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Previous Article | Next Article 
Molecular and Cellular Biology, August 2005, p. 7021-7032, Vol. 25, No. 16
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.16.7021-7032.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Pharmacology, The University of Texas Southwestern Medical Center, 6001 Forest Park Rd., Dallas, Texas 75390
Received 25 February 2005/ Returned for modification 18 March 2005/ Accepted 24 May 2005
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ABSTRACT |
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INTRODUCTION |
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Two related protein kinases, ATM and ATR, are key proximal signal transducers of the DNA damage response (20, 41, 51). They phosphorylate distinct, yet overlapping, sets of downstream substrates, including CHK1, CHK2, p53, BRCA1, and NBS1 (20, 41, 51). CHK1 and CHK2 are effector kinases and in turn phosphorylate the CDC25 family of phosphatases and p53, leading to cell cycle arrest or apoptosis (20, 41, 51). Upon DNA damage, many proteins involved in DNA damage sensing and repair are enriched in nuclear foci, which are thought to represent active cellular centers for DNA repair (2, 25, 26, 49).
The structural maintenance of chromosomes (SMC) family of proteins is essential for chromosomal architecture and organization (11, 14, 36). There are six known eukaryotic SMC proteins, SMC1 to -6, which form three types of heterodimers (14). The SMC1/3 heterodimer is a part of the cohesin complex that maintains sister chromatid cohesion (11, 14, 23, 36). SMC2/4 are components of the condensin complex that mediates chromosome condensation during mitosis (14, 44). A third SMC complex, containing SMC5/6, is mainly involved in the cellular response to DNA damage (7, 11, 14, 24, 34).
Most studies of the SMC5/6 complex have been conducted in fission and budding yeasts. The yeast SMC5/6 complex contains several non-SMC elements (NSE), including NSE1, MMS21/NSE2 (hereafter referred to as MMS21 for simplicity), NSE3, and NSE4 (8, 17, 29, 32, 35, 42, 50). SMC5, SMC6, NSE1, and MMS21 are all essential genes in yeasts (7, 8, 24, 29, 34, 35, 39). Cells harboring hypomorphic alleles of these genes show an increased sensitivity to a broad spectrum of DNA damage agents, including ionizing radiation, UV, and methyl methanesulfonate (MMS) (7, 8, 17, 24, 29, 34, 35, 38, 39). Genetic analysis has shown that SMC5/6, NSE1, and MMS21 function together with RAD51 in the repair of DNA DSBs through HR in yeasts (13, 29, 31, 34, 35). Recently, the SMC5/6 complex has also been shown to play a role in the segregation of repetitive sequences during mitosis in budding yeast (47). A similar SMC5/6 complex exists in human cells, although the non-SMC components of this complex have not been characterized (46).
Small ubiquitin-like modifier (SUMO) is a ubiquitin-like protein that can be covalently conjugated to target proteins (9, 18). Similar to ubiquitination, sumoylation of substrates is catalyzed by a cascade of enzymes: the E1 SUMO-activating enzyme (AOS1-UBA2), the E2 conjugating enzyme (UBC9), and the E3 SUMO ligases (9, 18). Several classes of SUMO ligases have been identified (9, 18). Among them, the PIAS class of SUMO ligases contains an SP-RING domain that is similar to the RING finger domain found in many ubiquitin ligases (9, 18). Unlike ubiquitination, sumoylation of substrates does not strictly require a SUMO ligase. UBC9 is sufficient to mediate sumoylation due to its ability to directly recognize 
KXE (, hydrophobic residues; X, any residue) motifs on substrates. SUMO ligases merely enhance the rate of sumoylation. Furthermore, sumoylation of substrate proteins does not generally lead to their degradation (9, 18). Instead, it appears to regulate the functions of target proteins by multiple, context-dependent mechanisms, such as altering their subcellular localization, increasing their stability, or mediating their binding to other proteins (9, 18).
