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1 Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan
2 Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki 2641, Chiba 278-8510, Japan
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Introduction |
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In contrast to DSBs, DNA single-strand breaks (SSBs) occur at a high frequency and are rapidly repaired by part of the base excision repair pathway (Caldecott 2003). When unrepaired SSBs at S phase encounter the DNA replication fork, they are converted to replication-mediated DSBs (Kuzminov 2001; Saleh-Gohari et al. 2005). Camptothecin (CPT) selectively inhibits DNA topoisomerase I (Topo I) and traps a Topo I-DNA cleavage complex resulting in the conversion to replication-mediated DSBs at S phase (Liu et al. 2000). Each "replication-mediated DSB" possesses only one DNA double-strand end (DSE), whereas DSBs induced independently of replication usually possess two adjacent DNA ends. Thus, the DSE may be an inappropriate substrate for NHEJ because there is no other end to join. The repair pathway for DSEs induced by collapse of the replication fork has been analyzed from yeasts to humans (Arnaudeau et al. 2001; Klein & Kreuzer 2002; Ferrara & Kmiec 2004; Saleh-Gohari et al. 2005). DSEs are preferentially repaired by the HR pathway, and the DNA replication fork is subsequently reestablished. Ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases are activated in response to DSEs induced by CPT and phosphorylate downstream factors in checkpoint signal transduction (Furuta et al. 2003; Pichierri et al. 2003; Shinozaki et al. 2003; Block et al. 2004a). DNA-PK has also been found to be activated in response to CPT and to participate in the phosphorylation of replication protein A2 (RPA2), histone H2AX, and Chk2 (Shao et al. 1999; Furuta et al. 2003; Li & Stern 2005). Furthermore, DNA-PK-deficient Chinese hamster cells and human malignant cells show sensitivity to CPT (Shao et al. 1999; Arnaudeau et al. 2001). These results imply that DNA-PK is required for reestablishment of the replication fork.
RPA is a single-strand DNA binding protein and is required for DNA replication, recombination, and repair (Wold 1997). RPA2, a component of heterotrimeric RPA, is hyperphosphorylated and forms nuclear foci in response to various types of DNA damage. The RPA2 focus formation in response to ionizing radiation (IR) has been reported to be dependent on ATR (Barr et al. 2003), whereas Balajee & Geard (2004) have shown that ATM is required for IR-induced RPA2 focus formation. The RPA2 hyperphosphorylation results from the phosphorylation of serine and threonine residues in the N-terminus of RPA2 (Zernik-Kobak et al. 1997). It has been reported that CPT-induced RPA2 hyperphosphorylation is not observed in DNA-PK-deficient M059J cells (Shao et al. 1999). In contrast, in cells treated with hydroxyurea (HU), which causes a replication fork stall mediated by ribonucleotide reductase inhibition with subsequent DSB induction, RPA2 is hyperphosphorylated in an ATR-dependent manner (Dodson et al. 2004). Recently, Vassin et al. (2004) reported that mimicked-hyperphosphorylated RPA2 is capable of forming nuclear foci in response to CPT-induced DNA damage, but is not associated with a replication center. Wu et al. (2005) reported that Rad51 and Rad52 preferentially interact with hyperphosphorylated RPA2. Furthermore, DNA-PK-deficient M059J cells exhibit sensitivity and no suppression of DNA synthesis in response to CPT (Shao et al. 1999). Taking these findings together, CPT-induced RPA2 hyperphosphorylation is likely to be concerned with S-phase checkpoint and DSE repair.
In the present study we focused on the roles of DNA-PK and ATR in RPA2 focus formation and hyperphosphorylation in the response to DSEs induced by CPT. We used inhibitors of phosphatidylinositol- 3-kinase (PI3K)-related protein kinases and siRNAs targeting DNA-PK and ATR to investigate the mode of action of these two kinases in the DSE response pathway, and we analyzed their involvement in RPA2 focus formation and hyperphosphorylation. The results suggested that DNA-PK is responsible for the RPA2 hyperphosphorylation following ATR-dependent RPA2 focus formation. This means that ATR and DNA-PK act differently, not redundantly, in response to DSEs. The results of a comparison of CPT with HU as a control implied that the DNA-PK dependency of RPA2 hyperphosphorylation is determined by DSEs induced directly by the DNA replication fork encountering SSBs.
