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Molecular and Cellular Biology, May 2007, p. 3793-3803, Vol. 27, No. 10
0270-7306/07/$08.00+0 doi:10.1128/MCB.02269-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Stuart Rulten,1,
Sherif F. El-Khamisy,1,2 and
Keith W. Caldecott1*
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, Sussex BN25 3EU, United Kingdom,1 Biochemistry Department, Faculty of Pharmacy, Aim Shams University, P.O. Box 11566, Cairo, Egypt2
Received 5 December 2006/ Returned for modification 4 January 2007/ Accepted 5 March 2007
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INTRODUCTION |
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DNA end processing is perhaps the most enzymatically diverse step of DNA strand break repair, primarily because of the broad range of termini that can arise. One enzyme implicated in the repair of oxidative DNA termini at both DNA single- and double-strand breaks is polynucleotide kinase (PNK) (10, 33). PNK associates with the DNA single-strand break repair (SSBR) and double-strand break repair (DSBR) protein complexes through direct interaction with casein kinase 2 (CK2)-phosphorylated XRCC1 and XRCC4, respectively (16, 21). These interactions are mediated through a divergent forkhead-associated (FHA) domain in PNK that can specifically target CK2-phosphorylated peptides. Intriguingly, the protein aprataxin, which is mutated in the neurodegenerative disease ataxia-oculomotor apraxia type 1, possesses a similar FHA domain and binds the same CK2-phosphorylated regions of XRCC1 and XRCC4 (6, 11, 12-14, 22, 25). Aprataxin is also an end processing enzyme, repairing abortive 5' AMP intermediates of DNA ligase activity (1). In the current study, we identify a third member of the aprataxin- and PNK-like FHA domain family which we denote APLF (aprataxin- and PNK-like factor). We demonstrate that APLF is a novel component of the cellular response to chromosomal DNA single- and double-strand breaks.
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MATERIALS AND METHODS |
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To create pRFP-C1-XRCC1, the XRCC1 ORF was amplified from pcD2EXH (8) by PCR using the primers 5'-AAGAATTCTATGCCGGAGATCCGCCTCCGCC-3' (the EcoRI site and the XRCC1 start codon are underlined) and 5'-TTGTCGACTCAGGCTTGCGGCACCACCCCATAGAG-3' (the SalI site and the stop codon are underlined) and subcloned into the EcoRI/SalI sites of pRFP-C1 encoding monomeric red fluorescent protein (mRFP) (9). To create pRFP-C1-XRCC4, the XRCC4 ORF was subcloned from pCI-neo-Myc-XRCC4 (kindly provided by Queti Riballo and Elaine Taylor) into pRFP-C1 by using EcoRI and XbaI. All recombinant histidine-tagged proteins expressed in Escherichia coli were expressed in BL21(DE3) and purified by metal chelate affinity chromatography using nickel-nitrilotriacetic acid agarose (QIAGEN).
Protein slot blots and far-Western blotting. Recombinant XRCC1-His (10 µg) expressed in and purified from bacteria (see above) was phosphorylated with CK2 in 90-µl reaction mixtures containing 10 mM MgCl2, 1 mM ATP, 13.3 mM HEPES, pH 8, 80 mM NaCl, and 0.67 mM dithiothreitol (DTT) at 30°C for 30 min. Mock phosphorylation reactions lacked CK2 or XRCC1, as indicated. For slot blot experiments, mock phosphorylation reactions and aliquots of CK2 phosphorylation reactions (0.125 to 1 µg of XRCC1-His as indicated) were slot blotted under a vacuum, in quadruplicate, onto Hybond-C nitrocellulose membranes. The membranes were air dried and one strip stained with amido black. The remainder were blocked in binding buffer (20 mM HEPES, 50 mM NaCl, 1 mM DTT) containing 2% (wt/vol) nonfat dried milk (NFDM) at room temperature for 4 h. Blots were then washed (three times for 5 min each) with binding buffer lacking NFDM and incubated with 5 µg of full-length recombinant His-APLF, His-APLF1-166, or His-APLF360-511 in binding buffer at 4°C overnight. Blots were then washed (three times for 5 min each) in binding buffer and incubated in binding buffer containing 2% NFDM and affinity-purified anti-APLF polyclonal antibody (SK3595) at a dilution of 1 in 500 at room temperature for 4 h. The blots were then washed (three times for 10 min each) in binding buffer and incubated in binding buffer containing 2% NFDM and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody at a dilution of 1 in 5,000 at room temperature for 1 h. Finally, following further washes in binding buffer (three 5-min washes), antibody complexes were detected by enhanced chemiluminescence (ECL Plus; Amersham Biosciences) and autoradiography. SK3595 was raised by Eurogentec and affinity purified against recombinant human APLF expressed and purified from E. coli.
