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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Oncogene. 2006 Jul 24;26(5):757–764. doi: 10.1038/sj.onc.1209828

ATR-Dependent Checkpoint Modulates XPA Nuclear Import in Response to UV Irradiation

Xiaoming Wu 1,*, Steven M Shell 1,*, Yiyong Liu 1, and Yue Zou 1,‡
PMCID: PMC3106104  NIHMSID: NIHMS61848  PMID: 16862173

Summary

In response to DNA damage, mammalian cells activate various DNA repair pathways to remove DNA lesions and, meanwhile, halt cell cycle progressions to allow sufficient time for repair. The nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint activation are two major cellular responses to DNA damage induced by UV irradiation. However, how these two processes are coordinated in the response is poorly understood. Here we showed that the essential NER factor XPA underwent nuclear accumulation upon UV irradiation, and strikingly, such an event occurred in an ATR (Ataxia-Telangiectasia Mutated and RAD3-Related) dependent manner. Either treatment of cells with ATR kinase inhibitors or transfection of cells with siRNA targeting ATR compromised the UV-induced XPA nuclear translocation. Consistently, the ATR-deficient cells displayed no substantial XPA nuclear translocation while the translocation remained intact in ATM (Ataxia-Telangiectasia Mutated)-deficient cells in response to UV irradiation. Moreover, we found that ATR is required for the UV-induced nuclear focus formation of XPA. Taken together, our results suggested that the ATR checkpoint pathway may modulate NER activity through the regulation of XPA redistribution in human cells upon UV irradiation.

Introduction

Nucleotide excision repair (NER) is a major DNA repair mechanism that cells use to remove a large variety of structurally unrelated bulky DNA lesions. In eukaryotic cells the process of NER requires more than 25 proteins for DNA damage recognition, incision, excision and repair re-synthesis to restore the original DNA structure (Thoma & Vasquez, 2003). Among the NER proteins, XPA (xeroderma pigmentosum group A) plays a unique role in DNA damage recognition as it is required for both global genome repair (GGR) and transcription-coupled repair (TCR). XPA is also involved in assembly of NER factors at DNA damage site through protein-protein interactions (Costa et al., 2003; Sancar et al., 2004).

In addition to the repair of DNA damage in response to genotoxic insults, cells also have evolved the mechanisms to coordinate repair processes with other cellular pathways. The DNA damage checkpoints are surveillance mechanisms that monitor the integrity of genome and, if activated, halt the replication of DNA to allow sufficient time for DNA repair (Zhou & Elledge, 2000). The checkpoint signaling cascades, conceptually, consist of three major biochemical components: damage sensors, signal mediators/transducers, and effector molecules. In mammalian cells, the ATR and ATM proteins which belong to the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and the Rad9-Rad1-Hus1/Rad17-Rfc2-5 checkpoint complex have been suggested to be involved in damage recognition and signaling (Kastan & Bartek, 2004; Wu et al., 2005a) (Abraham, 2001; Bartek et al., 2004). The ATM kinase seems to be activated primarily following generation of double-stranded DNA breaks (DSB), whereas ATR kinase is critical for cellular responses to a variety of DNA damage. When activated, these serine/threonine kinases phosphorylate and activate downstream checkpoint factors in DNA damage response network and eventually lead to the cell cycle arrest (Kastan & Lim, 2000; Kim et al., 1999; O'Neill et al., 2000). Recent evidences have indicated that checkpoint pathways may also modulate DNA repair processes, through the regulation of phosphorylation and intracellular redistribution of DNA repair proteins (Barr et al., 2003; Feng et al., 2004; Wu et al., 2006; Zhou & Elledge, 2000).

While NER and DNA damage checkpoint machineries are able to recognize DNA damage independently in vitro, it is believed that both pathways may act in a cooperative manner in cells. However, the cellular relationship between NER and the ATR/ATM-dependent checkpoints in DNA damage responses remains elusive. Several studies have suggested that NER may function in the upstream of cellular checkpoint response to UV irradiation as NER processing of UV damage was necessary for checkpoint activation in non-replicating cells (Giannattasio et al., 2004; Neecke et al., 1999; O'Driscoll et al., 2003). On the other hand, two checkpoint genes in budding yeast (RAD9 and RAD24, the yeast homologues of human BRCA1 and Rad17, respectively) have been shown to be required for the induction of NER (Yu et al., 2001). In mammalian system, the checkpoint abrogator UCN-01 inhibited the NER activity against cisplatin (Jiang & Yang, 1999). Given the central role of ATR/ATM kinases in the entire DNA damage response network, it is of particular interest to examine whether these kinases could directly regulate the cellular NER activity in response to UV damage. Previously, we have identified XPA as a phosphorylation substrate for checkpoint kinase ATR in response to UV irradiation (Wu et al., 2006). Here we found that XPA undergoes a dramatic cytoplasm-to-nucleus translocation upon treatment of cells with UV, and this event also was regulated by ATR. Our results presented herein provide evidence to support the functional link between NER and ATR-dependent checkpoint pathway and suggest a potential mechanism in which NER and ATR checkpoint function cooperatively in cellular responses to UV-induced DNA damage.

