APLF promotes the assembly and activity of non-homologous end joining protein complexes
2012; Springer Nature; Volume: 32; Issue: 1 Linguagem: Inglês
10.1038/emboj.2012.304
ISSN1460-2075
AutoresGabrielle J. Grundy, Stuart L. Rulten, Zhihong Zeng, Raquel Arribas-Bosacoma, Natasha Iles, Katie Manley, Antony W. Oliver, Keith W. Caldecott,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle23 November 2012free access APLF promotes the assembly and activity of non-homologous end joining protein complexes Gabrielle J Grundy Gabrielle J Grundy Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Stuart L Rulten Stuart L Rulten Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Zhihong Zeng Zhihong Zeng Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Raquel Arribas-Bosacoma Raquel Arribas-Bosacoma Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Natasha Iles Natasha Iles Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Katie Manley Katie Manley Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Antony Oliver Antony Oliver Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Keith W Caldecott Corresponding Author Keith W Caldecott Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Gabrielle J Grundy Gabrielle J Grundy Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Stuart L Rulten Stuart L Rulten Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Zhihong Zeng Zhihong Zeng Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Raquel Arribas-Bosacoma Raquel Arribas-Bosacoma Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Natasha Iles Natasha Iles Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Katie Manley Katie Manley Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Antony Oliver Antony Oliver Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Keith W Caldecott Corresponding Author Keith W Caldecott Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Author Information Gabrielle J Grundy1,‡, Stuart L Rulten1,‡, Zhihong Zeng1, Raquel Arribas-Bosacoma1, Natasha Iles1, Katie Manley1, Antony Oliver1 and Keith W Caldecott 1 1Genome Damage and Stability Centre, University of Sussex, Brighton, UK ‡These authors contributed equally to this work *Corresponding author. School of Biological Sciences, Genome Damage and Stability Centre, Science Park Road, Falmer, Brighton, Sussex BN1 9RQ, UK. Tel.:+44 (0) 1273 877519; Fax:+44 (0) 1273 678121; E-mail: [email protected] The EMBO Journal (2013)32:112-125https://doi.org/10.1038/emboj.2012.304 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein–DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway. Introduction DNA double-strand breaks (DSBs) are a major threat to genetic stability and hereditary defects in DSB repair result in a variety of disease pathologies (McKinnon and Caldecott, 2007). To date, two cellular pathways have been identified that repair DSBs; a mechanism in which undamaged sister chromatids and homologous recombination are employed and a mechanism denoted non-homologous end joining (NHEJ) in which the DNA ends are spliced together (Jackson and Bartek, 2009). Loss of homologous recombination-mediated repair is associated with increased predisposition to cancer, including hereditary breast cancer, whereas loss of NHEJ is typically associated with immunodeficiency and neurological dysfunction (McKinnon and Caldecott, 2007). During the classical and most commonly employed pathway for NHEJ, DSBs are bound by the Ku heterodimer, which comprises 70 kDa (Ku70) and 80 kDa (Ku80) subunits (Mahaney et al, 2009; Lieber, 2010). Ku then recruits a number of proteins to the DSB including DNA-PKcs, XLF/Cernunnos (from now on termed as XLF), and XRCC4-Lig4 complex. DNA-PKcs is a DNA-dependent protein kinase that is activated at DNA ends and promotes DNA end processing by Artemis (Ma et al, 2002; Goodarzi