For far-Western blotting, 3 µg of full-length recombinant His-APLF, His-APLF1-166, or His-APLF360-511 was fractionated in quadruplicate by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to a Hybond-C nitrocellulose membrane. The membrane was stained with Ponceau S to confirm equal loading and transfer and then destained. Proteins were denatured and renatured by sequential incubations (10 min each) with decreasing concentrations (6 M to 0.19 M) of guanidine-HCl essentially as described previously (23). Membranes were then incubated at 4°C overnight with 2 ml 1% NFDM-HYB100 containing recombinant XRCC1-His (10 µg) prephosphorylated in 90-µl reaction mixtures (at 30°C for 30 min) containing 10 mM MgCl2, 1 mM ATP, 13.3 mM HEPES, pH 8, 80 mM NaCl, 0.67 mM DTT, and rat liver CK2 (kindly provided by Flavio Meggio and Lorenzo Pinna). Mock phosphorylation reactions lacked CK2 or XRCC1-His, as indicated. Filters were then washed with 1% NFDM-HYB100 and the membranes incubated with anti-XRCC1 polyclonal antibody (SK3188) at a dilution of 1 in 1,000 in 1% NFDM-HYB100 at room temperature for 2 h. The membranes were then washed (three times for 10 min each) with 1% NFDM-HYB100 and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody at a dilution of 1 in 5,000 in 1% NFDM-HYB100 at room temperature for 30 min. Following further washes (three 10-min washes) with 1% NFDM-HYB100, antibody complexes were detected by enhanced chemiluminescence (ECL Plus; Amersham Biosciences) and autoradiography.
Anti-APLF (SK3595) and anti-XRCC1 (SK3188) polyclonal antibodies were raised by Eurogentec against recombinant human APLF and XRCC1 from E. coli and insect cells, respectively, and were affinity purified against the appropriate recombinant proteins from E. coli.
Yeast two-hybrid analyses. Saccharomyces cerevisiae Y190 cells were transformed with the indicated pACT and PGBKT7 constructs and transformants selected on minimal medium plates (glucose plus yeast nitrogen base without amino acids) additionally containing adenine and histidine. Pooled populations of transformants were streaked onto two minimal medium plates with adenine and histidine and one minimal medium plate with adenine and 50 mM 3-amino-1,2,4-triazole. The activation of the His3 reporter gene was indicated by comparative growth on minimal medium plates containing adenine and 3-amino-1,2,4-triazole and minimal medium plates containing adenine and histidine after 4 to 6 days at 30°C. The activation of the ß-galactosidase (ß-Gal) reporter gene was determined by qualitative X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) assays on colony filter lifts from minimal medium plates containing adenine and histidine. Quantitative ß-Gal assays were conducted essentially as described by Clontech (Yeast Protocols Handbook). Equivalent expression levels of DNA binding domain or activation domain fusion proteins were confirmed by Western blotting both for cell cultures employed for filter lift ß-Gal assays and for quantitative ß-Gal assays. Cell extracts were prepared in cracking buffer (8 M urea, 5% [wt/vol] SDS, 40 mM Tris-HCl [pH 6.8], 0.1 mM EDTA, 0.4 mg/ml bromophenol blue, 1% ß-mercaptoethanol, 1x protease inhibitor cocktail [Sigma]) and fractionated by SDS-PAGE, and Myc-tagged APLF (expressed from pGBKT7) was detected by immunoblotting with anti-Myc monoclonal antibody (MAb) (9B11; Cell Signaling Technology) and DNA binding domain fusion proteins by immunoblotting with polyclonal anti-GAL4 activation domain antibody (Upstate Biotechnology). Washed membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Dako) and bound antibodies visualized using enhanced chemiluminescence (Amersham Biosciences).
