a low UV dose that is needed for activation of UV-induced S-phase checkpoint in a normal human primary fibroblast cell line. The radioresistant DNA synthesis assay (12, 39) was performed to measure the S-phase checkpoint. While 0.015 J/m2 UV exposure has limited effect on down-regulating DNA synthesis, we observed dramatic inhibition of DNA synthesis after 0.03 to 0.12 J/m2 UV radiation. UV exposure of 0.03 J/m2 reduces DNA synthesis by almost 80% (Fig. 1A). In the time course experiment after 0.03 J/m2 UV exposure (Fig. 1B), we observed a decrease (50%) of DNA synthesis within 15 minutes. The inhibition of DNA synthesis reached a peak at 90 minutes. Cells released from arrest and began to reenter active S phase of the cell cycle 120 minutes after UV radiation. The inhibition of DNA synthesis in response to low-dose UV radiation is diminished by the presence of 1 mmol/L caffeine (Fig. 1A and B). The low-dose range of UV radiation has a limited effect on mitotic entry (the G-M checkpoint) performed by counterstaining of phosphohistone H3 and propidium iodide (Fig. 1C). Because low doses of UV radiation (generally <1 J/m2) are believed to have limited cytotoxicity and very little inhibition of DNA chain elongation (5), our data presented here demonstrate that low-dose UV radiation triggers the S-phase checkpoint that is transient and caffeine sensitive.



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  		FIGURE 1. Low-dose UV radiation activates the S-phase cell cycle checkpoint. A. Replicative DNA synthesis was assessed 30 minutes after various doses of UV radiation in normal human fibroblast (NHF) cells treated with or without 1 mmol/L caffeine. B. Replicative DNA synthesis was assessed at indicated time points after 0.03 J/m2 UV radiation in normal human fibroblast cells in the presence or absence of 1 mmol/L caffeine. C. UV-induced G2/M checkpoint: mitotic percentage change of normal human fibroblast cells 90 minutes after UV radiation. Mitotic cell percentage was determined by anti-phosphohistone H3 staining followed by flow cytometric analysis. Bars, SD of at least triplicate samples.

 
ATR, but not ATM, Is Required for the UV-Induced S-Phase Checkpoint
Because the UV-induced S-phase checkpoint is abrogated by caffeine, a well-known ATM/ATR kinase inhibitor, it was suspected that ATM and/or ATR kinase might play an important role in activation of the checkpoint. To fully understand the roles of ATM and ATR in the UV-induced S-phase checkpoint, we performed the radioresistant DNA synthesis assay in cells proficient or deficient in ATM or ATR. Human fibroblast cell lines with proficient (GM0637) or deficient (GM9607) ATM displayed a normal response to UV radiation (Fig. 2A), suggesting that the S-phase checkpoint induced by UV is an ATM-independent process. We used an established cell system that had been engineered to contain a conditional allele of ATR (ATR+/–; ref. 40). ATR was deleted by introducing the Cre recombinase after AdCre virus infection, resulting in ATR-null cells (ATR–/–). HCT116 (ATR+/+), which is the parental cell line of this system, was used as a positive control. We found that ATR–/– cells display an impaired S-phase arrest in response to UV, while parental cells arrest at S-phase normally (Fig. 2B). Interestingly, heterozygous ATR+/– cells, which normally only express 20% of the protein due to manipulation of the gene (40), have an intermediate response to UV radiation. These data support a previous report (5) that functional ATR is required for UV-induced S-phase checkpoint. This conclusion is further confirmed by our observation that overexpressing kinase-inactive form of ATR can abolish the checkpoint (data not shown). It is noted that AdCre virus infection and depletion of ATR do not change the basal level of DNA synthesis (data not shown).



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  		FIGURE 2. UV-induced S-phase checkpoint requires functional ATR. A. Replicative DNA synthesis was assessed 30 minutes after various doses of UV radiation in control cells (GM0637) and cells defective in ATM (GM9607). B. Replicative DNA synthesis was assessed 30 minutes after various doses of UV radiation in ATR+/+ (HCT116), ATR+/– (HCT flox), or ATR–/– (HCT flox infected with AdCre virus) cells. Bars, SD of at least triplicate samples.