Results

To determine the subcellular localization of XPA in cells treated with or without UV irradiation, A549 cells were irradiated with increasing doses of UV, followed by preparation of cytoplasmic and nuclear lysates 2 h after treatment (Wu et al., 2005a; Wu et al., 2005b; Zou et al., 2002). Western blot analysis of the samples revealed that the XPA was accumulated in nucleus upon UV irradiation in a dose-dependent manner up to 20 J/m2 (Fig.1A, left panel). The double bands of XPA in SDS-PAGE at the positions of ~38 and 40 kDa probably are probably due to the different reduction status of XPA related to the formation of disulfide bonds within the protein (Iakoucheva et al., 2001; Liu et al., 2005; Miura et al., 1991). Consistently, the XPA in cytoplasmic fraction was coincidently reduced, demonstrating that XPA was redistributed from cytoplasm to nucleus upon UV treatment. In this experiment, both PARP and β-actin were used as nuclear and cytoplasmic proteins, respectively, for subcellular fractionation and loading controls. The constant remaining of PARP and β-actin in nucleus and cytysol, respectively, during cytoplasmic and nuclear extract preparations validated our extraction procedures in which no protein leaking between nucleus and cytysol occurred. Furthermore, an increasing accumulation of XPA in nucleus in a dose dependent manner was also observed by immunofluorescence analysis of the treated whole cells (Fig 1A, middle panel). The anti-XPA antibody used in the present study was highly specific as demonstrated by Western blot analysis of whole cell lysates (Fig 1A, right panel). Results from this analysis also showed that the total levels of XPA in the cells remain essentially no change with increasing UV doses. In a time-course experiment, 10 J/m2 of UV irradiation resulted in a significant XPA nuclear accumulation in cells about 30 min after the exposure, which reached a maximum about 8 h later and decreased to the basal level 24 h after irradiation (Fig.1B, and data not shown). Consistent results were also obtained from the immunofluorescence analysis of the cells treated with UV (Fig 1B, right panel). Similar to the dose-dependent experiments in Fig 1A, the total levels of XPA in the cells did not change in the time-course experiments either (data not shown). Interestingly, such nuclear translocation of XPA was not observed in cells treated with camptothecin (CPT) (Fig. 1C), a topoisomerase inhibitor that induces double strand breaks, suggesting that the nuclear translocation of XPA is a NER-related or specific event. It should be mentioned that 5 uM of CPT treatment even had significantly higher cytotoxicity than 10 J/m2 UV irradiation for cells with over-expression of XPA protein. Finally experiments with other two cell lines (HeLa and MCF7) exhibited the similar UV-induced XPA nuclear accumulation (data not shown).

Figure 1. Induction of XPA nuclear accumulation by UV irradiation.

Figure 1

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(A) Exponentially growing A549 cells were treated with increasing doses of UV followed by a 2 h recovery. Proteins from the nuclear extracts, N, and cytoplasmic fractions, C, of the cells were separated on SDS-PAGE gels and probed with anti-XPA antibody (left panel). The UV-treated whole cells were also analyzed by immunofluorescence microscopy (middle panel). The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). Specificity of anti-XPA antibody used in these experiments and the total levels of XPA in the whole cell lysates versus UV doses were demonstrated by Western blotting in the right panel. GADPH was used as the loading control. (B) A549 cells were treated with 10 J/m2 UV irradiation, and then harvested at the indicated time after treatment. Cytoplasmic and nuclear extracts were prepared and probed with anti-XPA antibody. The UV-treated whole cells were also analyzed by immunofluorescence microscopy in a time-dependent manner (right panel). (C) A549 cells were treated with indicated doses of camptothecin (CPT) for 2 h, and then washed with PBS and further incubated for another 2 h. Cytoplasmic and nuclear extracts were prepared and probed with anti-XPA antibody.