Immunoprecipitations.
For immunoprecipitation experiments, 
3 x 107 HeLa cells were transiently transfected with pcD2E vector or pcD2E-His-Myc-APLF by using GeneJuice transfection reagent (Novagen) and pooled populations of transfected cells were selected in medium containing G418 (0.8 mg/ml) for 5 days. Alternatively, 3 x 107 A549 cells were stably transfected with pCD2E and either empty pSUPER or pSUPER encoding the APLF RNA interference (RNAi) sequence GAAGAAATCTGCAAAGATA (pSUPER-APLF) by selection with 0.8 mg/ml G418 for 4 weeks. Transfected cells were lysed in 3 ml lysis buffer (20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.5 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM ß-glycerophosphate, and 1x protease inhibitor cocktail [Sigma]) on ice for 5 min. Clarified extracts were diluted to 2 mg/ml in lysis buffer and 3 ml of each extract precleared for 1 h at 4°C with 60 µl protein G-Sepharose beads (Sigma). HeLa cell extract (1.5 ml) was incubated with 1 µl of either anti-Myc MAb (9B11) or anti-Flag MAb (M2; Sigma) and A549 cell extract (1.5 ml) with either 100 µl anti-XRCC1 polyclonal antibody (SK3188) or 0.01 µl rabbit immunoglobulin G (IgG; Dako) overnight at 4°C with gentle agitation. Each sample was then incubated with 30 µl protein G-Sepharose beads for 1 h at 4°C with gentle agitation. The beads were then washed three times with lysis buffer (300 µl each wash) and bound proteins eluted by heating with 55 µl of 2x SDS-PAGE sample buffer at 90°C for 5 min. Clarified protein samples were fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Myc MAb (9B11) or either anti-XRCC1 (SK3188) or anti-APLF (SK3595) polyclonal antibody.
Direct and indirect immunofluorescence experiments. For direct immunofluorescence experiments, XRCC1 mutant EM9 (30, 31) or XRCC4 mutant XR-1 (20, 28) CHO cells seeded onto coverslips were transfected with the indicated enhanced YFP or mRFP fusion protein constructs by using GeneJuice transfection reagent (Novagen). After 24 h, cells were washed with phosphate-buffered saline (PBS) and mock treated or treated with 10 mM hydrogen peroxide in PBS for 10 min at room temperature, washed with PBS, and then incubated in drug-free media at 37°C for 20 to 120 min. The coverslips were then washed in PBS and fixed (5 min in 4% paraformaldehyde in PBS). Fixed cells were then permeabilized in 0.2% Triton X-100 for 2 min, washed with PBS, and stained with 0.000025% DAPI (4',6'-diamidino-2-phenylindole) for 5 min. Coverslips were mounted in Vectashield (Vector Labs) and analyzed with a Zeiss Axioplan 2 fluorescence microscope. Photographs were taken at a magnification of x100 and YFP-APLF-transfected cells scored for subcellular localization of YFP-APLF.