et al, 2006), whereas XLF and XRCC4-Lig4 facilitate the final step of DNA ligation (Ahnesorg et al, 2006; Buck et al, 2006; Gu et al, 2007; Lu et al, 2007; Tsai et al, 2007). With the exception of DNA-PKcs, which evolutionarily is a relatively recent addition, these proteins comprise the core components of NHEJ. Importantly, while NHEJ involves multiple interactions between the core components of this process, the mechanisms by which these interactions are regulated and/or organised is unclear. Recently, Aprataxin-and-PNK-Like Factor (APLF; also denoted as PALF/C2orf13/XIP1) was identified as a novel component of NHEJ that promotes intracellular re-joining of transfected linear plasmid DNA molecules and accelerates the repair of chromosomal DSBs following γ-irradiation (Bekker-Jensen et al, 2007; Iles et al, 2007; Kanno et al, 2007; Macrae et al, 2008; Rulten et al, 2011). APLF is recruited to sites of DNA strand breakage via interaction of a C-terminal tandem zinc finger domain with poly (ADP-ribose); a nucleic acid-like structure synthesised at sites of DNA breakage by poly (ADP-ribose) polymerases (Ahel et al, 2008; Rulten et al, 2008; Eustermann et al, 2010; Li et al, 2010). Importantly, APLF appears to accelerate the rate of chromosomal NHEJ during the first few hours following γ-irradiation, suggesting that it is an accessory factor for NHEJ (Iles et al, 2007; Rulten et al, 2011). The molecular mechanism by which APLF accelerates NHEJ is unclear. One possibility is that APLF is an end processing factor, because it has been reported to possess nuclease activity in vitro, and thus may process incompatible DNA termini (Kanno et al, 2007; Li et al, 2011). However, we recently demonstrated that APLF-depleted cells exhibit reduced levels of XRCC4 in chromatin, and that overexpression of XRCC4-Lig4 complex can circumvent the requirement for APLF for rapid rates of NHEJ, suggesting that APLF accelerates NHEJ primarily by promoting DNA ligase activity (Rulten et al, 2011). It is possible that APLF promotes DNA ligation by modifying chromatin structure directly, since this protein interacts with histone H3/H4 and exhibits histone chaperone activity, in vitro (Mehrotra et al, 2011). However, APLF also possesses an amino-terminal fork-head associated (FHA) domain that interacts directly with the XRCC4 (Iles et al, 2007; Kanno et al, 2007; Macrae et al, 2008) and an MID domain that interacts directly with Ku heterodimer (Iles et al, 2007; Kanno et al, 2007; Macrae et al, 2008). The role and importance of these interactions remains unknown, but it seems likely that they will underpin the molecular mechanism by which APLF accelerates DNA ligation during NHEJ. Indeed, we show here that APLF promotes the assembly and activity of multi-protein Ku-DNA complexes containing all of the NHEJ factors required for DNA ligation, and describe the mechanism by which this is achieved. Collectively, our data identify APLF as a novel structural component of the NHEJ pathway, in mammalian cells. Results Identification of a conserved peptide motif in APLF sufficient for binding Ku80 In a yeast 2-hybrid (Y2H) screen employing full-length human APLF as bait we recovered multiple clones encoding XRCC1 and XRCC4, both of which interact with the amino-terminal FHA domain of APLF (Figure 1A). In addition, we recovered seven clones encoding the 80-kDa subunit of Ku (Ku80), but none encoding the 70-kDa subunit (Ku70). Additional Y2H experiments confirmed that the MID domain of APLF spanning residues 94–358 is sufficient for interaction with Ku80, consistent with a previously reported interaction of this region with Ku heterodimer (Macrae et al, 2008) (Figure 1B). Close inspection of the MID domain revealed a conserved peptide motif between residues 182 and 191, comprising a short patch of 3 or 