Interestingly, NSE1 contains a RING finger domain similar to ubiquitin ligases whereas MMS21 contains an SP-RING domain that is related to the PIAS family of SUMO ligases (29). It has recently been reported that both budding and fission yeast MMS21 proteins function as SUMO ligases (1, 50). Therefore, the SMC5/6 complex is a conserved chromatin-associated DNA repair complex that potentially has both ubiquitin ligase and SUMO ligase activities. Numerous studies have established multiple roles of sumoylation in regulating cellular responses for coping with DNA damage in both yeasts and mammals (12, 15, 16, 27, 28, 43, 45). Recently, using an in vitro expression cloning strategy, we have identified a large panel of human SUMO substrates (10). Several of these substrates are involved in DNA repair, including Ku80, XRCC1, and translin-associated factor X protein (TRAX) (10).
Our interest in sister chromatid cohesion and ubiquitin-like protein ligases prompted us to investigate the function of the SMC5/6 complex in human cells. We show that, similarly to the yeast MMS21, human MMS21 is a SUMO ligase that stimulates the sumoylation of hSMC6 and TRAX. RNA interference (RNAi)-mediated knockdown of hMMS21 increases the propensity of HeLa cells to undergo apoptosis in response to various DNA damage agents. Importantly, ectopic expression of the wild-type hMMS21, but not a ligase-inactive mutant of hMMS21, in hMMS21-RNAi cells rescues their hypersensitivity to DNA damage. This indicates that the SUMO ligase activity of hMMS21 is required for proper cellular response to DNA damage. Upon DNA damage, hMMS21-RNAi cells contained higher ATM/ATR activity and an increased number of phospho-CHK2 nuclear foci. Consistently, we observed an increased amount of unrepaired DNA lesions in hMMS21-RNAi cells as measured by the comet assay. These results suggest that hMMS21 and the hSMC5/6 complex play an important role in the repair of damaged cellular DNA.
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MATERIALS AND METHODS |
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In vitro sumoylation assay. Proteins were in vitro translated (IVT) from pCS2-myc plasmids in reticulocyte lysate (Promega) in the presence of [35S]methionine. IVT proteins were then subjected to in vitro sumoylation reactions with reaction mixtures that contained 2 µl of IVT product, 2 µg of AOS1-UBA2, 0.5 µg of UBC9, 1 µg of SUMO1, and 1 µl of energy mix (150 mM phosphocreatine, 20 mM ATP, 2 mM EGTA, 20 mM MgCl2, pH 7.7). Reaction mixtures were adjusted to 10 µl with XB buffer (10 mM HEPES, pH 7.7, 1 mM MgCl2, 0.1 mM CaCl2, 100 mM KCl, and 50 mM sucrose). After 2 h at 30°C, reactions were stopped with 10 µl of 2x sodium dodecyl sulfate (SDS) sample buffer, and reaction mixtures were boiled and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Reactions with reaction mixtures containing GST-hMMS21 were performed as described above except that 0.5 µg recombinant GST-hMMS21 purified from bacteria was used. Detection of GST-hMMS21 sumoylation was performed by incubating the SUMO reaction mixtures with glutathione-agarose beads for 1 h at 4°C. After washing, proteins bound to beads were eluted with 2x SDS sample buffer, boiled, and subjected to SDS-PAGE followed by immunoblotting with anti-SUMO1.
Human AOS1-UBA2, UBC9, and SUMO1 proteins were expressed and purified in bacteria as described previously (10). Briefly, pET28b-SUMO1 and pET28b-UBC9 were expressed as His6-tagged proteins in BL21(DE3) and purified using Ni2+-nitrilotriacetic acid beads per the manufacturer's protocols (QIAGEN). For the expression of AOS1-UBA2, pET11c-AOS1 and pET28b-UBA2 were cotransformed into BL21(DE3). The resulting AOS1-UBA2 complex was purified with Ni2+-nitrilotriacetic acid beads followed by a Superdex 200 gel filtration column (Amersham Biosciences) to remove the excess amount of AOS1. hMMS21 was expressed as a GST-tagged protein in BL21(DE3) and purified using glutathione-agarose beads (Amersham Biosciences).
