1 Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1, Sendai 980-8575, Japan
2 Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan
3 Experimental Immunology Branch, NIH, National Cancer Institute, Bethesda, MD 20892-1360, USA
4 GeneCare Research Institute, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan
5 Department of Pathology, University of Washington, Seattle, WA 98195-7470, USA
* Author for correspondence (e-mail: ayasui{at}idac.tohoku.ac.jp)
Accepted 6 June 2005
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Summary |
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Key words: Werner protein, Double-strand breaks, Laser irradiation, Damage accumulation, HRDC domain
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Introduction |
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It has been reported that many proteins physically and functionally interact with WRN. The interacting proteins appear to function at various levels in the mechanisms for maintaining the integrity of the genome and in the DNA damage response, suggesting that WRN plays one or more roles in DNA repair. WRN has been shown to interact with Ku and DNA-PKcs (Karmakar et al., 2002a; Karmakar et al., 2002b
; Li and Comai, 2000
; Orren et al., 2001
), and a recent report suggests that WRN may participate in non-homologous end-joining (Li and Comai, 2002
). Werner syndrome fibroblasts transformed with Simian Virus-40 (SV40) T antigen or immortalized by expressing human telomerase reverse transcriptase (hTERT) display a mild but distinct sensitivity to ionizing radiation when compared with appropriate control fibroblasts and Werner syndrome fibroblasts expressing exogenous WRN (Cheng et al., 2004
; Yannone et al., 2001
). This suggests that WRN may be involved in processing ionizing radiation-induced double-strand breaks. WRN also interacts physically with the Mre11-Rad50-NBS1 complex, which functions in homologous recombination for double-strand break processing (Cheng et al., 2004
). Other reports suggest that WRN may play a role in base excision repair because of a physical interaction between WRN and polymerase ß (POL ß) involved in base excision repair and the repair of single-strand breaks (Harrigan et al., 2003
). Furthermore, p53 has been shown to interact with the C-terminus of WRN and to inhibit WRN exonuclease activity in vitro (Blander et al., 1999
; Brosh et al., 2001
). Consistent with possible roles of WRN in DNA repair and genome stability, Werner syndrome cells show an attenuated p53-mediated apoptosis (Spillare et al., 1999
) and display extensive deletions at non-homologous joined ends as well as non-homologous chromosome exchanges (Oshima et al., 2002
). With regard to the localization of WRN in cells, various distribution patterns of the protein have been reported. WRN foci have been observed as diffuse nuclear, nucleolar or nuclear foci depending on the stage of the cell cycle (Gray et al., 1998
; Opresko et al., 2003
). Although the number of WRN-containing nuclear foci increases after replication fork arrest and upon DNA damage (Sakamoto et al., 2001
; Szekely et al., 2000
), the significance of the formation of these foci remains to be elucidated. Furthermore, in spite of many reports describing that WRN modified the enzymatic activity of the interaction partners and vice versa, it has not be shown whether WRN responds to DNA damage and whether WRN is involved in base excision repair, repair of single-strand breaks or double-strand breaks in living cells.
We recently established a laser micro-irradiation system for the localized production of single-strand breaks, double-strand breaks and oxidative base damage in a cell nucleus (Lan et al., 2004). Using this system we have analyzed the accumulation of endogenous and green-fluorescent-protein (GFP)-tagged WRN at various types of DNA damage and found that WRN accumulated via its HRDC domain at double-strand breaks within 1-2 minutes after irradiation and remained for a longer period at the site. Our results showed, for the first time, an immediate accumulation of WRN at double-strand breaks and suggest important roles for WRN in genome stability of living cells.
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Materials and Methods |
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Cell lines and transfection
Cell lines of HeLa, Parp1-(a cell line from mouse embryonic fibroblasts, a generous gift of Mitsuko Masutani), Susa/T-n cells (p53+/+, telomerase expressed, a generous gift of Kanji Ishizaki), 1022QVA (NBS1-deficient Nijmegen patient cells), CHO9 (WT cells), XR-1 (XRCC4 deficient cell line), XR-C1 (DNA-PKcs-deficient cell line), XRV15B (Ku80-deficient cell line), and H2AX-deficient MEF (Celeste et al., 2003) were used. All the above cell lines were propagated in D-MEM (Nissui) supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 38
(mouse Polß / cell line) and MB36.3 (mouse Polß /cell transfected with wild-type human POLß), generous gifts from Samuel H. Wilson, were grown at 34°C and 10% CO2. Cells were plated on glass bottom dishes (Matsunami Glass) at 50% confluence 24 hours before the transfection (Fugene-6, Life Technology) and irradiated by laser under a microscope 48 hours after transfection.