For indirect immunofluorescence experiments, A549 cells were seeded onto gridded coverslips (MatTek) in 10-cm dishes at 1 x 104 cells/cm2 and, after 2 days, preincubated for 10 min with 10 µg/ml Hoechst dye 33258 at 37°C. Selected cells were then irradiated with a 351-nm UVA laser focused through a 40x/1.2-W objective using a Zeiss Axiovert equipped with LSM 520 Meta. UVA (10.47 µJ) was introduced to an area of approximately 15 µm by 2 µm (approximately 0.35 µJ/µm2). After exposure, coverslips were incubated at 37°C for the times indicated and washed in ice-cold PBS, and cells were fixed in ice-cold methanol-acetone (1:1) for 5 min and then permeabilized for 5 min in PBS-0.1% Tween. Coverslips were incubated in PBS-5% NFDM for 30 min for blocking and then overnight at 4°C in PBS-1% NFDM containing anti-XRCC1 MAb (clone 33-2-5) and anti-APLF polyclonal antibody (both at 1/200 dilutions). Coverslips were then incubated with secondary labeling with Alexa Fluor 555 anti-mouse and Alexa Fluor 488 anti-rabbit antibodies (1/200 in PBS-1% milk; Invitrogen) for 1 hour at room temperature. DNA was stained with 0.1 µg/ml DAPI (Sigma). Tracks were then visualized using a Nikon Eclipse 50i microscope fitted with a x100 oil immersion objective.
Chromosomal DNA strand break repair assays.
To knock down APLF in SH-SY5Y cells, 2 x 106 cells were cotransfected with pCD2E vector (2 µg) and either pMAX-GFP vector (4 µg; Amaxa), pSUPER vector harboring the APLF RNAi sequence GAAGAAATCTGCAAAGATA (4 µg), or 160 pg of the XRCC1 small interfering RNA (siRNA) duplex GCCUGAAGUAUGUGCUAUAdTdT (sense strand; QIAGEN) by using Nucleofector and Nucleofector kit V (Amaxa). Twenty-four hours after nucleofection, cells were placed into a mild selection/differentiation medium containing 0.25 mg/ml G418 and 10 µM all-trans retinoic acid to induce neuronal differentiation. After 3 days of selection, the medium was replaced with medium containing 10 µM retinoic acid only. For alkaline comet assays, 5 x 105 SH-SY5Y cells were harvested 6 days after transfection and irradiated (20 Gy of gamma rays) before being returned to 37°C for 7.5, 15, or 30 min. At each time point, 1 x 105 treated cells were removed and DNA damage was measured by single-cell agarose gel electrophoresis, essentially as described previously (5). For 
-H2AX assays, A549 cells stably transfected with pCD2E and either empty pSUPER or pSUPER encoding the APLF RNAi sequence GAAGAAATCTGCAAAGATA, or SH-SY5Y cells 6 days after transient transfection with green fluorescent protein, pSUPER-APLF, or XRCC1 siRNA as described above, were grown on glass coverslips and exposed to 1 to 3 Gy of -radiation before being returned to 37°C. At the times indicated, coverslips were washed in PBS, fixed (5 min in 4% paraformaldehyde in PBS), permeabilized (0.2% Triton X-100 for 2 min), blocked (5% NFDM for 30 min), and incubated with anti--H2AX MAb (clone JBW301, 1/800 in 1% NFDM in PBS; Upstate). Cells were then washed (three times for 5 min each) in PBS containing 0.1% Tween 20 and 0.02% SDS and incubated for 1 h with Alexa Fluor 488 goat anti-mouse IgG secondary antibody (1/200 dilution in 1% milk-PBS). The cells were then washed (five times for 5 min each) as described above, mounted in Vectashield (Vector Labs), and scored for -H2AX foci by using a Nikon Eclipse 50i microscope.
DNA damage-induced modification of APLF. Subconfluent 1BR3, FD105 (AOA1), or AT7BI (A-T) primary fibroblasts were harvested, either exposed to gamma rays (3 Gy) or treated with 10 mM H2O2 on ice for 20 min, and then incubated at 37°C for 20 min (H2O2) or 30 min (gamma rays) in drug-free media. Cells were then harvested and lysed in SDS-PAGE sample buffer, and aliquots of cell extracts were fractionated by SDS-PAGE (7.5% gels) and immunoblotted with anti-APLF polyclonal antibody (SK3595).