Since the checkpoints play an important role in cellular DNA damage responses by regulating cell cycle progression, we were particularly interested in determining the relationship between the DNA damage checkpoints and NER. In particular, we wanted to address whether ATR/ATM plays a role in governing UV-induced XPA nuclear translocation. For this purpose, wortmannin and caffeine, two widely used checkpoint kinase inhibitors, were employed in our experiments (Sarkaria et al., 1999; Sarkaria et al., 1998). Cells were incubated in the presence or absence of wortmannin or caffeine after 20 J/m2 UV exposure. Efficiency of the ATR-signaling inhibition was demonstrated by the loss of UV-induced phosphorylation of the downstream signaling effector p53 at Ser-15 (Figure 2A, lower panel). Strikingly, Western blotting of the nuclear extracts did show that use of either agent led to a significant reduction in the UV-induced XPA nuclear accumulation (Figure 2). By contrast, the nuclear translocation of Hus1, a subunit of Rad9-Rad1-Hus1 checkpoint complex which has been suggested to be recruited to chromatin independently of ATR (Zou et al., 2002), remained uncompromised. Also interestingly, the nuclear accumulation of the recombinational repair factor Rad51 in response to UV was not affected by wortmannin or caffeine treatment (Figure 2A). The Hus1 and Rad51 nuclear accumulation which appeared to be independent of ATR/ATM was likely due to the cellular response to DSBs converted from cyclobutane pyrimidine dimer (CPD) lesions induced by UV in a replication-dependent manner (Garinis et al., 2005), further confirming the specificity of ATR modulation of XPA nuclear accumulation. As reported previously by other laboratories (Dart et al., 2004; Gately et al., 1998), the nuclear levels of ATR and ATM remained unchanged after UV irradiation and served as the excellent loading controls in the present experiment (Figure 2A).

Figure 2. UV-induced XPA nuclear import is wortmannin and caffeine sensitive.

Figure 2

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(A) A549 cells were mocked treated or treated with 20 J/m2 UV irradiation, and then incubated for 4 h in the presence of 100 μM wortmannin (Wort) (lanes 5 and 6) or 10 mM caffeine (Caff) (lanes 3 and 4) before harvesting. Nuclear extracts were analyzed by Western blotting probed with anti-XPA, anti-ATR and anti-ATM, respectively. Asterisk represents the phosphorylated form of XPA, which is also wortmannin and caffeine sensitive (Wu et al., 2006). (B) The UV-induced XPA nuclear accumulation was quantified using densitometry. The values (the mean ± SD of three independent experiments) were normalized to that for mock treated cells (as the value of 1).

To further investigate the role of ATR and ATM in this process, cells were transfected with siRNA targeting ATR or ATM prior to UV irradiation. As shown in Figures 3A and 3B, UV-induced XPA nuclear accumulation was abolished following transfection with ATR siRNA, while the accumulation was only slightly affected by ATM siRNA transfection. Transfection with either ATR or ATM siRNA generated little effects on UV-induced Hus1 and Rad51 nuclear translocation (Figure 3A), again indicating that the ATR regulation of XPA redistribution upon UV exposure was a specific cellular event. The critical role of ATR in the UV-induced XPA nuclear accumulation was further confirmed in similar experiments with ATR and ATM deficient cells (Figures 4A and 4B). These observations also were supported by our immunofluorescence microscopic determination. As shown in Figure 3C, XPA in unirradiated cells was overwhelmingly present in cytoplasm (subpanels a–c), while XPA in the UV-treated cells was largely translocated into nucleus (subpanels d–f). However, in the presence of ATR siRNA in cells, XPA failed to be re-distributed to the nucleus following UV irradiation (subpanel i), although cells transfected with GFP siRNA had no effect on XPA nuclear accumulation (subpanel l). The calreticulin (CAL) is an ER-lumen protein and was used as a cytosol protein control. The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). These results indicated that the UV-induced re-distribution of XPA to the nucleus was ATR kinase-dependent and the lack of re-distribution of XPA in ATR-siRNA transfected cells was not an artifact of transfection. To confirm that the XPA-required NER is impaired in the cells with ATR knocked down, we performed the immunofluorescence-based DNA repair assay by measuring the removal of UV-induced (6-4) photoproducts (6-4PPs) in cells transfected with ATR-specific siRNA in a time-course dependent manner (Figure 3D). The UV was irradiated through a filter containing 5 μm pores overlaid on the cells. Consistently, the DNA lesions in ATR-knocked down cells were significantly more persistent than those in the control cells (transfected with GFP siRNA).

