Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in double stranded breaks (DSBs) repair through the DNA damage response (DDR) signal transduction pathway. It acts as a mediator of the DDR pathway by facilitating the recruitment of additional repair proteins to DNA damage sites [1
]. MDC1 protein contains a forkhead-associated (FHA) domain at the N-terminus and two BRCA1 carboxyl-terminal (tBRCT) domains at the C-terminus. The central region consists of 14 repeated sequences of approximately 41 amino acids each that makes up the DNA-PKcs/Ku binding region [4
]. In the event of DNA damage, MDC1 is hyper-phosphorylated in an Ataxia-Telangiectasia Mutated (ATM)-dependent manner and re-localized to the damaged region. It then interacts with phosphorylated γ-H2AX via the tBRCT domain and recruits other DDR proteins [5
]. MDC1 is also required for events that take place subsequent to the recruitment of repair proteins, including phosphorylation and activation of the repair proteins [9
]. Homologous recombination (HR) functions predominantly when homologous sister chromatids occur during S/G2 phase and eliminate DSBs in an error-free manner [10
]. In addition to its major role as a platform for DDR, MDC1 also plays primordial roles in HR repair via promoting recruitment of Rad51, which is a key player of HR, to DBS sites [11
]. Any delay or impairment in the recruitment of MDC1 to the nucleus leads to overall deficient DDR signal transduction.
Karyopherin α-2 (KPNA2), also known as importin α-1, is one of seven members of the karyopherin/importin-α family. It contains an N-terminal hydrophilic domain which binds to importin-β, a central hydrophobic region, and a short acidic C-terminus. The central hydrophobic region is comprised of 10 armadillo (ARM) repeats, which bind to the nuclear localization sequence (NLS) of cargo proteins and transport them to the nucleus through a nuclear pore complex (NPC) by forming a heterodimeric complex with importin-β [12
]. This nuclear transport process takes place in two steps: firstly, energy-independent docking of cargo proteins to the nuclear membrane, and secondly, energy-dependent transport of cargo proteins through the NPC [15
In this study, we identified KPNA2 as a specific nuclear import adaptor for MDC1 and demonstrated that the nuclear localization of MDC1 is partly regulated by NLS motif located between residues 1989–1994 of the tBRCT domain. We then showed that depletion of KPNA2 decreased HR repair and impaired recruitment of MDC1, as well as its downstream signaling proteins to DNA damage sites. These findings establish that KPNA2 regulates MDC1-mediated DDR by promoting MDC1 nuclear import.
In this study, we have evaluated the role of KPNA2 in the MDC1-mediated DDR signal and DSB repair pathway. The key findings of this study are as follows: firstly, KPNA2 regulates MDC1 nuclear transport through interaction of NLS sequences in the MDC1 tBRCT domain. Secondly, KPNA2 depletion impaired MDC1 nuclear transport, without affecting MDC1 expression level, leading to a marked decrease in the recruitment of MDC1 and its downstream proteins to DNA damage sites after irradiation and decreased HR-mediated DSB repair.
Accumulating data suggest that DDR proteins are transported to the nucleus via NLS-dependent interaction with importin [20
]. Although MDC1 is a well-known large nuclear protein that requires an active nuclear import mechanism to participate in DNA repair [2
], the mechanism of its nuclear translocation remained unknown to date. In this study, we identified KPNA2 as the specific member of the importin-α adaptor family that facilitates MDC1 recognition by the import machinery. KPNA2 belongs to a family of transporter proteins known to translocate NLS-containing cargo proteins from the cytoplasm into the nucleus [21
]. Selectivity for specific KPNA2 adaptor is mediated by specific NLS sequences in combination with structural features of cargo [26
]. We thus hypothesized that KPNA2 may recognize the nuclear targeting signal of MDC1. Using the NLStradamus program, we were able to identify a putative NLS in the tBRCT domain of MDC1. Our data showed that putative NLS (amino acid residues 1989–1994) was critical for binding of MDC1 to KPNA2 and also as concomitant targets of positive regulation for MDC1 nuclear import. The depletion of the NLS of MDC1 or KNPA2 knockdown led to a significant decrease in MDC1 protein in the nucleus, suggesting that KPNA2 is the transport adaptor for the MDC1 nuclear import. To the best of our knowledge, this is the first experiment to show how MDC1 is transported into the nucleus.