 
ATR Is Required for UV-Induced SMC1 Phosphorylation
We and others reported previously that SMC1 participates in the DNA damage response by ATM phosphorylation at Ser957 and Ser966 in response to IR (23, 24). We also reported an ATM-independent SMC1 phosphorylation after UV radiation (23); however, the mediator of this UV-induced SMC1 phosphorylation was not elucidated. To determine whether ATR is responsible for UV-induced SMC1 phosphorylation, we performed Western blot analysis on cell lysates formed from the conditional ATR knockout cells 30 minutes after treatment with 0.06 J/m2 UV radiation or 6 Gy IR. A phosphospecific antibody recognizing Ser957 phosphorylation of SMC1 was used to study SMC1 phosphorylation. We found that SMC1 is phosphorylated 30 minutes after UV or IR exposure, and the ATR–/– cells show Ser957 phosphorylation after IR, suggesting that ATR is not required for IR-induced SMC1 phosphorylation. However, depletion of ATR totally abrogates UV-induced SMC1 phosphorylation (Fig. 3A), demonstrating that ATR is specifically required for UV-induced SMC1 phosphorylation.



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  		FIGURE 3. ATR phosphorylation of SMC1 is required for UV-induced S-phase checkpoint. A. Immunoblot analyses using a phosphoserine-specific antibody against SMC1-Ser957 phosphorylation (top) or an anti-SMC1 antibody (K1; bottom) on SMC1 protein from ATR+/+ (HCT116), ATR+/– (HCT116 flox), or ATR–/– (HCT flox infected with AdCre virus) cells 30 minutes after treatment with 0.06 J/m2 UV radiation. B. 293T cells transiently transfected with empty vector or wild-type (WT) or mutant (2S/A, Ser957 and Ser966 to alanine mutant) of SMC1 were assessed for inhibition of DNA synthesis 30 minutes after exposure to 0.06 J/m2 UV radiation. Bars, SD of at least triplicate samples. Expression of transduced Myc-tagged SMC1 was assessed by immunoblotting with an anti-myc antibody.

 
ATR Phosphorylates SMC1 to Activate the S-Phase Arrest After UV Radiation
Because SMC1 is phosphorylated by ATM in response to IR in the S-phase checkpoint pathway, we hypothesized that ATR phosphorylation of SMC1 plays a similar role in activation of the UV-induced S-phase checkpoint. To test this hypothesis, we overexpressed vector-only, wild-type, or serine-to-alanine mutant SMC1 constructs into 293T cells. We found that cells transiently transfected either with an empty vector or with wild-type SMC1 exhibited a normal UV-induced S-phase arrest (Fig. 3B). In contrast, cells transiently transfected with vectors expressing SMC1 containing the serine-to-alanine mutation at Ser957 and Ser966 exhibited substantially impaired S-phase arrest after UV radiation (Fig. 3B). These observations demonstrate that, similar to its role in IR response, SMC1 phosphorylation by ATR in response to UV is involved in activation of UV-induced S-phase checkpoint. It is noted that 293T cells have functional ATR and have an intact S-phase arrest in response to UV radiation. Overexpression of the exogenous SMC1 proteins does not affect the basal level of DNA synthesis (data not shown).

The ATPase Activity of Rad17 Is Crucial for Its Binding to Chromatin
Our next question concerned how ATR gets activated by UV radiation. Rad17 is believed to serve as a DNA damage sensor due to its ability to bind chromatin and its similarity to the five replication factor C subunits (32, 33). Rad17 is also phosphorylated by ATR at Ser635 and Ser645 in response to DNA damage (22). A recent article suggests that Rad17 is required for ATR function in response to DNA damage (36). Because Rad17 has weak ATPase activity that is stimulated by primed DNA and ssDNA (33, 34), it is likely that ATPase activity is required for Rad17 to bind chromatin and, consequently, will affect the function of its downstream targets. To study whether the ATPase activity of Rad17 is involved in ATR-dependent S-phase arrest, we constructed a series of Rad17 cDNA, including wild-type, phosphorylation site mutant (Ser635 and Ser645 to alanine mutant, 2S/A), and ATPase activity mutant (Lys132 to glutamic acid mutant, K132E). These constructs were expressed in 293T cells to investigate whether these mutants affected its chromatin binding. By fractionating extracts of asynchronously growing 293T cells, we obtained fractions of cytoplasmic proteins, soluble nuclear proteins, and a fraction enriched for chromatin-bound proteins. A portion of wild-type Rad17 is detected in the chromatin fraction (Fig. 4). We observed that the phosphorylation site mutant of Rad17 still binds to chromatin, suggesting that the phosphorylation status of Rad17 is independent of its chromatin association. Interestingly, the ATPase activity mutant (K132E) loses the ability to bind to chromatin, indicating that the ATPase activity of Rad17 is required for chromatin association. It is noted that neither the phosphorylation site mutant nor the ATPase mutant affected chromatin association of the nuclear protein SMC1 (Fig. 4).