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Results |
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Shao et al. (1999) reported that CPT-induced RPA2 hyperphosphorylation with DNA-PK-deficient M059J cells is dependent on DNA-PK. However, since this cell line has abnormal ploidy and mutations in DNA damage response genes, such as p53 and ATM (Anderson & Allalunis-Turner 2000; Anderson et al. 2001; Tsuchida et al. 2002), there may be unidentified genetic abnormalities in M059J cells. We therefore first investigated the DNA-PK dependency of RPA2 hyperphosphorylation in response to DNA damage induced by CPT by using human embryonic kidney 293 cells and DNA-PK inhibitors. Wortmannin inhibits PI3K-related protein kinases, including DNA-PK and ATM (Sarkaria et al. 1998), whereas LY294002 is a relatively specific inhibitor of DNA-PK (Izzard et al. 1999; Stiff et al. 2004). To inhibit DNA-PK, wortmannin or LY294002 was added to 293 cells for 1 h prior to exposure to CPT. After exposure to CPT for 2 h, hyperphosphorylated RPA2 was analyzed by Western blotting. The results showed that RPA2 hyperphosphorylation was suppressed in the wortmannin- and LY294002-treated cells (Fig. 1A), but phosphorylation of histone H2AX on Ser139 (
-H2AX) as a DSB marker was unaffected by LY294002 (Fig. 1A). Since the major checkpoint kinase ATR is known to be activated in response to DNA damage induced by Topo I inhibitor topotecan (Cliby et al. 2002), we investigated whether the suppression of RPA2 hyperphosphorylation was attributable to ATR inhibition by LY294002. The effect of LY294002 on ATR activity was verified by the phosphorylation on the Ser345 of Chk1, a known substrate of ATR (Cliby et al. 2002; Cortez 2003), by CPT and another replication inhibitory agent, HU. LY294002 slightly suppressed the Chk1 phosphorylation induced by CPT in the indicated concentrations of LY294002, and RPA2 hyperphosphorylation induced by HU was insensitive to LY294002 (Fig. 1B). To determine whether DNA-PK is involved in RPA2 hyperphosphorylation, we knocked down the expression of DNA-PK in 293 cells with siRNA targeting DNA-PK. RPA2 hyperphosphorylation by CPT was reduced with decrease in the DNA-PK protein level (Fig. 1C). These results indicate that DNA-PK is specifically involved in CPT-induced RPA2 hyperphosphorylation, and is not involved in HU-induced RPA2 hyperphosphorylation.
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The Thr21 and Ser33 of RPA2 are phosphorylated by DNA-PK in vitro (Niu et al. 1997), and CPT-induced RPA2 hyperphosphorylation was suppressed by LY294002 (see Fig. 1). Based on these findings, we considered that phosphorylation of the Thr21 and/or Ser33 of RPA2 by DNA-PK is likely to trigger hyperphosphorylation. We constructed expression vectors of mutant RPA2 fused with the FLAG tag, in which Thr21, Ser33, or both were replaced by alanine, and transfected the vectors into 293 cells to investigate the effect of the mutations on RPA2 phosphorylation status. The CPT-induced phosphorylated band of T21A RPA2 mutant showed slightly higher mobility, but there were hardly any changes in the S33A RPA2 mutant (Fig. 2), indicating that phosphorylation of the Thr21 and Ser33 of RPA2 is not the trigger of the RPA2 hyperphosphorylation in response to CPT.
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Next, U2OS cells were treated with caffeine to investigate the dependency of RPA2 focus formation on PI3K-related protein kinases. RPA2 foci were markedly abrogated by treatment with caffeine prior to the addition of CPT (Fig. 3A). Caffeine has been reported to inhibit ATR and ATM and to partially impair DNA-PK (Sarkaria et al. 1999; Block et al. 2004b). Usual CPT-induced RPA2 focus formation was observed in M059J (DNA-PK deficient) cells and GM05849C-mock (ATM deficient) cells (Fig. 3B,C), indicating that a caffeine-sensitive kinase other than ATM or DNA-PK, probably ATR, is required for RPA2 focus formation in response to CPT.