MDC1 is recruited to DSB sites and functions as an assembly platform to trigger the recruitment of additional DDR factors to DNA damage sites, including RNF8, RNF168, BRCA1, and 53BP1 [4
]. Thus, we looked for the effect of KPNA2 depletion on foci formation and found that IR-induced MDC1 nuclear foci were significantly lower in KPNA2 knockdown cells compared to control cells. Moreover, the recruitment of RNF8, 53BP1, BRCA1, and RNF168 to DNA damage sites was also compromised in KPNA2 knockdown cells. In contrast, γ-H2AX, a protein that acts upstream of MDC1 in the DDR, was unaffected by the presence or absence of KPNA2. These findings demonstrate that KPNA2 regulates DDR via MDC1 nuclear import.
HR is a repair pathway thought to be error-free because it uses undamaged sister chromatids as the template for repair [28
]. As MDC1 plays an important role in HR [11
], we also looked for the role of KPNA2 in HR. An assay for HR-mediated DNA repair using I-SceI-induced DSBs showed significantly lower rates of HR in KPNA2-depleted cells compared to control cells. Notably, depletion of both KPNA2 and MDC1 did not further decrease HR activity as observed with MDC1 depletion alone. Moreover, KPNA2 is known to be involved in the nuclear translocation of Nijmegen breakage syndrome 1 (NBS1 [20
], a key regulator of the MRE11-RAD50-NBS1 (MRN) complex, which plays an important role in the DSB repair pathway. Thus, the role of KPNA2 in HR repair may be to promote the nuclear import of both MDC1 and NBS1.
Human importin-α family is classified into three subfamilies on the basis of their sequence homology: the α1 subfamily includes KPNA1, KPNA5, and KPNA6; the α2 subfamily includes KPNA2 and KPNA7; the α3 subfamily includes KPNA4 and KPNA3 [29
]. The recently identified KPNA7 belongs to the same subfamily as KPNA2 and has a high amino acid similarity to KPNA2 [30
]. Thus, KPNA7 may exhibit functional redundancy with KPNA2 regarding transport of MDC1 to the nucleus. However, a striking feature of KPNA7 is its localization to the nucleus under steady state conditions, while KPNA2 is predominantly cytoplasmic [30
]. The difference in the intracellular distribution of these two proteins suggest that nucleocytoplasmic shuttling of KPNA7 differs from that of KPNA2. Importin-α isoforms contribute to distinctive physiological roles due to differential expression in tissues and differential binding affinity for these isoforms with NLS [32
]. Nevertheless, it does not exclude the possibility that other subfamily proteins, including α1 and α3, are involved in the nuclear transfer of MDC1, which would be good topics for future study.
In conclusion, this study establishes the role of KPNA2 as a novel regulator of the DDR function of MDC1, as it controls MDC1 nuclear import. Furthermore, we demonstrated that nuclear import of MDC1 is controlled by its NLS interacting with KPNA2, which is essential for the MDC1-mediated DDR pathway. Our work provides evidence for a novel connection between KPNA2 and MDC1, and evidence for the interaction of KPNA2 and MDC1 as a mechanism for regulating the response of MDC1 to DNA damage.
4. Materials and Methods
4.1. Cell Culture
The human cervix carcinoma HeLa cells, human embryonic kidney HEK293T cells, and DR-GFP U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). All cells were maintained in a humidified incubator containing 5% CO2 at 37 °C. Upon reaching 70–80% confluency, cells were digested with 0.5% trypsin-EDTA before being passaged. Cells in exponential growth were harvested for subsequent experiments. To induce DNA double strand breaks, exponentially growing cells were irradiated at 5 Gy from 137Cs source (Gammacell 3000 Elan irradiator, Best Theratronics, Ottawa, Canada) and allowed to recover at 37 °C incubator for various times.
4.2. Transfection of Small-Interference RNA (siRNA)
The five siRNA sequences against KPNA2 (NM_002266.2) are as follows: KPNA2 siRNA#1, 5’-GCAGCUAAGAAAGUACAUAdTdT-3′; KPNA2 siRNA #2, 5′-GCAUCAUGAUGAUCC AGAA dTdT-3′; KPNA2 siRNA #3, 5′-ACGAAUUGGCAUGGUGGUGAAdTdT-3′; KPNA2 siRNA #4, 5′-CCGGGUGUUGAUUCCGAAdTdT-3′; KPNA2 siRNA #5, 5′-CAGAUACC UG CUGGGCUAUUUCCU AdTdT-3′; MDC1 siRNA, 5′-UCCAGUGAAUCCUUGAGGUdTdT-3′; Negative control siRNA (Bioneer, Daejeon, Korea), 5′-CCUACGCCACCAAUUUCGUdTdT-3′. The control or KPNA2 siRNA were transiently transfected into the cells using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. At 48 h post transfection, the inhibition of KPNA2 or MDC1 was judged by western blot analysis.