Microscopy and laser irradiation
Fluorescence images were obtained and processed using a FV-500 confocal scanning laser microscopy system (Olympus). A 365 nm pulse laser micro-irradiation apparatus combined with the confocal microscope was used as previously described (Lan et al., 2004). We used two irradiation doses, a lower dose (0.75 µJ) or a higher dose (2.5 µJ), which were obtained by passing lasers through either an F20 or an F25 filter, respectively, in front of the lens. By using this system, various types of DNA damage, such as single-strand breaks (produced by lower dose and higher dose irradiation), double-strand breaks and oxidative base damage (produced by higher dose irradiation), were produced at restricted nuclear regions of mammalian cells. We also used a 405 nm pulse laser system (Olympus) for irradiation of cells in the epi-fluorescence path of the microscope system. The power of the laser scan can be controlled by the number of scans used or by laser dose. One scan of the laser light at full power delivers energy of around 1600 nW. We used only a full power scan from the 405 nm laser in this study and regulated the dose by changing the number of scans. Both 365 nm and 405 nm lasers were focused through a 40x objective lens. Cells were incubated with Opti-medium (Gibco) in glass-bottom dishes, which were placed in chambers to prevent evaporation, on a 37°C hot plate. The energy of fluorescent light at the irradiated site was measured with a laser power/energy monitor (Orion, Ophir Optronics, Israel). The mean intensity of each focus was obtained after subtraction of the background intensity in the irradiated cell. Each experiment was performed at least three times and the data presented here are mean values obtained in a given experiment. Local UVC-light irradiation were performed as previously described (Okano et al., 2003
).
Cell-cycle synchronization
Cell synchronization was performed by the double thymidine block method. In all, 5x104 cells were seeded in a 3.5 cm dish and grown for 2 days. Thymidine was then added to 2.5 mM final concentration and cells were further incubated for 22-24 hours. Thymidine-containing medium was removed and cells were washed three times with Hank's buffer and fresh medium was added. After 10 hours, cells were treated with hydroxyurea at 1 mM final concentration and incubated for 14-16 hours. Under these conditions, cells accumulate at the G1/S border. Cells were then washed three times with Hank's buffer. Synchronization of the cell cycle was analyzed by a FACSCalibur (Becton Dickinson). Cells were incubated for 3 hours, 8 hours and 16 hours in fresh medium to obtain S-phase, G2-M-phase and G1-phase cells, respectively.
Immunocytochemistry and chemicals
Cells were irradiated and stained with antibodies raised against human H2AX, WRN and XRCC1. Cells were fixed 15 minutes after irradiation. Immunofluorescence was performed as described previously (Okano et al., 2003
). Anti-phosphorylated H2AX (
H2AX) (1:200 dilution; Upstate Biotechnology), anti-WRN (1:20) and anti-XRCC1 (1:200 dilution, Abcam) were used. For photosensitization treatment of cells, Ro-19-8022 (Roche) at a final concentration of 250 nM was added into the medium and cells were incubated at 37°C for 5 minutes. For 1,5-dihydroxyisoquinoline (DIQ) treatment, cells were incubated with DIQ (Sigma) in the medium at a final concentration of 500 µM for 1 hour before irradiation. Camptothecin (Sigma) at a final concentration of 1 µM was added to the medium and cells were incubated at 37°C for 6 hours. 5-Bromo-2'-deoxyurine (BrdU; Roche) at a final concentration of 10 µM was added to the medium 8 hours before laser irradiation.
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Results |
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To exclude the possibility that the accumulation of WRN is an artifact due to overexpression of WRN tagged with GFP, we examined the accumulation of native WRN protein using antibody against WRN. Accumulation of WRN was not detected at irradiated sites in cells not pre-treated with BrdU (Fig. 2E left), but was detected 15 minutes after various numbers of scans using 405 nm laser irradiation following pre-treatment with BrdU in HeLa cells at the narrow irradiated lines (Fig. 2E right). GFP-tagged WRN accumulated at the irradiated site even without pre-treatment with BrdU, which may be explained by the fact that the antibody recognizes the endogenous human WRN with a very low affinity. Accumulation of WRN was observed by the antibody only at the irradiated sites in cells, further suggesting that WRN accumulated only at the region where double-strand breaks are present.