Figure 3. ATR is required for XPA nuclear accumulation following UV irradiation.

Figure 3

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(A) A549 cells were transfected with indicated siRNA, and then treated with 20 J/m2 UV 72 h after transfection. Nuclear extracts were isolated 4 h after UV treatment and immunoblotted with the antibodies indicated above. Asterisk represents the phosphorylated form of XPA (Wu et al., 2006). (B)The quantities of nuclear XPA after UV treatment were estimated using densitometry and normalized to those obtained from mock treated cells, which were designated as 1.0. (C) A549 cells were transfected with siRNA targeting either ATR or GFP, or mock-transfected and then irradiated with 20 J/m2 UV 72 hours after transfection. Cells were then fixed and stained with antibodies for either XPA (red) or calreticulin (green), an ER-lumen protein; nuclei were stained with DAPI. Subpanels c and f illustrate the cellular distribution of XPA in normal (mock-transfected) cells with XPA located primarily in the cytosol prior to UV irradiation (subpanel c) and accumulating in the nuclei following irradiation (subpanel f). Cells transfected with siRNA for ATR did not show similar translocation of XPA to the nucleus following UV irradiation (subpanel i). However, cells transfected with GFP siRNA had no effect on XPA nuclear accumulation (subpanel l). (D) Removal of UV-induced (6-4)PPs over time were assayed in the cells transfected with siRNAs for ATR and GFP, respectively, as analyzed by immunofluorescence with anti-(6-4)PPs antibody. The UV was irradiated through a filter containing 5 μm pores overlaid on the cells. The foci represent the (6-4)PPs lesions.

Fig. 4. UV-induced XPA nuclear accumulation is defective in ATR deficient cells.

Fig. 4

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(A) A549 cells, ATR and ATM deficient cells were treated with indicated doses of UV, and nuclear extracts were prepared 4 h after treatment for Western blotting with anti-XPA antibody. Asterisk represents the phosphorylated form of XPA (Wu et al., 2006). (B) The quantitative data of Figure 4A, which represent the mean ± SD of three independent experiments.

We also examined the dependence of XPA nuclear focus formation on ATR using the method of immunofluorescence. Exposure of GFP siRNA-transfected cell cultures to UV resulted in more than 85% of cells exhibiting ATR and XPA foci (Figure 5). XPA foci have been previously shown to colocalized with UV-induced photolesions (Volker et al., 2001; Wang et al., 2006). As predicted, siRNA-mediated repression of ATR expression led to a reduction of ATR nuclear foci in UV-treated cells to levels comparable to untreated controls (Figure 5). Strikingly, however, knockdown of ATR resulted in a corresponding decrease in XPA foci induced by UV treatment (Figure 5). These results, in combination with the above data (Figures 3 and 4), suggest that ATR may exert effects on NER pathway through regulation of the UV-induced XPA intracellular redistribution and thus recruitment to DNA lesions.

Fig. 5. ATR is required for the UV-induced XPA foci formation.

Fig. 5

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(A) A549 cells were transfected with ATR siRNA or GFP siRNA as described in Materials and Methods, and then treated with 20 J/m2 UV, 72 h after transfection. Cells were then subjected to immunofluorescence assays with anti-XPA and anti-ATR antibodies. Subpanels B and F (green) are anti-XPA stained cells; Subpanels C and G (red) are anti-ATR stained cells. Subpanels D and H are the merged images of the anti-XPA and anti-ATR stained cells. Subpanels A and E are DAPI (4',6-diamidino-2-phenylindole)-stained nuclei. (B) and (C) The UV-induced ATR and XPA nuclear foci were scored from a total of randomly picked at least 100 cells in two independent experiments. The cells containing more than 10 foci/cell were defined as positively stained cells. The data represented the percentage of positively stained cells.