Cell culture, transfections, and treatments. HeLa cells were grown in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 100 µg/ml penicillin and streptomycin (Invitrogen). At 40 to 50% confluence, plasmid or small interfering RNA (siRNA) transfection was performed using the Effectene reagent (QIAGEN) and the Oligofectamine reagent (Invitrogen), respectively. The siRNA oligonucleotides against hMMS21 (5'-CUCUGGUAUGGACACAGCUTT-3') and hSMC5 (5'-GCAGUGGAUUCAGGGUUGATT-3') were chemically synthesized at an in-house facility. The annealing and transfection of the siRNAs were performed as previously described (6). After 48 h, cells were treated with either 0.015% MMS or 40 µg/ml etoposide for the desired duration.
Immunoblotting and immunoprecipitation. At 24 h post-transfection of the desired plasmids, HeLa cells were lysed in SDS sample buffer, sonicated, boiled, separated by SDS-PAGE, and blotted with the indicated antibodies. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (Amersham Biosciences) was used as secondary antibody, and immunoblots were developed using the ECL reagent (Amersham Biosciences) per the manufacturer's protocols. For immunoprecipitation of overexpressed proteins, whole-cell lysate was made by lysing cells in NP-40 lysis buffer (50 mM Tris-HCl, pH 7.7, 150 mM NaCl, 0.5% NP-40, 1 mM dithiothreitol, and 1x protease inhibitor cocktail) on ice for 15 min and passing them through a 25-gauge needle 10 times, followed by centrifugation at 16,000 x g for 15 min at 4°C. Antibodies against myc and HA were covalently coupled to Affi-Prep Protein A beads (Bio-Rad) and incubated with the supernatants for 2 h at 4°C. The beads were then washed three times with the NP-40 lysis buffer. The proteins bound to the beads were dissolved in SDS sample buffer, boiled, separated by SDS-PAGE, and blotted with the indicated antibodies.
For immunoprecipitation of endogenous proteins, whole-cell lysate was made by lysing cells in buffer A (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.5% Triton X-100, 5 mM EDTA, 5 mM MgCl2, 50 mM NaF, 80 mM ß-glycerophosphate, 1 mM dithiothreitol, and 1x protease inhibitor cocktail) on ice for 10 min and then sonicated for 30 s. The lysate was then treated with 150 U/ml DNase I at 25°C for 1 h to help solubilize chromatin-bound proteins (46). The lysate was cleared by centrifugation at 16,000 x g for 15 min at 4°C. Antibodies against hSMC5, hBUBR1, and GST were covalently coupled to Affi-Prep Protein A beads (Bio-Rad) and incubated with the supernatants for 2 h at 4°C. After being washed with buffer A, the proteins bound to the beads were dissolved in SDS sample buffer, boiled, separated by SDS-PAGE, and blotted with the indicated antibodies.
Cell death quantification. Cells were treated with the appropriate conditions, and cell death was quantified in the following ways. Cells were stained with Hoechst 33258 (1:100; Molecular Probes) for 30 min at 37°C. All cells (floating and adherent) were collected and washed in phosphate-buffered saline (PBS). A portion of the cells was stained with trypan blue for 5 min. The number of blue cells (dead cells) was counted with a hemacytometer. At least 100 cells were counted for each condition in each experiment. The percentage of dead cells was then plotted. The stained nuclei were also imaged for nuclear condensation. The rest of the cells were lysed in sample buffer, sonicated, boiled, and separated by SDS-PAGE. The samples were immunoblotted with anti-Asp214 cleaved PARP.