Accumulation kinetics of GFP-tagged WRN at the sites of double-strand breaks
Having established the accumulation of WRN at the site of laser-induced double-strand breaks, we characterized the kinetics of the accumulation of WRN and compared it with those of other repair proteins. Fig. 3 depicts the accumulation kinetics of GFP-tagged WRN and other proteins after the higher dose of 365 nm laser irradiation. WRN accumulated rapidly at the irradiated sites and the fluorescence at the sites reached a plateau 3 minutes after irradiation. NBS1, an early response protein to double-strand breaks (Celeste et al., 2003), accumulated at irradiated sites with very similar kinetics as for WRN, whereas BRCA1 accumulated much more slowly. Once they had accumulated at the irradiated sites, all these proteins remained there up to 4 hours after irradiation. In contrast to these proteins, LIGIII
, which functions in a final step in the repair of single-strand breaks, dissociated from the irradiated site around 1 hour after irradiation (Fig. 3). Like NBS1 and BRCA1, which are involved in the repair of double-strand breaks via homologous recombination, WRN remained at the irradiated site for a long time, suggesting that the response of WRN might also be an early event in homologous recombination repair.
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Accumulation of WRN at irradiated sites is not enhanced by photosensitization, which increases the production of oxidative base damage
As WRN has been reported to enhance the repair of oxidative base damage in vitro (Harrigan et al., 2003), we wanted to examine the accumulation of WRN in cells pre-treated with RO-19-8022, a photosensitizer, which increases the production of oxidative base damage by absorption of light around 400 nm (Will et al., 1999
). We have previously shown that treatment of cells with RO-19-8022 before laser irradiation enhanced the accumulation of various glycosylases for oxidized bases, such as NTH1, OGG1, NEIL1 and NEIL2, as well as other repair proteins, such as POL ß and PCNA, involved in base excision repair at irradiated sites (Lan et al., 2004
). This indicates that the amount of various types of produced base damage is increased by the photosensitization. However, in contrast to the glycosylases and the other proteins, the accumulation of WRN was not enhanced at all in cells pre-treated with RO-19-8022 (Fig. 4A) under the same irradiation condition as that of GFP-NTH1 shown here (Fig. 4B), suggesting that the observed accumulation of WRN is different from that of the glycosylases and that WRN is not directly involved in the repair of oxidative base damage.
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The accumulation of WRN at irradiated sites is independent of DNA replication
A previous study showed that, 6 hours after treatment of cells with camptothecin, WRN formed distinct foci, which partially colocalized with RPA and RAD51 (Sakamoto et al., 2001). We therefore investigated the dependence of the accumulation of WRN at sites of laser irradiation on DNA replication. HeLa cells were synchronized at the G1/S border by the double thymidine block method and released by removing HU-containing medium; they were then irradiated with the 365 nm laser in each phase of the cell cycle. WRN accumulated at the laser-induced damage site in all the cells of different cell-cycle phases shown in Fig. 5. There was no significant difference in the kinetics of accumulation of WRN at different cell-cycle phases (not shown). These data suggest that accumulation of WRN at sites of DNA damage is independent of DNA replication.
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Interacting proteins do not influence WRN accumulation at double-strand breaks
Many proteins involved in DNA repair interact with WRN. To identify whether the accumulation of WRN at sites of double-strand breaks is dependent on the double-strand breaks-repair proteins that have been reported to interact with WRN, we first examined the accumulation of WRN in cells deficient in the proteins. Because WRN has been reported to interact with Ku, DNA-PKcs, and NBS1 (Cheng et al., 2004), we checked the accumulation of WRN in cells derived from the Chinese hamster ovary (CHO) cell lines of XR-C1 (DNA-PKcs-deficient), XR-V15B (Ku80-deficient) and XR-1 (XRCC4-deficient), as well as in a human cell line 1022QVA (Nijmegen patient cells, NBS1-deficient). To examine whether the modification of H2AX, an initial signal of double-strand breaks, influences the accumulation of WRN, its accumulation in H2AX/ MEF cells was also analyzed. WRN accumulated in all of the cell lines listed above in the same way as it accumulated in their corresponding wild-type cells after a higher dose of irradiation with the 365 nm laser (Fig. 7), which indicates that the accumulation of WRN at double-strand breaks is independent of these proteins. WRN is phosphorylated in an ATM/ATR-dependent manner on production of stalled replication fork (Pichierri et al., 2003
). Because pre-treatment of cells with caffeine has been reported to interfere efficiently with ATM-dependent events such as H2AX phosphorylation, we also examined the accumulation of WRN in cells treated with caffeine. Accumulation of WRN was not affected by caffeine (not shown), supporting the above result of H2AX-independent WRN accumulation.