4.3. Preparation of Plasmid Construction
The plasmids encoding with type, ΔFHA, ΔtBRCT-MDC1 were obtained from Zhenkun Lou and HA-FHA and HA-tBRCT-MDC1 construct were prepared by subcloning previously in our lab [1
]. Full-length human KPNA2 cDNA was amplified from HeLa cDNA, and the PCR products were cloned into pEGFP-N3 vector. To construct the expression vector encoding MDC1-ΔNLS (PARERR), we performed mutagenesis using GENEART® Site-Directed Mutagenesis System (Invitrogen) according to the manufacturer’s instructions. The all constructs were confirmed by automated DNA sequencing.
4.4. Western Blot Analysis
For total cell lysates extraction, cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 10 μg/mL leupeptin and 10 μg/mL aprotinin) for 30 min on ice. The supernatants were collected by centrifugation at 13,200 rpm for 20 min at 4 °C and then quantified using Bio-Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA USA). For cellular fractionation, cells were harvested and lyzed in cytosol extraction buffer (CEB; 10 mM HEPES pH 7.5, 3 mM MgCl2, 14 mM KCl, 5% glycerol, 1mM dithiothreitol, 1mM phenylmethanesulfonyl fluoride, 10 μg/mL leupeptin and 10 μg/mL aprotinin) for 10 min on ice. For complete lysis, 0.2% NP-40 was added followed by vortexing for 10 s. After centrifugation at 86,000× g for 2 min, the supernatant which is cytosolic fraction, was transferred to a new tube. The pellet was washed three times in CEB and then lysed in nuclear extraction buffer (NEB; 10 mM HEPES, 3 mM MgCl2, 400 mM NaCl, 5% glycerol, 1mM DTT, 1mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin) for 30 min at 4 °C, followed by centrifugation at 13,200 rpm for 30 min. The supernatant is nuclear fraction. Equal amounts of protein were separated by 6–12% SDS-PAGE, followed by electrotransfer onto a polyvinylidene difluoride membrane (PALL life sciences). The membranes were blocked for 1 h with TBS-t (10 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween-20) containing 5% nonfat milk and then incubated with indicated primary antibodies overnight at 4 °C. The blots were washed four times for 15 min with TBS-t and then incubated for 1 h with peroxidase-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch Inc, West Grove, PA, USA). The blots were washed four more times with TBS-t and developed using an enhanced chemiluminescence detection system (ECL; intron).
The protein extracts were precleared with protein A-Sepharose beads (GE Healthcare, Chicago, IL, USA) prior to adding the antibody. Next, after removing the protein A-Sepharose by centrifugation, the supernatant was incubated at 4 °C overnight with appropriate antibodies. After the addition fresh protein A-Sepharose bead, the incubation was continued for an additional one hours, and then beads were washed five times with RIPA buffer. The immune complexes were further analyzed by immunoblotting.
4.6. Immunofluorescence Microscopy
To visualize DNA damage foci, cells cultured on cover slips coated with poly-L-lysine (Sigma, Saint Louis, MO, USA) were irradiated at 5 Gy and allowed to recover at 37 °C for adequate times. Cells were fixed with 4% paraformaldehyde for 10 min and ice-cold 98% methanol for 5 min, followed by permeabilization with 0.3% Triton X-100 for 10 min at room temperature. After blocking using 5% BSA (Sigma), cells were single or double immunostained with primary antibodies against the indicated proteins and appropriate Alexa Fluor 488- (green, Molecular Probe, Eugene, OR, USA), Alexa Fluor 594- (red, Molecular Probe) conjugated secondary antibodies. Fluorescence images were taken using a confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) and analyzed with Zeiss microscope image software ZEN (Carl Zeiss). The foci number per cells was counted at least 100 cells.