Immunofluorescence. Cells were plated in four-well chambered slides (Lab-Tek) and treated according to experimental conditions. Cells were washed with PBS followed by fixation with 4% paraformaldehyde at 4°C for 30 min. The fixed cells were washed with PBS and blocked and permeabilized in Tris-buffered saline (TBS) containing 5% normal donkey serum and 0.03% Triton X-100 for 30 min at room temperature. After blocking, the cells were incubated in the appropriate primary antibodies overnight at 4°C in TBS containing 1% normal donkey serum and 0.03% Triton X-100. The cells were then washed with TBS and incubated with fluorescent secondary antibodies (Molecular Probes) in TBS containing 1% normal donkey serum and 0.03% Triton X-100 for 2 to 6 h at 4°C. After incubation, cells were washed with TBS and nuclei were stained with Hoechst 33258 (1:10,000) for 20 min at 4°C. Cells were again washed with TBS and mounted. Cells were viewed with a 63x objective on a Zeiss Axiovert 200M fluorescence microscope. Images were acquired with a charge-coupled device camera using Slidebook imaging software (Intelligent Imaging Innovations). Images examining the number of phospho-CHK2 (T68) foci were taken at 0.2-µm intervals, deconvolved using the nearest neighbor algorithm, and stacked to better resolve the number of foci. ATM/ATR phosphosubstrate antibody intensities were measured using the Slidebook imaging software. For both measurements, at least 100 cells were counted for each condition in each experiment.
Comet assay. Comet assays were performed according to the manufacturer's protocol (Trevigen). Briefly, cells were collected after the indicated treatments, mixed with low-melting-point agarose, and spread onto slides. The cell/agarose pad was then allowed to harden at 4°C for 30 min. Cells were then lysed by immersing slides in cold lysis solution for 30 min at 4°C. After lysis, slides were immersed in alkaline solution (200 mM EDTA, pH 13) for 20 min at room temperature to unwind and denature the DNA and hydrolyze sites of damage. Slides were then washed two times with Tris-buffered EDTA (TBE) for 5 min. Slides were then placed in a horizontal electrophoresis apparatus, equidistant from the electrodes in TBE. Voltage was applied for 10 min at 12 V (1 V per cm measured electrode to electrode). Slides were then immersed in 70% ethanol for 5 min and allowed to air dry at room temperature overnight. Slides were stained with SYBR Green I (Molecular Probes) for 10 min and then washed once in TBE. All incubations were performed in the dark to prevent unintended DNA damage. Slides were viewed using a 20x objective as described above. The percentage of cellular DNA in the comet tails was calculated by subtracting the nuclear florescence intensity from that of the entire cell. At least 100 cells were scored in each condition over three separate experiments.
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RESULTS |
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hMMS21 is a SUMO ligase in vitro and in vivo. We then tested whether hMMS21 has SUMO ligase activity in vitro. Because several known SUMO ligases, such as PIAS1 and RanBP2, undergo efficient autosumoylation (19, 37), we first tested whether hMMS21 was sumoylated in an in vitro sumoylation assay. Briefly, the 35S-labeled hMMS21 protein translated in rabbit reticulocyte lysate was incubated with purified, bacterially expressed AOS1-UBA2, UBC9, and SUMO1 proteins in the presence of ATP. hMMS21 efficiently formed high-molecular-weight conjugates on SDS-PAGE (Fig. 2A). The appearance of these high-molecular-weight conjugates was dependent on the presence of ATP, AOS1-UBA2, UBC9, and SUMO1, since the removal of each of these components abrogated the laddering (Fig. 2A). This suggested that hMMS21 was sumoylated in this assay. Addition of recombinant GST-PIAS1 or the SUMO ligase domain of RanBP2, two known E3 SUMO ligases, did not enhance the sumoylation of hMMS21 (see Fig. S1 in the supplemental material). In fact, addition of PIAS1 partially suppressed hMMS21 sumoylation (see Fig. S1 in the supplemental material), presumably due to competition between hMMS21 and PIAS1 for certain components in the SUMO reaction.