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Although we showed that WRN did not accumulate at single-strand breaks and base damage, WRN has been reported to interact with poly(ADP)ribose polymerase-1 (PARP1) and POL ß (von Kobbe et al., 2003a), which play important roles in single-strand breaks and base excision repair. Because these proteins may also influence the accumulation of WRN at double-strand breaks, we tested the accumulation of WRN in PARP1/ or POL ß/ MEF cells and found that accumulation of WRN at the damage site in POL ß/ MEF cells and PARP1/ MEF cells was the same as that in the parental cells after the higher dose of 365 nm laser irradiation (Fig. 7A). Because PARP2 is known to act as a backup for PARP1, a potent inhibitor for both PARPs, 1,5-dihydroxyisoquinoline (DIQ) was used in HeLa cells. DIQ treatment significantly suppressed the accumulation of XRCC1 at the irradiated sites (Lan et al., 2004
) but did not influence the accumulation of WRN (not shown). Thus, PARP1, PARP activation and POL ß did not influence the accumulation of WRN at the irradiated sites. It has also been reported that an interaction between WRN and p53 may be involved in cellular responses to DNA damage (Blander et al., 1999
; Brosh et al., 2001
). Because the presence of functional p53 may influence the accumulation in WRN at the irradiated site, we investigated the accumulation of WRN at laser-induced double-strand breaks in hTERT-immortalized human fibroblast cells (Susa/T-n) with functional p53 (Nakamura et al., 2002
). WRN accumulated at double-strand breaks in Susa/T-n cells as well as in other cell lines, suggesting that the functional p53 does not influence the accumulation of WRN at double-strand breaks (Fig. 7).
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Discussion |
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By comparing the accumulation kinetics of WRN with other proteins, we found that WRN showed similar kinetics to NBS1, a protein exhibiting an immediate response to double-strand breaks and playing a central role in the repair of double-strand breaks. These results suggest that the response of WRN and NBS might cooperate with each other in related repair pathways. Although the interaction between WRN and double-strand breaks repair-related Ku proteins has been reported (Cooper et al., 2000; Li and Comai, 2001
), our results showed that deletion of the Ku-interaction domain of WRN (N-terminal of WRN) did not prevent its accumulation at double-strand breaks and that WRN accumulated at double-strand breaks even in Ku86-deficient cells, suggesting that the accumulation of WRN at double-strand breaks is not dependent on Ku molecules. A recent study showed that TRF2 accumulated at laser-induced double-strand breaks independently of the presence of Ku70, DNA-PKcs, MRE11/RAD50/NBS1 complex and WRN as an early response to DNA damage (Bradshaw et al., 2005
). The accumulation of WRN at double-strand breaks is quite similar to that of TRF2, as the accumulation of WRN at sites of double-strand breaks is also independent of the above proteins (Fig. 7) and WRN responds to double-strand breaks as early as TRF2 does. Both proteins associate with telomeres as well as with double-strand breaks, indicating that they may join and cooperate in the repair of double-strand breaks as well as in the processes for protecting telomere ends.