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  		FIGURE 4. The ATPase activity of Rad17 is required for its chromatin association. Extracts of 293T cells, which were transfected with empty vector, wild-type (WT), or mutant (K132E or 2S/A) of Rad17, were fractionated as described in Materials and Methods. The resultant fractions were resolved on SDS-PAGE and immunoblotted with the indicated antibodies.

 
Chromatin Association of Rad17 Is Required for the ATR-Mediated S-Phase Checkpoint in Response to UV Radiation
Because we found that the ATPase activity of Rad17 affects its chromatin binding, we further hypothesized that this ATPase ability is required for ATR function in response to UV. To test this hypothesis, we overexpressed mutant Rad17 cDNAs into 293T cells and looked for the dominant negative activity in the UV-induced S-phase checkpoint (Fig. 5A). To assess the transfection efficiency in each experiment, we transfected with a green fluorescent protein reporter vector and analyzed for green fluorescent protein expression by flow cytometric analysis 36 hours after transfection. The efficiency of transfection in multiple assessments was always between 90% and 97% (data not shown). Our data show that cells expressing empty vector or wild-type Rad17 have an optimal SMC1 phosphorylation (Fig. 5B) and a normal S-phase arrest (Fig. 5C) in response to IR and UV radiation. Overexpression of serine-to-alanine mutant of Rad17 in 293T cells does not affect UV-induced SMC1 phosphorylation (Fig. 5B) and the S-phase arrest (Fig. 5C), while it affects DNA damage–induced G2-M checkpoint,3 indicating that ATR/ATM phosphorylation of Rad17 is independent of UV-induced S-phase checkpoint. However, we found that overexpressing the ATPase mutant form of Rad17 significantly diminishes the SMC1 phosphorylation and the S-phase arrest, leading to radioresistant DNA synthesis (the radioresistant DNA synthesis phenotype; Fig. 5C). It is interesting that overexpressing this DNA binding site mutant not only affects UV response but also abolishes IR-induced SMC1 phosphorylation and the S-phase arrest. These results indicate that Rad17 DNA binding activity is essential for both UV- and IR-induced S-phase cell cycle checkpoints. It is noted that expression of these mutant constructs affected only the DNA replication after UV or IR exposure and had no effects on the basal level of DNA replication.



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  		FIGURE 5. The chromatin binding activity of Rad17 is required for IR- and UV-induced S-phase checkpoint. 293T cells transiently transfected with empty vector or wild-type (WT) or mutant (K132E or 2S/A) of Rad17 were assessed for (A) expression of Flag-tagged Rad17 proteins and total Rad17 proteins, (B) SMC1-Ser957 phosphorylation 30 minutes after exposure to IR (6 Gy, 30 minutes) or UV light (0.06 J/m2, 30 minutes), and (C) inhibition of DNA synthesis 30 minutes after exposure to IR (6 Gy, 30 minutes) or UV light (0.06 J/m2, 30 minutes). Bars, SD of at least triplicate samples.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Cell cycle checkpoints activated by environmental DNA damage agents, such as IR or UV, delay cell cycle progression at the G1, S, and G2 phases of the cell cycle. These mechanisms ensure that DNA replication and chromosomal segregation during each cell cycle are completed in order and with fidelity. The fundamental importance of these cell cycle checkpoints is underscored by the genetic instability that results from the loss of checkpoint functions and the involvement of such defects in the evolution of normal cells to cancer (41, 42).