4 basic amino acids followed by a hydrophobic patch of 6–9 amino acids that includes an invariant tryptophan (Figure 1C; Supplementary Figure 1). Mutation of the three basic residues RKR (182–184) resulted in an APLF protein that constitutively transactivated Y2H reporter genes, and so was uninformative (data not shown). However, mutation of the invariant tryptophan (W189) ablated Ku80-dependent activation of Y2H reporter genes, suggesting that W189 is critical for the interaction of APLF with Ku80 (Figure 1B; Supplementary Figure 2A). Figure 1.Identification of a novel peptide motif in APLF that binds Ku80. (A) Recovery of human cDNA clones encoding XRCC1, XRCC4, or Ku80 in a yeast 2-hybrid (Y2H) screen employing APLF as bait. The number and relative representation of each clone type is indicated. (B) Ku80 interacts with APLF residues 94–358, and requires W189 for this interaction. Y190 budding yeast harbouring pACT-Ku80 (library clone 5) and empty pGBKT7, pGBKT7-APLF94–358, or pGBKT7-APLFW189G was examined for activation of the His3 and LacZ reporter genes by Y2H analysis. (C) Schematic of APLF depicting a conserved Ku80-binding peptide motif in the MID domain. Top, APLF cartoon depicting the interaction domains; FHA domain that interacts with XRCC1 and XRCC4 (FHA), MID domain that interacts with Ku80, PBZ domain that interacts with poly (ADP-ribose), and C-terminal (CT) domain that interacts with histone H3/H4. Bottom, ClustalW alignment of the putative Ku80-binding peptide motif (grey box) from the indicated organisms. The NCBI accession numbers employed are NP_775816, NP_001163960, XP_419335, NM_001113131, XP_788055, XP_001635068, and XP_002117261. (D) Mutation of the conserved peptide motif in APLF prevents Ku binding, in vitro. One microgram of wild-type APLF, APLFW189G, APLFRKR(182–184)EEE, or APLF347–511 was slot blotted onto nitrocellulose and individual membrane strips stained with Amido black as a loading control (AB) or mock incubated (−) or incubated (+) with 100 nM of recombinant XRCC1, XRCC4-Lig4 heterodimer (LX), or Ku70/80 as indicated. (E) The APLF conserved peptide motif is sufficient to bind Ku. The indicated fluorescein-labelled peptides spanning the wild-type (WT) or mutant APLF conserved peptide motif were examined for binding to Ku70/80ΔC by fluorescence polarisation. Data points are the average of three independent experiments, with error bars representing one standard deviation. Download figure Download PowerPoint To confirm the importance of the conserved peptide motif for Ku80 binding, we slot-blotted wild-type and mutant recombinant APLF proteins onto nitrocellulose and measured Ku binding in solution. Whereas Ku heterodimer (Ku70/80) bound wild-type APLF in these experiments, it failed to bind a C-terminal fragment of APLF in which the MID domain was missing (APLF347–511) or full-length APLF in which RKR (182–184) or W189 was mutated (Figure 1D). Neither of the point mutations disrupted binding of APLF to XRCC1 or XRCC4-Lig4 (LX), which are known to interact with the amino-terminal FHA domain of APLF (Bekker-Jensen et al, 2007; Iles et al, 2007; Kanno et al, 2007; Macrae et al, 2008). Next, we examined whether the conserved peptide motif is sufficient for interaction with Ku heterodimer, using fluorescence polarisation. We employed Ku heterodimer containing Ku80ΔC for these experiments, and for some of the experiments described later, because this protein lacks the highly flexible C-terminal 141 residues and appears to be more homogenous in structural conformation (Walker et al, 2001). Notably, whereas an 18-mer synthetic peptide spanning the conserved peptide motif bound Ku with a Kd of ∼0.6 μM, Ku binding by a mutant peptide in which RKR (182–184) and W189 were mutated was too low to quantify (Figure 1E). Together, these data identify