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The caffeine-induced abrogation of RPA2 focus formation suggested that ATR also contributes to CPT-induced RPA2 hyperphosphorylation. To investigate this possibility, we analyzed caffeine-treated cells for CPT-induced RPA2 hyperphosphorylation, and RPA2 hyperphosphorylation was found to be markedly suppressed in cells treated with caffeine for 1 h prior to the addition of CPT (Fig. 4A). Since DNA-PK activity is also affected by caffeine, we specifically knocked down expression of ATR with siRNA. We constructed a pSUPER inserted with the sequence for siRNA targeting ATR and transfected the pSUPER-siATR construct into 293 cells. Transfected cells were harvested 72 h later and analyzed for RPA2 hyperphosphorylation in 293 cells in which DNA damage had been induced with CPT. The transfection with pSUPER-siATR reduced the protein level of ATR (Fig. 4B), and exhibited no effect on that of DNA-PK (data not shown). RPA2 hyperphosphorylation was appreciably suppressed in the pSUPER-siATR transfected cells (Fig. 4B). The cell cycle distribution pattern was not significantly changed at least 72 h after pSUPER-siATR transfection (data not shown), although ATR is essential for cell survival (Brown & Baltimore 2000). In addition, we analyzed CPT-induced RPA2 focus formation in ATR knocked down cells. According to the decrease in ATR protein level and RPA2 hyperphosphorylation, RPA2 focus formation was suppressed in ATR knocked down cells (Fig. 4C). These findings suggest that both RPA2 focus formation and hyperphosphorylation induced by CPT are dependent on ATR.
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Since DNA-PK has DNA-end-binding activity dependent on Ku protein (Smith & Jackson 1999), we assumed that DSB induction is a key element in the DNA-PK dependency. To investigate whether DNA-PK is required for the response to replication-mediated DNA damage, we compared the response to CPT and HU. Since HU causes a replication fork stall mediated by deoxyribonucleotide depletion, DSBs are not thought to be directly caused by HU (Lundin et al. 2002). We analyzed the time-dependent changes in RPA2 hyperphosphorylation and -H2AX as a DSB marker. After adding CPT or HU to 293 cells, the cells were harvested at the times indicated and phosphorylated Chk1, RPA2, and H2AX were analyzed by Western blotting (Fig. 5). In the CPT-treated cells -H2AX was strongly expressed early and the expression peaked at 60 min. RPA2 hyperphosphorylation was observed from 60 min onward, and peaked at 90 min. -H2AX detection in the HU-treated cells, on the other hand, was weak at 15 min and gradually increased until 120 min. Hyperphosphorylated RPA2 was also weakly detected from 90 min onward, but it had not peaked by 120 min. These phosphorylation patterns remained unchanged at up to 5 mM HU (data not shown). Common features of the response to CPT and HU were that Chk1 Ser345 phosphorylation occurred immediately, and that RPA2 hyperphosphorylation occurred following -H2AX expression. These results indicate that ATR is immediately activated after the addition of CPT and HU, and that RPA2 is hyperphosphorylated following DSB induction. The rapid induction of -H2AX and hyperphosphorylated RPA2 by CPT suggest that DNA-PK participates in RPA2 hyperphosphorylation when DSEs are directly induced by replication fork progression.
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To determine whether there are any differences in PRA2 focus formation between CPT- and HU-treated cells, we analyzed RPA2 focus formation and hyperphosphorylation earlier after CPT and HU addition. U2OS cells were cultured on coverslips and exposed to CPT or HU for the times indicated. RPA2 focus formation peaked at 30 min in the CPT-treated cells and at 60 min in the HU-treated cells (Fig. 6A). RPA2 hyperphosphorylation was observed from 1 h onward in the CPT-treated cells (Fig. 6B), whereas RPA2 was still not hyperphosphorylated in the HU-treated cells at 2 h (Fig. 6B). These results indicate that RPA2 focus formation occurs prior to hyperphosphorylation after exposure to these drugs, and that RPA2 focus formation is insufficient for RPA2 hyperphosphorylation. We also compared -H2AX focus formation in CPT- and HU-treated cells. In contrast to the HU-treated cells, -H2AX foci were observed in CPT-treated cells immediately after the addition of the drug (Fig. 6C), suggesting that RPA2 hyperphosphorylation is required for the presence of DSBs at the site of replication fork stall.