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RESULTS |
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(Fig. 2A). We showed previously that neither XRCC1 nor XRCC4 transactivates the His3 or ß-Gal reporter genes in these assays by itself (11). In addition, endogenous XRCC1 was immunoprecipitated from Myc-APLF-transfected HeLa cell extracts by anti-Myc antibodies but was not immunoprecipitated by control anti-FLAG antibodies (Fig. 2B, upper panel) or by anti-Myc antibodies from untransfected cells (Fig. 2B, lower panel). More importantly, immunoprecipitation of endogenous XRCC1 from the human A549 cell extract, which we detected using both standard anti-XRCC1 polyclonal antibodies and antibodies specific for CK2-phosphorylated XRCC1(pS485/pT488) (21, 22), corecovered endogenous APLF, confirming that APLF is a bona fide component of the single-strand break repair machinery (Fig. 2C, left panel). That it was APLF that was recovered and detected in these experiments was confirmed by its absence from A549 cells in which APLF was depleted (Fig. 2C, middle and right panels). Interestingly, we noted in these and other experiments (Fig. 3A) that APLF migrates much more slowly than expected during SDS-PAGE, at a position expected for a polypeptide of 75 kDa, most likely due to the acidic C-terminal tail.
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To further examine whether APLF is a component of the DNA damage response, we compared the subcellular localization patterns of endogenous XRCC1 and APLF in human A549 cells following UVA laser irradiation. As expected, XRCC1 rapidly accumulated (within 3 min) at sites of UVA damage (Fig. 4A). More significantly, APLF colocalized and accumulated with XRCC1 at these sites, confirming that APLF is a component of the response to UVA-induced cellular damage. To examine the relationship between XRCC1 and APLF in more detail, we compared the subcellular localization patterns of YFP- and RFP-tagged human APLF and XRCC1 before and after treatment with H2O2 in transiently transfected XRCC1 mutant CHO cells (EM9 cells). Whereas RFP-XRCC1 was largely or entirely nuclear in all transfected EM9 cells, YFP-APLF was largely nuclear or pancellular in 60% of cells in the presence of RFP-XRCC1 and largely cytosolic or pancellular in 90% of cells in the absence of RFP-XRCC1 (Fig. 4B and C, left panel). The fraction of cells with largely nuclear YFP-APLF transiently increased after H2O2 treatment in an RFP-XRCC1-dependent manner (Fig. 4C, middle and right panels). The presence of a large proportion of cells with largely nuclear YFP-APLF was also dependent on a functional FHA domain, because the YFP-APLFR27A protein harboring a mutated FHA domain was largely cytoplasmic or pancellular in most cells both before and after H2O2 treatment, even in the presence of RFP-XRCC1 (Fig. 4C). We conclude that RFP-XRCC1 promoted the nuclear import or retention of YFP-APLF in these experiments in a manner that was stimulated by oxidative DNA damage and which required the APLF FHA domain.
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83%) of cotransfected cells, YFP-APLF and RFP-XRCC1 colocalized in subnuclear foci after H2O2 treatment (Fig. 4D and 5A, left panel). In contrast, far fewer focus-positive cells (15%) were observed in EM9 cells transfected with either YFP-APLF alone or a combination of YFP-APLFR27A and RFP-XRCC1 (Fig. 5A). These data suggest that RFP-XRCC1 stimulates the accumulation of YFP-APLF at oxidative DNA strand breaks but that it is not essential for this process. To examine whether this effect of RFP-XRCC1 reflected its impact on the nuclear localization of YFP-APLF, we created YFP-nls-APLFR27A, a derivative of YFP-APLFR27A in which we engineered an SV40 nuclear localization signal (NLS). As expected, YFP-nls-APLFR27A was largely or entirely nuclear and, moreover, accumulated extensively at oxidative DNA strand breaks (Fig. 5A, right panel). These data suggest that XRCC1 facilitates the accumulation of APLF at oxidative SSBs by promoting the nuclear import of APLF, in part at least.