a conserved peptide motif in APLF that is both required and sufficient for interaction with Ku80. The Ku80-binding motif is an autonomous module that recruits proteins into Ku-DNA complexes To examine whether the Ku-binding motif is required to recruit APLF into DNA complexes containing Ku heterodimer (Ku70/80), we employed electrophoretic mobility shift assays (EMSAs). Whereas full-length APLF and a truncated derivative harbouring the MID domain (APLF1–469) super-shifted DNA complexes containing Ku (Figure 2A, lanes 7–12), N-terminal (APLF1–164) and C-terminal (APLF360–511) fragments lacking the MID domain did not (Figure 2A, lanes 13–18). None of the APLF proteins bound DNA substrate in the absence of Ku, confirming that APLF did not bind DNA independently of Ku under the conditions employed (Figure 2A, lanes 1–5). More importantly, mutation of the Ku-binding motif ablated the ability of APLF to super-shift Ku-DNA complexes, confirming that this motif is required for recruitment of APLF into DNA complexes containing Ku (Figure 2B). Figure 2.The Ku-binding motif is a novel autonomous module for recruiting proteins into Ku-DNA complexes. (A) The MID domain is required for APLF recruitment into DNA complexes containing Ku. A Cy3-labelled 30-bp duplex oligonucleotide (10 nM) harbouring blunt-ended termini was incubated with (+) or without (−) recombinant Ku70/80 (20 nM) in the absence (−) or presence of 150, 50, or 17 nM of the indicated recombinant APLF protein and employed in EMSA. Where a single concentration of APLF is indicated, the protein was employed at 150 nM. The composition of the protein DNA complexes is indicated on the right (arrows). (B) The Ku-binding motif is required for APLF recruitment into DNA complexes containing Ku. EMSAs were conducted as above. Where indicated, Ku70/80 was present at 20 nM and the indicated wild-type and mutant APLF proteins were employed at 150, 50, 17, 6 and 2 nM. (C) The Ku-binding motif is an autonomous module for protein recruitment into Ku-DNA complexes. Top left, alignment of the peptide sequences from APLF, XLF, and WRN that were fused to GST and analysed in the bottom panels, by EMSA. The conserved basic and hydrophobic patches that characterise the Ku-binding motif are indicated in blue and green, respectively. Bottom left, Cy3-labelled 30-bp duplex oligonucleotide (10 nM) was incubated with Ku70/80 (10 nM) in the presence of 0.5 μM of GST, the indicated GST peptides, full-length His-APLF (lane 2), or full-length XLF (lane 6), and employed in EMSAs as described above. Bottom right, Cy3-labelled 30-bp duplex oligonucleotide was incubated as above in the presence of GST (3 μM), His-APLF (0.5 μM), GST-XLF (1.6 μM), or the indicated GST-APLF (1.3 μM), GST-XLF (1.6 μM), or GST-WRN (3 μM) peptides. Download figure Download PowerPoint We next examined whether the Ku-binding motif can function autonomously, and thus target an unrelated protein into Ku-DNA complexes in vitro. Indeed, GST fusion proteins harbouring short peptides that span the Ku-binding motif in APLF were efficiently recruited into Ku complexes on a 30-bp duplex DNA substrate, whereas GST lacking the motif was not (Figure 2C, bottom left, lanes 1–5). Similar results were observed on a shorter, 19-bp, duplex substrate that is approximately the same size as the footprint of a single Ku heterodimer, suggesting that the GST peptides assemble into Ku-DNA complex by direct interaction Ku (Supplementary Figure 2). Indeed, none of the GST peptides bound the 19-bp duplex in the absence of Ku (Supplementary Figure 2). Intriguingly, sequence analyses revealed a similar motif at the C-terminus of XLF and two similar motifs in tandem at the C-terminus of Werner (WRN) protein (Figure 2C, top left). Both XLF and WRN are involved in NHEJ and have been reported to interact