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Discussion |
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Although ATR and DNA-PK are likely to act sequentially in the same pathway in response to DSE, it is unknown whether DNA-PK activity is regulated by ATR. DNA-PK-dependent RPA2 hyperphosphorylation has been reported to be indirectly abrogated by treatment with a Chk1/Chk2 inhibitor, UCN-01 (Shao et al. 1999). Since this finding suggests that Chk1 or Chk2 is involved in DNA-PK-dependent RPA2 hyperphosphorylation downstream of ATR, we knocked down the expression of Chk1 or Chk2. However, the reduction in Chk1 or Chk2 expression had no appreciable effect on CPT-induced RPA2 hyperphosphorylation (data not shown), and thus downstream factors of ATR other than Chk1 and Chk2 may be involved in the regulation of RPA2 hyperphosphorylation.
DNA damage-induced RPA2 hyperphosphorylation alters its affinity for both single-strand and double-strand DNA in vitro, and affects its interaction with p53 in vivo and with DNA polymerase 
in vitro (Abramova et al. 1997; Binz et al. 2003; Patrick et al. 2005). However, the biological significance of DNA damage-induced RPA2 hyperphosphorylation has not yet been clarified. If induction of DSB is required for RPA2 hyperphosphorylation at S phase, RPA2 hyperphosphorylation may be necessary for the DSB repair pathway to reestablish the replication fork, but not checkpoint response. It has been reported that Rad51 and Rad52, which are involved in HR, preferentially interact with hyperphosphorylated RPA2 (Wu et al. 2005). However, spontaneous HR increases in DNA-PK-deficient Chinese hamster V3 cells (Allen et al. 2002), and the spontaneous HR is thought to be triggered by the replication-mediated DSE (Saleh-Gohari et al. 2005). Combined with these reports, a portion of the DSEs is probably repaired by DNA-PK-dependent pathway, although the DSE is presumably an inappropriate substrate of NHEJ. Thus, it remains to be demonstrated how RPA2 hyperphosphorylation is related to the DSE repair pathway in vivo.
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Experimental procedures |
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Human embryonic kidney 293 cells and osteosarcoma U2OS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Human malignant glioma cell lines M059K and M059J (Lees-Miller et al. 1995) were maintained in RPMI 1640 medium with 10% FBS. GM05849C cells transfected with pcDNA3 (vector only) or pcDNA3-YZ5 (ATM complemented) were cultured in DMEM containing 200 µg/mL hygromycin (Invitrogen, Carlsbad, CA, USA) (Oguchi et al. 2003). Wortmannin (Wako, Osaka, Japan), LY294002 (Calbiochem, San Diego, CA), and camptothecin (Wako) were dissolved in DMSO at 10 mM, and caffeine (Wako) was dissolved in water at 200 mM. Hydroxyurea (Sigma, St. Louis, MO, USA) was dissolved in water at 1 M. Cells were treated with wortmannin (Wako), LY294002, and caffeine for 1 h prior to exposure to DNA-damaging agents.
Expression of small interfering RNAs
To construct small interfering RNA (siRNA) expression vectors, oligonucleotides targeting the coding region of each gene were annealed and inserted into the BglII/HindIII sites of pSUPER (Oligoengine, Seattle, WA, USA). The sequences targeting each gene were: ATR, cga gac ttc tgc gga ttg c (Wang & Qin 2003); DNA-PK, agg gcc aag ctg tca ctc t (Feng et al. 2004). A puromycin-resistance gene derived from pCre-Pac (Toyobo, Osaka, Japan) was inserted into SalI site of constructed siRNA expression vectors, and the resulting constructs were transfected into 293 cells with FuGENE6 (Roche, Mannheim, Germany). Transfection was performed according to the manufacturer's protocol. pSUPER-siRNA-Puro transfected cells were maintained in DMEM containing 6 µg/mL puromycin (Sigma).