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40%) of XR-1 cells possessed YFP-APLF or YFP-APLFR27A foci after H2O2 treatment (data not shown). This most likely reflects the XRCC1-independent recruitment/accumulation of recombinant APLF at DNA strand breaks that was observed, albeit to a lesser extent, in EM9 cells. We considered the possibility that the XRCC1-independent recruitment/accumulation of APLF at sites of oxidative DNA damage might be mediated via the putative C2H2 ZnF motifs. To examine this possibility, we expressed derivatives of APLF in which the two putative zinc coordinating cysteine residues in the first (YFP-APLFzfm1) or second (YFP-APLFzfm2) ZnF were mutated to alanine. Neither of the ZnF mutations reduced XRCC1-dependent nuclear import/retention of YFP-APLF in untreated EM9 cells, although the ZFM1 mutation did reduce the XRCC1-dependent increase in nuclear YFP-APLF observed after H2O2 treatment (Fig. 5B, right panel). The ZFM1 mutation also reduced the formation of YFP-APLF nuclear foci in the presence of XRCC1, by 30% (Fig. 5C, left panel). More significantly, however, the XRCC1-independent accumulation of YFP-APLF in nuclear foci was reduced by the ZFM2 mutation and ablated by the ZNF1 mutation (Fig. 5C, right panel). These data confirm that the tandem ZnF motif is required for XRCC1-independent recruitment/accumulation of APLF at sites of oxidative DNA strand breakage. To examine whether the tandem ZnF promoted XRCC1-independent accumulation of YFP-APLF at sites of oxidative DNA damage through an impact on nuclear localization, we employed derivatives of YFP-APLF and YFP-APLFZnF1 that harbored an SV40 NLS, denoted YFP-nls-APLF and YFP-nls-APLFZnF1, respectively. While both YFP-nls-APLF and YFP-nls-APLFZnF1 were able to assemble into subnuclear foci in the presence of XRCC1, only YFP-nls-APLF did so in the absence of RFP-XRCC1 (Fig. 5D, left and middle panels). Thus, the addition of an NLS did not circumvent the requirement for the tandem ZnF, suggesting that the ZnF fulfills a function other than increasing the nuclear import/retention of YFP-APLF. Interestingly, we noted in these experiments that RFP-XRCC1 stimulated the assembly of YFP-nls-APLF nuclear foci, despite the presence of an NLS, and did so in a manner that was dependent on an intact FHA domain (Fig. 5D, middle and right panels). This suggests that the impact of the XRCC1-APLF interaction extends beyond its ability to promote APLF nuclear import/retention. In summary, we conclude from these experiments that the accumulation of YFP-APLF at oxidative DNA strand breaks is promoted by at least three mechanisms: two that are dependent on FHA domain-mediated interaction with XRCC1, including one that involves increased nuclear import/retention of APLF, and one that is independent of XRCC1 but dependent on the tandem ZnF motif.
To further examine the involvement of APLF in the response to DNA damage, we compared its electrophoretic mobilities before and after treatment with gamma rays or H2O2. Notably, the mobility of endogenous APLF during SDS-PAGE decreased after treatment with either H2O2 or ionizing radiation (IR) in wild-type cells and in aprataxin-defective AOA1 primary fibroblasts (Fig. 6A). However, this shift in electrophoretic mobility was not detected in ataxia telangiectasia fibroblasts lacking the DSB-responsive protein kinase ATM. These data support the idea that APLF is involved in the cellular response to DSBs. To examine whether APLF is required for chromosomal DNA single- and double-strand break repair, we mock depleted or depleted APLF or XRCC1 from differentiated/noncycling SH-SY5Y neuroblastoma cells by transient siRNA transfection (Fig. 6B, left panel). Whereas the type of siRNA did not affect the level of DNA strand breaks induced by IR, as measured by alkaline comet assays, the rate at which DNA strand breaks declined was significantly delayed in both XRCC1- and APLF-depleted cells (Fig. 6C). It is likely that the delay observed here reflects a defect in SSBR, because >95% of the total breaks induced by IR are SSBs (4). To examine the rate of DSBR, we quantified the level of -H2AX, an established marker of DSBs (26, 27), in gamma ray-treated SH-SY5Y neuroblastoma cells that were depleted of APLF as described above or in A549 cells that were stably depleted of APLF (Fig. 5B, right panels). We consistently observed in these experiments that -H2AX foci declined at significantly reduced rates in both APLF-depleted neuroblastoma cells and A549 cells following 1 to 3 Gy of IR (Fig. 6D). In contrast, repair rates were normal in XRCC1-depleted SH-SY5Y cells, consistent with the absence of a measurable DSBR defect in these cells (Fig. 6D, right panel).