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We next performed in vitro sumoylation reactions using bacterially purified GST-hMMS21. GST-hMMS21 formed a high-molecular-weight ladder on SDS-PAGE in reactions with reaction mixtures containing ATP, AOS1-UBA2, UBC9, SUMO1, and GST-hMMS21 (Fig. 2C). These high-molecular-weight species of GST-hMMS21 were absent in reaction mixtures lacking any given one component (Fig. 2C). To confirm that these high-molecular-weight GST-hMMS21 species were indeed GST-hMMS21-SUMO1 conjugates, the same reaction mixtures were blotted with anti-SUMO1. A similar high-molecular-weight ladder of SUMO1 was observed when all components of the SUMO reactions were included (Fig. 2D). These results establish that hMMS21 is sumoylated in vitro and this sumoylation is dependent on an intact SP-RING domain.
We next tested whether hMMS21 was sumoylated in HeLa cells. myc-tagged hMMS21 was modified in the presence of untagged or green fluorescent protein (GFP)-tagged SUMO1 overexpression (Fig. 3A). As reported previously (10), GFP-SUMO1 was expressed at much higher levels and was thus conjugated more efficiently to substrates than untagged or endogenous SUMO1 (Fig. 3A). Modification of myc-hMMS21 was significantly reduced by expression of SENP2, a SUMO isopeptidase (Fig. 3A and B). In addition, modification of myc-hMMS21 was not observed with the overexpression of SUMO1 
GG, a SUMO mutant incapable of conjugation (18) (Fig. 3B). Finally, we performed immunoprecipitations of myc-hMMS21 in HeLa cells followed by immunoblotting with anti-SUMO1. Expectedly, the major modified form of hMMS21 contained SUMO1, indicating that hMMS21 was indeed sumoylated in HeLa cells (Fig. 3C). Furthermore, the myc-hMMS21 C215A mutant was sumoylated to a much lesser extent in HeLa cells, as was observed in vitro (Fig. 3B and C). These findings establish that hMMS21 is sumoylated in vivo and sumoylation of hMMS21 requires an intact SP-RING domain, consistent with hMMS21 being an SP-RING-type SUMO ligase.
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To determine whether the increased cell death of hMMS21-RNAi cells following MMS treatment was due to apoptosis, we examined the nuclear morphology of these cells. As expected, after MMS treatment, hMMS21 RNAi caused an increased number of cells with hypercondensed nuclei (data not shown), a hallmark of apoptotic cells. The number of cells with condensed nuclei was greatly reduced by expression of the wild-type hMMS21 but not the ligase-inactive mutant hMMS21 C215A (data not shown). To further confirm that the observed cell death was indeed apoptosis, we examined the cleavage of a known caspase 3 substrate, PARP. PARP cleavage was observed in hMMS21-RNAi cells treated with MMS (Fig. 5D). Cleavage of PARP was prevented by expression of the wild-type hMMS21, but not hMMS21 C215A, in hMMS21-RNAi cells (Fig. 5D). These results indicate that hMMS21-RNAi cells are more prone to undergo apoptosis in the presence of DNA damage, and the SUMO ligase activity of hMMS21 is required for preventing DNA damage-induced apoptosis.
RNAi-mediated depletion of hMMS21 results in hyperactivation of ATM/ATR following DNA damage. ATM and ATR are related protein kinases that are activated upon DNA damage and phosphorylate many cellular targets at Ser/Thr-Gln sites (22). To determine whether hMMS21 is required for the DNA damage response through ATM/ATR, we stained control and hMMS21-RNAi cells with an antibody that specifically recognizes phosphorylated Ser/Thr-Gln motifs (5) (Fig. 6A). The intensity of the antibody staining was quantified and used as a measure for the activity of ATM/ATR (5) (Fig. 6B). As expected, the level of phosphorylated ATM/ATR substrates increased upon DNA damage (Fig. 6A and B). In the absence of MMS, the basal level of ATM/ATR-mediated phosphorylation was higher in hMMS21-RNAi cells. After DNA damage, the hMMS21-RNAi cells contained threefold-more phosphorylated ATM/ATR substrates than did control cells (Fig. 6A and B).