with Ku via their C-termini, suggesting that the APLF-like motifs similarly might function as Ku binding modules in these proteins (Cooper et al, 2000; Karmakar et al, 2002; Yano et al, 2011). Consistent with this idea, GST fusion proteins harbouring the putative Ku-binding motif from XLF or the first of the two motifs from WRN (residues 1403–1418) were recruited into DNA complexes containing Ku, albeit less efficiently than the APLF motif (Figure 2C bottom panels, lanes 7–9). Ku80 binding promotes APLF accumulation at chromosome damage APLF is recruited and/or retained at cellular DNA strand breaks via PBZ and FHA domain-mediated interactions with poly (ADP-ribose) and XRCC1/XRCC4, respectively (Bekker-Jensen et al, 2007; Iles et al, 2007; Kanno et al, 2007; Ahel et al, 2008; Macrae et al, 2008; Rulten et al, 2008). To address the contribution of the Ku-binding motif in this process, we compared YFP-tagged wild-type and mutant derivatives of APLF for ability to accumulate at sites of UVA laser damage. As expected, YFP-APLFR27A and YFP-APLFZFD proteins harbouring mutated FHA and PBZ domains, respectively, were recruited and/or retained at sites of UVA laser damage to a lesser extent than was wild-type YFP-APLF (Figure 3A, bottom; 'R27A' and 'ZFD'). However, YFP-APLFW189G ('WG') harbouring a mutated Ku-binding motif also exhibited reduced retention at such sites, and the accumulation of YFP-APLFZFD/W189G or YFP-APLFZFD/R27A/W189G harbouring mutations in both the PBZ and Ku80-binding domains or in all three domains was reduced to ∼15 and 5% of wild type, respectively (Figure 3A, bottom). We noted during these experiments that mutation of the Ku-binding motif affected the subcellular distribution of YFP- or mRFP-tagged APLF (Supplementary Figure 3A). However this did not account for the impact of W189G on APLF accumulation at sites of UVA laser damage, because only cells harbouring similar levels of nuclear YFP-APLF or YFP-APLFW189G were scored in these experiments. Moreover, YFP-nls-APLFW189G protein, which harboured an engineered SV40 nuclear localisation signal and was entirely nuclear, similarly exhibited reduced retention at sites of UVA laser damage (Supplementary Figure 3B). Figure 3.The Ku80-binding motif promotes APLF accumulation at chromosome damage. A549 cells were transiently transfected with constructs encoding wild-type YFP-APLF (WT) or YFP-APLF harbouring point mutations in the PBZ domain (YFP-APLFZFD; 'ZFD'), FHA domain (YFP-APLFR27A; 'R27A'), Ku-binding motif (YFP-APLFW189G; 'WG'), or in combinations of these ('ZFD/WG', 'R27A/WG/ZFD'). mRFP-XRCC1 and GFP-XRCC4 were used as markers of recruitment to single- and double-strand breaks, respectively. Cells were irradiated with 4.36 J/m2 (A) or 0.22 J/m2 (B) using a UVA laser (arrow). Images were captured at 15 s intervals after laser irradiation. For each data point, data are normalised to the YFP fluorescence intensity prior to irradiation (set to 100%). Data are the mean (±s.e.m.) of 10 or more individual cells per data point. Representative examples of YFP-APLF, GFP-XRCC4, and mRFP-XRCC1 accumulation at sites of UVA laser damage are shown (top). Download figure Download PowerPoint The amount of UVA damage introduced in the above experiments was sufficient to induce both single-strand breaks (SSBs) and DSBs, as indicated by the accumulation of both the SSB repair protein XRCC1 and the DSB repair protein, XRCC4 (Figure 3A, top). Consequently, since APLF interacts with both of these proteins and is involved in the repair of both SSBs and DSBs, we wished to confirm that the Ku80-binding motif was required specifically for accumulation of APLF at the latter. To do this, we employed a 20-fold lower level of UVA damage; an experimental condition in which the level of SSB damage was still sufficient to