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DISCUSSION |
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Direct evidence of a role for APLF in the cellular response to DNA damage emerged from the observations that endogenous APLF accumulates at sites of UVA laser damage in human A549 cells and that recombinant YFP-APLF accumulates in subnuclear foci in H2O2-treated CHO cells. Whereas the UVA laser tracts most likely contain sites with both single- and double-strand breaks (data not shown), the H2O2-induced foci most likely reflect just SSBs. This is because they colocalize and are stimulated by RFP-XRCC1 and because they do not significantly colocalize with sites of -H2AX (data not shown). In addition, we failed to detect accumulation of the DSBR protein XRCC4 in H2O2-induced subnuclear foci under the same experimental conditions. It is also noteworthy that, in collaborative experiments with Akira Yasui, we observed the accumulation of YFP-APLF at UVA laser-induced damage under conditions where SSBs are selectively induced (19; unpublished observations).
The ability of RFP-XRCC1 to stimulate the accumulation of YFP-APLF in subnuclear foci most likely reflected, at least in part, the impact of recombinant XRCC1 on the nuclear import or retention of YFP-APLF. This is because the addition of an NLS to YFP-APLF circumvented the requirement of RFP-XRCC1 for high levels of YFP-APLF nuclear foci. However, these experiments do not rule out an additional role(s) for XRCC1 during the accumulation of YFP-APLF at sites of DNA damage. This is because the NLS had a much greater impact on the nuclear import/retention of YFP-APLF than did recombinant RFP-XRCC1, perhaps thereby masking additional roles for XRCC1. Indeed, consistent with this notion, the coexpression of RFP-XRCC1 stimulated the appearance of YFP-nls-APLF nuclear foci, albeit to a lesser extent than YFP-APLF foci (fivefold versus twofold), despite the presence of an NLS. The nature of the alternative mechanism by which XRCC1 might promote the accumulation of YFP-APLF at sites of oxidative DNA damage is not known. However, since this mechanism required an intact FHA domain in APLF, one attractive possibility is that it reflects the scaffolding role of XRCC1. A similar explanation has been suggested for the observation that XRCC1 also promotes the accumulation of aprataxin, PNK, and DNA polymerase ß at sites of H2O2-induced DNA damage (21; unpublished observations).
H2O2-induced YFP-APLF foci were observed in 15 to 40% of CHO cells in the absence of XRCC1, and a similar fraction of YFP-APLF focus-positive cells was observed when RFP-XRCC1 was coexpressed with YFP-APLF lacking an intact FHA domain. Thus, although greatly stimulated by the coexpression of recombinant XRCC1, the recruitment/accumulation of YFP-APLF at sites of oxidative DNA damage can occur independent of XRCC1. Intriguingly, this XRCC1-independent mode of APLF recruitment/accumulation required the two tandem ZnF motifs, supporting the notion that these structures represent a novel class of DNA strand break-responsive motifs. The amino acid sequences of the tandem ZnFs in APLF appear to be distinct from those present in other SSBR/DSBR proteins, such as aprataxin and poly(ADP-ribose) polymerase 1, as indicated by the limited number of proteins detected as containing this motif in database searches (Fig. 1C). It is not yet clear what function these putative ZnFs fulfill, but the presence of this structure in a variety of other DNA repair and DNA metabolic proteins suggests that it is an important one. The impact of the ZnFs on the XRCC1-independent recruitment/accumulation of YFP-APLF at sites of oxidative DNA damage was not mediated via increased nuclear import or retention of APLF, because the addition of an NLS did not compensate for the absence of intact ZnFs. We suggest that the ZnF may bind to DNA strand breaks directly, thereby promoting XRCC1-independent recruitment of APLF at sites of chromosomal damage.