Construction of mutated RPA2 expression vectors
Base replacements were introduced into cloned human RPA2 cDNA in pGEM-T vector, resulting in T21A or S33A amino acid replacement by the site-directed mutagenesis method based on PCR. To express a fusion protein with the FLAG tag, a fragment containing the FLAG sequence derived from pIRES-hrGFP-1a (Stratagene, La Jolla, CA, USA) was inserted into BamHI/AvrII sites of pIREShyg2 (Clontech Laboratories, Palo Alto, CA, USA), resulting in a pIRES-FLAG-hyg construct. Mutated RPA2 cDNA was inserted into NheI/BamHI sites of pIRES-FLAG-hyg. These mutated RPA2 expression vectors were transfected into 293 cells with FuGENE6.
Western blotting
Cells were lyzed in an SDS sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, 0.1% bromophenol blue), and the DNA was sheared by sonication. Sonicated cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences, Chalfont St. Giles, UK). The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS), and then incubated in primary antibodies against DNA-PK (1/1000, Kamiya Biomedical Company, Seattle, WA, USA), ATR (1/1000, GeneTex, San Antonio, TX, USA), phosphorylated Chk1 (Ser-345) (1/500, Cell Signaling Technology, Beverly, MA, USA), RPA2 (100 ng/mL, Oncogene Research Products, Cambridge, MA, USA), phosphorylated H2AX (Ser139) (1/20 000, Upstate Biotechnology, Lake Placid, NY, USA), and ß-actin (1/10 000, Sigma). The membrane was then incubated with appropriate secondary antibodies conjugated with HRP. After washing the membrane with TTBS, specific proteins were visualized by chemiluminescence with ECL (Amersham Biosciences). All data presented were confirmed in independent experiments.
Immunofluorescence microscopy
Cells were grown on 22-mm coverslips overnight prior to drug treatment. They were then washed with PBS, treated with PBS containing 0.5% Triton X-100 for 5 min, and fixed with 10% neutral buffered formalin for 15 min. In 293 cells, cells were grown on chamber slide, and fixed with 7.5% neutral buffered formalin containing 0.25% Nonidet P-40 after washing with PBS. Next, the cells were permeabilized with PBS containing 0.5% Triton X-100 for 5 min, and the fixed cells were incubated for 1 h with anti-RPA2 antibody (250 ng/mL) or anti--H2AX antibody (1/5000) diluted in PBS containing 2% BSA, washed with PBS and incubated with FITC-conjugated anti-mouse IgG for 1 h. Nuclei were stained with Cellstain DAPI solution (Dojindo, Kumamoto, Japan). Immunofluorescence images were captured with a Zeiss LSM510 confocal microscope. Cells containing more than 10 foci were counted as positive.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: hteraoka.pbc{at}mri.tmd.ac.jp
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Received: 13 November 2005
Accepted: 12 December 2005
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 P. Huertas and S. P. Jackson Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair J. Biol. Chem., April 3, 2009; 284(14): 9558 - 9565. [Abstract] [Full Text] [PDF]
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 A. A. Cohen, N. Geva-Zatorsky, E. Eden, M. Frenkel-Morgenstern, I. Issaeva, A. Sigal, R. Milo, C. Cohen-Saidon, Y. Liron, Z. Kam, et al. Dynamic Proteomics of Individual Cancer Cells in Response to a Drug Science, December 5, 2008; 322(5907): 1511 - 1516. [Abstract] [Full Text] [PDF]
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R. Sakasai and R. Tibbetts RNF8-dependent and RNF8-independent Regulation of 53BP1 in Response to DNA Damage J. Biol. Chem., May 16, 2008; 283(20): 13549 - 13555. [Abstract] [Full Text] [PDF]
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E. Fanning, V. Klimovich, and A. R. Nager A dynamic model for replication protein A (RPA) function in DNA processing pathways Nucleic Acids Res., September 10, 2006; 34(15): 4126 - 4137. [Abstract] [Full Text] [PDF]
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