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Knockdown of hMMS21 decreases repair of damaged DNA. To determine whether knockdown of hMMS21 resulted in an increased amount of unrepaired DNA lesions following DNA damage, we performed a single-cell gel electrophoresis assay (comet assay) (4). The comet assay is simple and widely used to evaluate the amount of DNA damage in cells. It is based on the ability of denatured, cleaved DNA fragments to migrate out of the cell when current is applied, whereas undamaged DNA migrates slower and remains within the confines of the nucleus (4).
We employed this assay to measure the amount of unrepaired DNA damage in control or hMMS21-RNAi cells after MMS treatment. As expected, control and hMMS21-RNAi cells had no comet tails without treatment of MMS (Fig. 7A). To measure the ability of cells to repair damaged DNA, we treated cells with 0.015% MMS for 1 h and then washed out MMS and allowed cells to repair the damaged DNA for 3 h. The hMMS21-RNAi cells, unlike control cells, were incapable of repairing their DNA lesions (Fig. 7A). The amount of DNA that migrates out of the nucleus and forms a comet tail provides a quantitative measure of the amount of DNA damage in the cell (4). Quantitation of the amount of DNA in the comet tails showed a dramatic increase in the amount of unrepaired DNA lesions in the hMMS21-RNAi cells (95%) compared to control cells (25%) (Fig. 7B). To rule out the possibility that the increase in the amount of unrepaired DNA lesions was due simply to an increased number of apoptotic cells in the hMMS21-RNAi culture, we performed the experiment in the presence of a pan-caspase inhibitor, zVAD-fmk. Although zVAD-fmk could inhibit MMS-induced cell death in hMMS21-RNAi cells (see Fig. S2 in the supplemental material), addition of zVAD-fmk had no effect on the amount of unrepaired DNA present in hMMS21-RNAi cells (Fig. 7A and B). Finally, to rule out the possibility that hMMS21-RNAi cells were more susceptible to DNA damage than control cells, we treated cells with 0.015% MMS for 1 h without a period for repair. Treatment of both control and hMMS21-RNAi cells with MMS without recovery resulted in comet tails with similar, large amounts of DNA damage lesions (Fig. 7A and B). These results suggest that knockdown of hMMS21 causes a decreased ability of cells to repair DNA lesions.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SUMO ligase activity of MMS21. Studies of both S. cerevisiae MMS21 and S. pombe NSE2 have identified MMS21 as a SUMO ligase that autosumoylates. Our results concur with these previous findings and show that hMMS21 is a SUMO ligase that undergoes autosumoylation in vitro and in vivo. The S. cerevisiae MMS21 has been shown to stimulate the sumoylation of two substrate proteins, SMC5 and Ku70 (50). The S. pombe NSE2 stimulates the sumoylation of SMC6 and NSE3, but not SMC5 or NSE1 (1). Consistent with the results in S. pombe, we show that hMMS21 enhances sumoylation of hSMC6. In addition, we also identify TRAX as a substrate of hMMS21. The differences with respect to MMS21-mediated sumoylation of SMC5/6 in S. pombe, S. cerevisiae, and Homo sapiens could reflect interesting differences in the mechanism and regulation of the SMC5/6 complex in these organisms.
In fission yeast, SMC6 sumoylation is dependent on NSE2, indicating that SMC6 is a physiologically relevant substrate of NSE2 (1). Like many SUMO substrates in mammalian cells, we were unable to detect the sumoylation of SMC6 or TRAX in the absence of SUMO1 overexpression, presumably due to the low steady-state levels of SUMO conjugates of these proteins. Upon overexpression of SUMO1, the SUMO1 conjugates of SMC6 and TRAX were observed in HeLa cells. Overexpression of hMMS21 further enhanced the sumoylation of SMC6 and TRAX. However, we did not detect a decrease in the sumoylation of SMC6 or TRAX in hMMS21-RNAi cells. This could be due to the incomplete knockdown of hMMS21 by RNAi. Alternatively, sumoylation of SMC6 and TRAX in the presence of SUMO1 overexpression does not strictly require hMMS21. It is also possible that other SUMO ligases can mediate the sumoylation of hSMC6 and TRAX under these conditions. Therefore, our results indicate that hMMS21 is capable of stimulating the sumoylation of SMC6 and TRAX in living cells. It remains to be established whether and how sumoylation of SMC6 and TRAX is important for DNA repair in human cells.