In contrast to RFP-XRCC1, RFP-XRCC4 did not promote the redistribution of APLF to nuclei or the accumulation of APLF in H2O2-induced subnuclear foci. This may reflect the nature of the cellular response to DSBs because accumulation in discrete subnuclear foci does not appear to be a general feature of nonhomologous end-joining enzymes such as XRCC4 and Ku following oxidative stress. However, further evidence that APLF is involved in the response to DSBs was suggested by the change in the electrophoretic mobility of this protein induced by IR or H2O2. Significantly, this phenomenon was dependent on the DSB sensor protein kinase ATM, suggesting that APLF is phosphorylated in an ATM-dependent manner in response to DSBs, though we cannot rule out that ATM is activated in this context by some other type of lesion or event. It also remains to be determined whether APLF is a substrate for ATM or whether APLF is modified via ATM indirectly.
Alkaline comet and -H2AX assays suggested that the depletion of APLF by siRNA retards the rate of DNA single- and double-strand break repair at early times after IR. We observed a DSBR defect in both proliferating A549 lung carcinoma cells and noncycling SH-SY5Y neuroblastoma cells, as measured by -H2AX staining. However, using alkaline comet assays, we detected a DNA strand break repair defect only in noncycling SH-SY5Y cells (data not shown). Since alkaline comet assays measured primarily SSBs in these experiments, due to the abundance of SSBs relative to DSBs induced by IR (20:1), we suggest that this reflects a difference in the redundancies of APLF during SSBR in proliferating versus noncycling cells. Differences in polypeptide requirements during SSBR in S-phase versus nonreplicating cells have been reported previously (24, 29). The impact of APLF depletion on DNA strand break repair rates was subtle, though in the case of SSBR, it was no less subtle than in cells depleted of XRCC1, which is the archetypal SSBR protein (7, 32). The relatively small impact on repair rates observed in these experiments could thus be due to the incomplete depletion of XRCC1 and APLF by siRNA. Alternatively, it is possible that APLF is required for the rapid repair of only a subset of DNA strand breaks, such as those with specific types of DNA termini. This is also most likely the case for aprataxin, which removes rare 5' AMP adducts from DNA strand breaks in vitro but does not have a measurable impact on global rates of DNA strand break repair in vivo (1).
What role might APLF play during DNA strand break repair? Given that APLF most likely binds the same regions of XRCC1 and XRCC4 that are bound by PNK and aprataxin, both of which are end processing enzymes, it is tempting to speculate that APLF fulfills or facilitates a similar activity. This suggests a model in which CK2-phosphorylated XRCC1 or XRCC4 can recruit one of at least three different end processing activities to an SSB or DSB, depending on the nature of the damaged termini. The unusual nature of the tandem ZnFs may be informative in this respect. Combined sequence/structure database analyses assign this motif as a tandem ZnF most closely related to the CCCH tandem ZnFs present in the tristetrapolin family of mRNA binding proteins (3, 17). Interestingly, these tandem ZnFs appear to be required to facilitate the deadenylation of 3' poly(A) tails of specific mRNAs, most likely through the recruitment of an RNase (18). Although they share some similarities, the APLF tandem ZnFs and tristetraprolin family ZnFs are clearly distinct, most notably in the spacing between the zinc coordinating residues and the absence of specific residues critical for mRNA binding. It is thus tempting to speculate that the tandem putative ZnFs in APLF target DNA rather than RNA, thereby facilitating the processing of a specific type of DNA 3' terminus.
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ACKNOWLEDGMENTS |
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We thank Roger Phillips in the Sussex Centre for Advanced Microscopy for help with the UVA laser experiments.
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FOOTNOTES |
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Published ahead of print on 12 March 2007.
These authors contributed equally to this work.
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