trigger XRCC1 accumulation but in which the level of DSB damage was too low to trigger significant recruitment of XRCC4 or Ku (Figure 3B, top, and data not shown). The accumulation of APLF at sites of UVA laser damage remained dependent on the PBZ domain under these conditions, consistent with APLF recruitment at chromosomal SSBs, but was largely independent of W189 (Figure 3B, bottom). Together, these data suggest that the Ku-binding motif is required to promote the retention of APLF at sites of chromosomal DSBs. APLF interacts with the von Willebrand-like domain of Ku80 The seven Ku80 cDNA clones recovered by APLF in our Y2H screen encoded only the amino-terminal 258–278 amino acids, and thus just the von-Willebrand Factor A (WA)-like domain (Aravind and Koonin, 2001) (Figure 4A). This observation is significant, because while this putative protein–protein interaction domain is highly conserved in eukaryotic Ku80, its protein partner/s have not yet been identified and its molecular function is unknown. Consequently, we attempted to map the site of APLF interaction in more detail. Given the partially hydrophobic character of the Ku-binding motif in APLF, we considered it likely that the corresponding interface in Ku80 is also hydrophobic. We therefore identified and mutated nine highly conserved hydrophobic residues predicted to be present on the surface of the vWA domain (Supplementary Figure 4), and examined the impact of these mutations on the interaction of Ku80 with the APLF MID domain in Y2H experiments. Whereas six of the nine mutations did not measurably impact on the interaction with the APLF MID domain, three of the mutations (Ku80L68R, Ku80Y74R, and KuI112R) greatly reduced or ablated it (Figure 4B). Strikingly, these three residues co-localise in Ku80 on the surface of the vWA domain (Walker et al, 2001), consistent with them forming part of a hydrophobic interface for the Ku-binding motif (Figure 4C). Indeed, in contrast to wild-type Ku (Ku70/80ΔC), KuL68R harbouring a mutant Ku80 vWA domain (Ku70/80ΔCL68R) failed to recruit APLF into DNA complexes in vitro (Figure 4D, compare lanes 3–5 with 7–9). This was not a non-specific impact of L68R on Ku folding, because the mutant heterodimer bound DNA in vitro (Figure 4D, lane 6) and was recruited to sites of chromosome damage in cells (Figure 4E). To confirm the impact of mutating the vWA domain on APLF behaviour in cells, we exploited our observation that the nuclear retention of mRFP-APLF is increased by co-expression with recombinant Ku80 (see Supplementary Figure 3A). Whereas co-transfection with GFP-Ku70/GFP-Ku80 increased the fraction of A549 cells harbouring predominantly nuclear mRFP-APLF, co-transfection with GFP-Ku70/GFP-Ku80L68R failed to do so (Figure 4F). Importantly, neither GFP-Ku70/GFP-Ku80 nor GFP-Ku70/GFP-Ku80L68R promoted the nuclear retention of mRFP-APLFW189G, confirming that the vWA domain promoted mRFP-APLF nuclear retention only if the Ku-binding motif was intact. Collectively, these results reveal that APLF interacts directly with the vWA domain of Ku80, thereby identifying a protein partner and molecular role for this domain. Figure 4.APLF interacts with the vWA domain of Ku80. (A) Cartoon of Ku70 and Ku80, depicting the von Willebrand-like ('vWA') domains, heterodimerisation domains ('Het'), Ku70 SAP domain ('S'), and Ku80 C-terminal domain ('CTD'). The regions of Ku80 recovered by APLF in the Y2H screen depicted in Figure 1A are shown (bottom). (B) Mutation of the Ku80 vWA domain disrupts interaction with APLF. Y190 cells harbouring empty pGBKT7 or pGBKT7-APLF94–358 (encoding the MID domain) and either empty pACT2 or the indicated wild-type or mutant derivative of pACT-Ku801–258 (clone 5) were examined for His3 and LacZ reporter gene expression. (C) Residues required for APLF