Role of MMS21 and the SMC5/6 complex in DNA repair. Down-regulation of the SMC5/6 complex in yeasts and in mammalian cells (this study) renders cells more sensitive to a wide spectrum of DNA-damaging agents (7, 8, 17, 24, 29, 34, 35, 38, 39). It is possible that the SMC5/6 complex is directly involved in the repair of multiple types of DNA lesions. On the other hand, components of the SMC5/6 complex are epistatic with RAD51 and RAD52, suggesting that the SMC5/6 complex may function in the repair of DSB through the RAD51- and RAD52-dependent HR pathway (1, 29, 32, 34, 35). Because many types of DNA-damaging agents can indirectly lead to the generation of DSBs, it is also possible that a major function of the SMC5/6 complex is the repair of DSB through HR.
In addition to DNA repair, cohesin and condensin play roles in the DNA damage checkpoint that delays cell cycle progression to allow time for DNA repair (11). Though the SMC5/6 complex is not required for the initiation of the DNA damage checkpoint response in S. pombe, it is involved in the maintenance of the checkpoint (13). In this study, we show that ATM/ATR are activated to a greater extent in hMMS21-RNAi cells, which is more consistent with SMC5/6 playing a role in DNA repair. On the other hand, hMMS21 RNAi does not completely deplete the cellular pool of the hMMS21 protein. It is possible that the residual amount of hMMS21, though insufficient for DNA repair, is sufficient for DNA damage checkpoint signaling. In addition, hMMS21-RNAi cells might be defective in checkpoint pathways that do not involve ATM/ATR.
Role of sumoylation in DNA repair. Through its reversible, covalent modification of target proteins, SUMO regulates diverse cellular processes, including transcription, chromatin remodeling, and DNA repair (9). Several proteins involved in DNA repair are SUMO substrates, including PCNA, WRN, XRCC1, KU70, KU80, and TRAX (9, 10, 50). We show that a subunit of the hSMC5/6 complex, hMMS21, is a functional SUMO ligase. Furthermore, the SUMO ligase activity of hMMS21 is essential for its function in protecting cells from apoptosis induced by DNA damage, thus further establishing a role of sumoylation in DNA repair. At present, we do not know the mechanism by which the ligase activity of MMS21 regulates DNA repair. However, we envision two nonexclusive possibilities. First, hMMS21 might stimulate the sumoylation of other DNA damage checkpoint and repair proteins, such as TRAX, and mediate their recruitment to the sites of DNA damage. Consistent with this notion, sumoylation has been shown to alter the subcellular localization and/or kinetics of nucleocytoplasmic trafficking of certain target proteins (9, 18). However, we did not observe failure in the recruitment of DNA damage checkpoint and repair proteins, such as BRCA1, BRCA2, CHK1, CHK2, or RAD51, to nuclear foci after DNA damage in hMMS21-RNAi cells (Fig. 6C and data not shown). In a second possibility, sumoylation of SMC6 by MMS21 might regulate its ATPase activity, thus affecting the loading and unloading of the SMC5/6 complex onto chromatin. It will be interesting to examine the possibility that SMC5/6 loading and unloading are required for DNA repair and how the SUMO ligase activity of hMMS21 may regulate this process during the cellular DNA damage response.
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ACKNOWLEDGMENTS |
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-SMC5 antibody; C. Gocke for GST-hMMS21 and for a preliminary experiment that identified TRAX as an hMMS21 substrate; and J. Bembenek for preparation of recombinant AOS1-UBA2, SUMO1, and UBC9 proteins. We also thank X. Zhao and G. Blobel for communicating results prior to publication and H. Zou, C. Gocke, and J. Kang for helpful discussions and for reading the manuscript critically. H.Y. is the Michael L. Rosenberg Scholar in Biomedical Research. This work is supported by the Packard Foundation and the W. M. Keck Foundation.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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