Domain analysis indicated that the HRDC domain is essential and sufficient for the accumulation of WRN at sites of double-strand breaks. The function of the HRDC domain in mammalian WRN has not been elucidated. The threedimensional structure of the HRDC domain has been determined for the Saccharomyces cerevisiae RecQ helicase Sgs1p, the yeast homologue of WRN, by nuclear magnetic resonance (NMR) spectroscopy (Liu et al., 1999). Structural similarities of Sgs1p to bacterial DNA helicases suggest that the HRDC domain of Sgs1p may function as an auxiliary DNA-binding domain. However, most of the amino acids in the basic patch of the HRDC domain in Sgs1p are not conserved in the HRDC domains of other helicases including human WRN, and a structural model of the HRDC domain of human WRN shows different surface properties when compared with that of Sgs1p (Liu et al., 1999
). A previous study indicated that the HRDC domain of WRN (a.a. 1072-1432) binds to the forked duplex and Holliday junction with high affinity but to the 5'-overhang duplex with lower affinity, whereas exonuclease and helicase domain of WRN also contain these binding activities in vitro (von Kobbe et al., 2003b
). In addition, full-length WRN showed a very low binding affinity to DNA in a non-sequence-specific, structure-dependent manner, and the HRDC domain is not a specific DNA-binding domain in vitro. However, our study showed very clearly that only the HRDC domain provides the protein with the ability to accumulate at DNA damage. Although the exonuclease and helicase domains showed binding activities to specific DNA structure in vitro (von Kobbe et al., 2003b
), these two domains did not respond to double-strand breaks in our assay. This may suggest a difference in the DNA damage response between in vivo and in vitro. Our data showed that the HRDC domain of WRN is able to assemble at double-strand breaks and may function in guiding the whole WRN to DNA damage in cells. Because the HRDC domain was shown to be an independently folded structural domain, the HRDC domain may either bind directly to double-strand breaks or interact with other protein(s) present at double-strand breaks, which remain to be identified.
All the mutations identified so far in patients with Werner syndrome result in a truncated WRN protein that lacks the C-terminus and the NLS. The inability of WRN to be transported into the nucleus has been thought to be crucial for the pathogenesis of WRN. Although Werner syndrome has been associated with mutations in the HRDC domain, the deleted WRN protein in the patients lost both the complete HRDC domain and NLS (Moser et al., 2000; Moser et al., 1999
; Oshima, 2000
). Therefore, the importance of HRDC might have been concealed because of the simultaneous loss of NLS, and it is possible that the response to double-strand breaks via the HRDC domain of WRN is actually important for the functions of WRN in cells.
Because camptothecin-induced WRN foci formation was inhibited by aphidicolin, the formation of foci is thought to be related to replication (Sakamoto et al., 2001). We have tested the ability of deletion mutants of WRN to form foci. Although the helicase mutant formed foci as efficiently as the full-length WRN in cells treated with camptothecin, all the other deletion mutants including HRDC domain alone failed to form foci (Fig. S4 in supplementary material). From previous reports, MRE11-RAD50-NBS1 complex seems to be a key factor for repair of DNA double-strand break and blocked replication fork by modulating related proteins, including BRCA1 and WRN. In response to
-irradiation, BRCA1 and MRE11-RAD50-NBS1 cooperate with each other to form irradiation-induced foci (IRIF) (Wu et al., 2000
; Zhong et al., 1999
). WRN also interacts with the complex, and hydrourea-induced foci of WRN was shown to be dependent on NBS1 (Cheng et al., 2004
; Franchitto and Pichierri, 2004
; Pichierri and Franchitto, 2004
). Autonomous accumulation of WRN at laser-induced damage sites through its HRDC domain suggests that the accumulation of WRN at double-strand breaks is an initial response of WRN to double-strand breaks in living cells. Because the accumulation of WRN at sites of laser-induced double-strand breaks is independent of replication and other interacting proteins (Figs 5, 7), the HRDC domain-dependent accumulation of WRN at double-strand breaks is followed by interaction with other proteins at the site of replication. Further analysis is necessary to identify the relationship between double-strand breaks accumulation and replication-dependent foci formation of WRN in cells and the steps after accumulation of WRN at double-strand breaks for forming IRIF.
Werner syndrome cells show replication defects and altered telomere dynamics leading to the shortening of telomeres. A mouse model with both Wrn and Terc (encoding the telomerase RNA component) deficiencies was shown to exhibit accelerated replicative senescence of cells with Werner-like premature aging phenotypes (Chang et al., 2004). Because the telomere exhibits a double-strand breaks-like structure, it is tempting to suppose that WRN accumulates via the HRDC domain at shortened telomeres after replication in late S phase and may contribute to the recovery of telomere length together with other proteins. Further studies of the functions of WRN at double-stand breaks in cells will help us to understand the molecular basis of the phenotype of WS patients.
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Acknowledgments |
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Footnotes |
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References |
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