Discussion

NER and DNA damage checkpoints are two major pathways in cellular DNA damage responses. However, how these two processes are coordinated in cells in response to DNA damage remains unclear. Although it was previously reported that the cellular expression level of XPC was regulated by p53 (Adimoolam & Ford, 2002; Ford, 2005), a downstream effector in G1/S checkpoint (Sancar et al., 2004), XPC is a NER factor only for GGR subpathway. Importantly, in intra-S phase checkpoint lockout of ATR kinase activity (kinase-dead) in cells did not result in blocking the response of p53 to DNA damage (Nghiem et al., 2002). In this study, we presented evidence that ATR actively targeted the XPA, rather than XPC (data not shown), for regulation of its nuclear import in response to DNA damage. Such regulation appears to be particularly important as the XPA, an indispensable factor in NER for both GGR and TCR has been widely considered to be a representative protein for NER. Cells with XPA deficiency are extremely sensitive to killing by UV irradiation. Unlike XPC which is a molecular matchmaker, XPA is required for NER from damage recognition all through the completion of dual incisions (Riedl et al., 2003; Sancar et al., 2004). Targeting other NER proteins for general regulation of NER by ATR also seems to be unlikely. Although RPA is a DNA damage recognition protein in NER, it is the major ssDNA binding protein in cells and a major player in replication and many other DNA metabolic pathways unrelated to DNA damage and repair. Thus, it is very unlikely and there is no evidence that RPA (except for its phosphorylation which is not required for NER) is the target for regulation of NER by ATR in response to UV damage. While TFIIH is a major component of NER for both GGR and TCR, it is also an essential complex for normal transcription which is independent of DNA repair, making it an unlikely target for regulation of NER. In addition, recent evidence indicated that recruitment of XPF/ERCC1 NER nuclease depends on XPA (Guzder et al., 2006). Finally, it was recently reported that XPA becomes a limiting factor for NER at < 10% level of XPA in whole cells (Koberle et al., 2006), which is consistent to the % level of XPA in nucleus. All these suggest a general mechanism in which the NER could be regulated by ATR checkpoint upon DNA damage via modulation of XPA nuclear import. In further support, an increased UV-sensitivity was observed in ATR-mutant cells (Cliby et al., 1998; Wright et al., 1998), suggesting that the NER efficiency is likely reduced in these cells. This may, at least in part, attributed to the defect in ATR-dependent nuclear translocation of XPA as loss of cell cycle checkpoints themselves did not result in decreased cell viability after DNA damage (Xu et al., 2001; Xu et al., 2002).

There are two possible scenarios regarding the underlying mechanism by which ATR regulates XPA nuclear import. Phosphorylation of XPA is unlikely to mediate this process as these two events did not correlate with each other in the time course experiments (Wu et al., 2006). Given that ATR interacted significantly more efficiently and co-localized to DNA damage sites with XPA in cells upon UV irradiation (as the nuclear foci demonstrated) (Figure 5) (Wu et al., 2006), it is possible that formation of the ATR-XPA complex at the damage sites on chromatin may significantly reduce the concentration of free XPA in the nucleus. This may consequently disrupt the balanced XPA concentration across the nuclear membrane separating the nucleus from the cytoplasm. It is speculated, therefore, that a significant concentration gradient of XPA across the membrane could form, which may serve as a driving force for efficient cellular transportation of XPA from cytoplasm to nucleus.

Alternatively, ATR may regulate the nuclear translocation of XPA via other proteins. Since XPA and RPA form complex in vitro and ATR kinase activity was found to be necessary for efficient intranuclear relocalization of RPA to DNA damage sites (Barr et al., 2003), raising the possibility of RPA involvement in the XPA nuclear localization. Nevertheless, knockdown of RPA in cells by siRNA did not reduce the UV-induced XPA nuclear import (data not shown). In addition, a recent study reported that XPA and RPA were loaded separately onto DNA lesions in vivo (Rademakers et al., 2003). Notably, XPA contains a potential nuclear localization sequence (NLS) of residues 30–42 (Miyamoto et al., 1992). The localization of XPA from cytoplasm to nucleus is likely mediated by its interactions with nuclear transport proteins via the NLS. Recently a cytoplasmic GTPase, XAB1, was reported to interact with XPA, and deletion of the residues 30–34 of XPA abolished this interaction (Nitta et al., 2000). Evidently, the potential role of this interaction or related XPA-protein interactions in the regulation of XPA nuclear import deserves further investigations.

Materials and Methods

Cell culture and treatments

Human lung adenocarcinoma cells A549 were obtained from American Type Culture Collection and maintained at 37 °C and 5% CO2 in Dubelco's Modified Eagles Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Two mutant human fibroblast cell lines, ATR (GM18366) and ATM (GM09607), were purchased from Coriell Cell Repositories (Camden, NJ). For UV exposure, cells were irradiated with various doses of UV and further incubated for indicated time at 37 °C before harvesting.