Using very low dose UV radiation, which can minimize the structural limitations on DNA replication imposed by UV-induced bulky DNA adducts, our data presented here provide direct evidence that ATR is specifically involved in UV-induced S-phase checkpoint through phosphorylating the downstream target SMC1. However, how phosphorylation of SMC1 inhibits DNA replication is still not understood. There was speculation that phosphorylation of SMC1 after IR might interfere with the replication elongation process by interfering with the establishment of cohesion between the template and the sister chromatid that is being elongated, thus slowing down the progression of the replication fork (24). Paradoxically, we have found that low-dose UV radiation, which does not inhibit DNA chain elongation, still activates the ATR-SMC1 pathway. Therefore, SMC1 phosphorylation by ATR in response to UV may directly affect cell cycle regulators, possibly cyclin E/Cdk2. The detailed mechanisms remain to be elucidated.

There is evidence that ATR participates in the UV-induced S-phase checkpoint through activation of Chk1 (5). Other components, including Rad9 (37) and claspin (38), are also reported in UV-induced S-phase response. It is conceivable that, in response to UV radiation, ATR may simultaneously phosphorylate several proteins, such as Chk1 and SMC1, to regulate the S-phase checkpoint. We found that although heterozygous ATR cells (ATR+/–) have an intermediate S-phase checkpoint (Fig. 2B), SMC1 phosphorylation is intact in the cells (Fig. 3A), suggesting that multiple pathways act in concert to cause S-phase arrest after UV radiation.

Rad17 is one of the proteins that may function as an upstream component of ATR in the pathway. This model is supported by the fact that Rad17 binds to chromatin to recruit DNA damage protein complexes in the presence of DNA damage (43) and that depletion of Rad17 leads to a defective ATR function (36). Our data presented here clearly demonstrate that the ATPase activity of Rad17 is essential for its chromatin association and for the ATR-mediated UV response. How this model fits into a recently reported replication protein A-ATR interacting protein-ATR model (44) for DNA damage sensing is still unknown. It is quite likely that both Rad17 and replication protein A recruitment to the sites of DNA damage is required for the activation of the checkpoint kinases, such as ATM and ATR, although Rad17 and ATR can be recruited to DNA damage independently (43). Our observations that Rad17 DNA binding mutant abrogates the IR-induced S-phase checkpoint suggest that, in addition to its role in ATR-dependent UV response, Rad17 may also play an important role in ATR-independent IR response.

Our results, together with the known essential replication checkpoint role of ATR and Rad17, support the current model that ATR plays a central role in regulation of cellular response to UV radiation. Future direction is to examine how SMC1 phosphorylation signals to the replication fork to halt replicon initiation in response to UV radiation.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Plasmids
To construct the Flag-tagged Rad17 expression vector, we amplified the entire coding region of Rad17 by performing a PCR. The PCR product was cloned into a pSG5 vector (Stratagene, La Jolla, CA) that encoded c-Flag tag. The Quick-Change Site-Directed Mutagenesis kit (Stratagene) was used to generate the following Rad17 mutants: 2S/A and K132E.

Cell Lines
SV40-transformed human fibroblast lines from a healthy ataxia telangiectasia heterozygote or from a patient with ataxia telangiectasia (GM0637 or GM9607, respectively; Human Mutant Cell Repository, National Institute of General Medical Sciences) and 293T and HCT116 cells (both from American Type Culture Collection, Manassas, VA) were all grown as monolayers in DMEM supplemented with 10% fetal bovine serum. Diploid normal human fibroblasts were established from neonatal circumcision specimens collected at the Medical Center of Louisiana (New Orleans, LA; ref. 45). Normal human fibroblasts were cultured in DMEM containing 10% fetal bovine serum and 100 units/mL penicillin/streptomycin and generally used at passage 6. ATRflox/– cells, which allow for Cre/lox-mediated removal of ATR, were obtained from Dr. Stephen J. Elledge (Baylor College, Houston, TX) and maintained in MEM{alpha} medium + 10% fetal bovine serum + 100 µg/mL G418. Expression of Cre recombinase in these cells was accomplished through infection with adenovirus AdCre1, which was obtained from Dr. Jakob Reiser (Vector Core Laboratory, Louisiana State University Health Sciences Center). All cell lines were grown at 37°C in a humidified atmosphere containing 5% CO2.

Delivery of Radiation
IR was delivered from a 137Cs source at a rate of ~120 cGy/min. UV radiation was delivered using a UV light lamp, and the dosage was measured using a UV meter (Control Company, Friendswood, TX). For UV radiation experiments, cells were plated into 60 mJ/m2 dishes. Prior to treatment with UV, culture medium was removed and reserved. Cultures were washed once with conditional PBS buffer and placed uncovered under the UV source. Following radiation, reserved medium was replaced, and the cultures were incubated for the indicated periods of time.