4.7. Laser Microirradiation
To measure accumulation of GFP-MDC1 at microirradiated genomic regions, GFP-MDC1 transfected-control, and KPNA2-depleted HeLa cells were cultured onto a 35-mm round glass dish and 10 μM 5-bromo-2’-deoxyuridine (BrdU, Sigma) was added to the medium for 24 h. DSBs were then induced by microirradiation with a 405 nm laser 2 s irradiation time (100 lines/susing an A1 confocal microscope (Nikon, Japan). Images were acquired every 1 min for 10 min.
The following antibodies were used for western blot analysis: anti-MDC1 rabbit polyclonal antibody (R2, manufactured in our lab) [1
], anti-KPNA2 mouse monoclonal antibody (sc-55538, Santa cruz, Dallas, TX, USA), anti-HA mouse monoclonal antibody (sc-7392, Santa Cruz), anti-GFP mouse monoclonal antibody (sc-5286, Santa Cruz), anti-Histone3 rabbit polyclonal antibody (ab1791, Abcam, Cambridge, UK), and anti-α-Tubulin mouse monoclonal antibody (sc-5286, Santa Cruz). For immunoprecipitation assay, anti-HA rabbit polyclonal antibody (F-7, Santa Cruz), anti-GFP mouse monoclonal antibody (sc-5286, Santa Cruz), anti-MDC1 rabbit polyclonal antibody (R2, manufactured in our lab), and anti-KPNA2 mouse monoclonal antibody (sc-55538, Santa cruz) were used. The following antibodies were used for immunofluorescence staining: anti-MDC1 rabbit polyclonal antibody (R2), anti-HA mouse monoclonal antibody (sc-7392, Santa Cruz), anti-RNF8 goat polyclonal antibody (ab15850, Abcam), anti-53BP1 rabbit polyclonal antibody (sc-22760, Santa Cruz), anti-BRCA1 mouse monoclonal antibody (sc-6954, Santa cruz), anti-γH2AX mouse monoclonal antibody (05-636-1, Millipore, Burlington, MA, USA), and anti-RNF168 rabbit polyclonal antibody (ABE367, Millipore).
4.9. Neutral Comet Assay
Cells were left untreated or treated with 5 Gy γ-irradiation, followed by the incubation in culture medium at 37 °C for adequate times. Cells were harvested (20 μL, 1 × 105 cells per pellet), mixed with 200 μL low-melting temperature agarose, and layered onto agarose-coated glass slides. The slides were maintained in the dark at 4 °C for all of the remaining steps. Slides were immerged in lysis solution (Cat. #4250-050-01, TREVIGEN® Instructions, Gaithersburg, MD, USA) for 1 h at 4 °C and then placed into a horizontal electrophoresis apparatus filled with fresh neutral electrophoresis solution (100 mM Tris, 300mM Sodium Acetate at pH 9.0) for 30 min. After electrophoresis (~30 min at 1 V/cm tank length), slides were air-dried and stained with 30–50µl of SYBR green (Lonza, Basel, Switzerland). The slides were analyzed at × 400 magnification using a fluorescence microscope (Nikon). The microscope images revealed circular shapes indicating undamaged DNA, or comet-like shapes indicating the DNA had migrated out from the head to form a tail (damaged DNA). Average comet tail moment was scored for 40–50 cells/slide using a computerized image analysis system (Komet5.5, Andor Technology, Belfast, UK).
4.10. Clonal Survival Assay
After treatment with irradiation, 5 × 102 cells were immediately seeded on 60 mm dish in triplicate and grown for 2–3 weeks at 37 °C to allow colony formation. Colonies were stained with 2% methylene blue/50% ethanol and were counted. The fraction of surviving cells was calculated as the ratio for the plating efficiency of treated cells over untreated cells.
4.11. DR-GFP Assay
To measure the HR repair, U2OS-DR-GFP cells were transfected with control, KPNA2, MDC1 siRNA using lipofectamine RNAimax, and then infected with I-SceI-carrying adenovirus at an estimated MOI of 10. After 72 h, GFP-positive cells were measured by fluorescence-activated cell sorting (FACSCalibur, BD Bioscience, Franklin Lakes, NJ, USA). The acquired data was analyzed using CellQuest Pro software (BD Bioscience).
4.12. Statistical Analysis
Data in all experiments were represented as mean ± standard deviation (SD) for three independent experiments. Statistical comparisons were carried out using two-tailed paired Student’s t-test, where p < 0.01 (**) was considered statistically significant.