Subcellular fractionation

The cellular protein fractionation was performed essentially as described (Wu et al., 2005a; Zou et al., 2002). Briefly, cells were first lysed in Buffer A (10 mM HEPES at pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100, 1 mM Na3VO4, 10 mM NaF, β–glycerophosphate, 1 mM PMSF, and protease cocktail) on ice for 5 min. Cytoplasmic proteins were separated from nuclei by low-speed centrifugation (1,300×g for 5 min). Isolated nuclei were washed once with Buffer A and then further lysed in Buffer B (50 mM Tris-HCl, pH 7.8, 420 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.34 M sucrose, 10% glycerol, 1 mM Na3VO4, 10 mM NaF, β–glycerophosphate, 1 mM PMSF, and protease cocktail from Roche) at 4°C for 30min. Nuclear proteins were isolated by centrifugation at 14,000×g for 30 min. Protein concentration was determined by Bradford assay (Bio-Rad).

Western blotting

Cell lysates were separated on 8% SDS-polyacrylamide gels and transferred to PVDF membrane. The membranes were blocked with TBST buffer containing 5% powered milk and probed using the following primary antibodies: anti-XPA, anti-Rad51and anti-Hus1 (Santa Cruz) or anti-ATR and anti-ATM (GeneTex, Inc). The membranes were then incubated with horseradish peroxidase-linked secondary antibodies and bound antibodies were visualized using the ECL chemiluminescent method.

Immunofluorescence

For measurement of nuclear foci formation, cells were grown on 18 mm coverslips overnight prior to treatment. After treatment, cells were extracted with PBS containing 0.5% NP-40, fixed with 100% methanol, and blocked in PBS containing 15% FBS. Primary antibody dilutions used are as follows: rabbit anti-XPA (Santa Cruz) 1:500, mouse anti- ATR 1:2000 (GeneTex, Inc). Cells were then stained with fluorescence dye-linked secondary antibodies and visualized by fluorescent microscopy. Secondary antibody dilutions are as follows: anti-rabbit Alexa Fluor 488 1:250 and anti-mouse Alexa Fluor 568 1:250 (Molecular Probes). Images were captured with a Nikon inverted fluorescence microscope with attached CCD camera at 100 × magnification and processed using Photoshop 6.0 (Adobe) software.

For nuclear accumulation immunofluorescence, cells were grown on 18mm coverslips and transfected with or without siRNA as described for 72 hours. Cells were then irradiated with 20 J/m2 of UV and further incubated for 2 hours in standard culture conditions. The cells were then washed with PBS, immediately fixed using 100% methanol, and blocked with 15% FBS in PBS. Primary antibody dilutions are as follows: Mouse-anti-XPA (Kamyia Biomedical) 1:500, and Rabbit-anti-Calreticulin (Stressgen) 1:1000. Fluorescent dye-linked secondary antibody dilutions are as follows: Goat-anti-mouse Alexa Fluor 568, 1:250; Donkey-anti-Rabbit Alexa Fluor 488, 1:250 (Molecular Probes). Nuclei were stained using 300 nM DAPI (4',6-diamidino-2-phenylindole) (Molecular Probes). Obtained fluorescence microscopic images were processed using Photoshop software.

Small interfering RNA (siRNA) transfections

The siRNA transfection experiments were carried out using TransIT-TKO Transfection Reagent (Mirus) by following the manufacturer's instructions. Transfection-ready siRNA duplexes were purchased from Dharmacon. siRNA sequences used in this study were: ATR: 5'-CCU CCG UGA UGU UGC UUG A-3', and SMART pool siRNA for ATM.

Immunofluorescence DNA repair assay

Cells were grown on 18mm coverslips and transfected with siRNA as described for 72 hours. Cells were then irradiated with 20 J/m2 of UV through a 5 μm polycarbonate isopore filter (Millipore) and further incubated for indicated amounts of time. Cells were then washed with PBS and fixed in 2% formaldehyde/0.5% Triton X-100 followed by incubation in 2 M HCl, and blocked with 15% FBS in PBS. Fixed cells were stained with Mouse-anti-[6-4]-photoproduct (Clone 64M-2) antibody (MBL) and Goat-anti-Mouse Alexa Fluor 568 fluorescent dye-linked secondary antibody (Molecular Probes). Images were obtained and processed as described above.

Acknowledgments

We thank Dr. Priscilla B. Wyrick for her generous assistance in immunofluorescence measurements.

This study was supported by NCI grant CA86927 (to Y.Z.)

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