Chromatin Fractionation
The chromatin fractionation was performed as described by Mendez and Stillman (46). About 2 x 106 cells transfected with either Flag-tagged wild-type, K132E, mutant 2S/A form of Rad17, or vector were washed with PBS and resuspended in 200 µL of solution A [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.34 mol/L sucrose, 10% glycerol, 1 mmol/L NaF, 1 mmol/L Na2VO3, 0.1% Triton X-100, and protease inhibitors]. The cells were incubated on ice for 5 minutes, and cytoplasmic proteins were separated from nuclei by low-speed centrifugation at 1,300 x g for 4 minutes. The nuclei were washed with solution A and lysed in 200 µL of solution B (3 mmol/L EDTA, 0.2 mmol/L EGTA, 1 mmol/L DTT, and protease inhibitors) followed by incubation on ice for 10 minutes. The lysate was centrifuged at 1,700 x g for 4 minutes to separate soluble nuclear proteins from chromatin. The isolated chromatin was washed with solution B and pelleted by centrifugation at 10,000 x g for 1 minute. The chromatin was resuspended in 200 µL of SDS sample buffer and sheared by sonication.

Antibodies
Immunoblotting and immunoprecipitation studies used the following antibodies: anti-Flag M2, M5 (Sigma Chemical Co., St. Louis, MO), anti-SMC1 rabbit polyclonal antibody (Bethyl Laboratories, Houston TX), and an anti-phospho-Ser957 of SMC1 that is an affinity-purified antibody from keyhole limpet hemocyanin–conjugated phosphopeptide SQEEGS[PO3]SQGEDS.

Western Blot Analyses
Logarithmically growing cells were seeded at 106 per 60 mm dish, irradiated as described above, and incubated in reserved medium for 30 minutes at 37°C. Cells were harvested by trypsinization, washed with PBS, and resuspended in Tris-Glycine-NP40 (TGN) lysis buffer. Protein concentrations were determined using the detergent-compatible protein assay (Bio-Rad Laboratories, Richmond, CA). Samples containing equal amounts of protein were mixed with an equal volume of 2x Laemmli sample buffer, boiled, and separated by SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted with the antibodies listed above.

S-Phase Checkpoint Assay
Inhibition of DNA synthesis after UV radiation was assessed as described previously. Cells were prelabeled with 10 nCi of [14C]thymidine (NEN Life Science Products, Inc., Boston, MA) for 24 hours. Cells were UV radiated, incubated for 30 minutes, and pulse labeled with 2.5 µCi of [3H]thymidine (NEN Life Science Products) per milliliter. Cells were harvested and fixed with 70% methanol. The amount of radioactivity was assayed in a liquid scintillation counter. The measure of DNA synthesis was derived from resulting ratios of 3H to 14C counts per minute, corrected for counts that resulted from channel crossover.

G2-M Checkpoint Assay
Cells were harvested at the indicated time points after 6 Gy IR and fixed in 70% ethanol at –20°C. The cells were suspended in 100 µL of PBS containing 1% bovine serum albumin and 0.75 µg of a polyclonal antibody that specifically recognizes the phosphorylated form of histone H3 (Upstate Biotechnology, Lake Placid, NY) and incubated for 3 hours at room temperature. The cells were rinsed with PBS containing 1% bovine serum albumin and incubated with FITC-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted at a ratio of 1:30 in PBS containing 1% bovine serum albumin. After 30-minute incubation at room temperature in the dark, the cells were stained with propidium iodide (Sigma Chemical), and cellular fluorescence was measured by a FACSCalibur flow cytometer.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. Steven Elledge for providing the ATR flox cell, Dr. Jacob Reiser for providing the AdCre virus, and Dr. Kevin Brown for reading the manuscript.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Cancer Association of Greater New Orleans (B. Xu) and NIH (M.B. Kastan).

Note: The editor-in-chief of Molecular Cancer Research is a coauthor of this paper. In keeping with the AACR's Editorial Policy, a member of the AACR's Publications Committee had the paper reviewed independently of the journal's editorial process and made the decision whether to accept the paper. Back

3 Unpublished data. Back

Received February 26, 2004; revised April 30, 2004; accepted May 17, 2004.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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