interaction co-localise in a hydrophobic interface on the surface of the Ku80 vWA domain. The location of L68, Y74, and I112 within the Ku heterodimer (RCSB PDB entry; 1JEY) (Walker et al, 2001) is shown. Blue and red denote basic and acidic regions, respectively. (D) Mutation of the Ku80 vWA domain prevents recruitment of APLF into DNA complexes containing Ku. A Cy3-labelled 30-bp duplex (10 nM) was incubated with (+) or without (−) 10 nM wild-type Ku (Ku70/80Δ) or mutant Ku (Ku70/80ΔL68R) in the absence (−) or presence of 700, 350, or 175 nM of the indicated recombinant APLF protein and employed in EMSA. (E) Normal accumulation of Ku70/GFP-Ku80L68R at sites of chromosome damage. A549 cells were transiently co-transfected with His-Ku70 and either GFP-Ku80 or GFP-Ku80L68R prior to UVA laser irradiation. Images were captured at 30 s intervals after laser irradiation. (F) Impact of mutations in the vWA domain on the subcellular localisation of APLF. mRFP localisation following co-transfection with mRFP-APLF or mRFP-APLFW189G and GFP vector, GFP-Ku70/GFP-Ku80, or GFP-Ku70/GFP-Ku80L68R. Data are the mean of three independent experiments (±s.e.m.). Download figure Download PowerPoint APLF promotes the stability of NHEJ protein complexes, in vitro The data described above define the mechanism by which APLF and Ku interact, and demonstrate that this interaction is important for APLF recruitment into Ku complexes and for retention at chromosome damage. However, these results do not explain how APLF accelerates DNA ligation during NHEJ. To address this question, we examined the impact of APLF on the stable assembly of the DNA ligase heterodimer, XRCC4-Lig4, into DNA complexes containing Ku. Surprisingly, despite the established ability of XRCC4-Lig4 to interact directly with Ku (Nick McElhinny et al, 2000; Hsu et al, 2002; Costantini et al, 2007), we observed little if any incorporation of XRCC4-Lig4 into DNA complexes containing Ku heterodimer (Ku70/80ΔC) in the absence of APLF, under the conditions employed (Figure 5A, lanes 1 and 2). In contrast, in the presence of APLF, all of the DNA complexes containing Ku were super-shifted by XRCC4-Lig4 into a single protein complex of lower mobility (Figure 5A, lanes 3 and 4). Importantly, both anti-XRCC4 and anti-APLF antibodies were able to super-shift these Ku–DNA complexes, confirming the presence of both APLF and XRCC4-Lig4 (Figure 5B, lanes 4–6). In contrast, neither antibody super-shifted Ku-DNA complexes in the absence of APLF, confirming their specificity (Figure 5B, lanes 7–9). Note that while Ku heterodimer harbouring Ku80ΔC was employed for the above experiments, similar results were observed for Ku heterodimer harbouring full-length Ku80 (Supplementary Figure 6A). Figure 5.APLF promotes NHEJ complex assembly. (A) Co-assembly of APLF and XRCC4-Lig4 into 19-bp DNA complexes containing Ku. A Cy3-labelled 19-bp duplex (10 nM) was incubated with Ku70/80ΔC (20 nM), in the absence or presence of APLF (0.4 μM) and/or XRCC4-Lig4 (LX; 0.4 μM) and then employed in EMSA. (B) NHEJ protein complexes assembled by APLF are super-shifted by anti-APLF and anti-XRCC4 antibodies. A Cy3-labelled 19-bp duplex was incubated with Ku70/80ΔC as described above in the presence or absence of APLF (0.4 μM) and/or XRCC4-Lig4 (LX; 0.55 μM) and/or the indicated anti-XRCC4 (Santa Cruz; sc-8285) or anti-APLF (Abmart 2G11) antibody and employed in EMSA. (C) Co-assembly of APLF and XRCC4-Lig4 into 60-bp DNA complexes containing Ku. A Cy3-labelled 60-bp duplex (10 nM) was incubated with full-length Ku70/80 (20 nM) in the presence and absence of APLF (0.4 μM) and 0–140 nM XRCC4-Lig4 (LX), as indicated. (D) APLF promotes assembly of both XRCC4-Lig4 and XLF into Ku-DNA complexes and functions as a molecular scaffold. A Cy3-labelled 19-bp duplex (10 nM) was incubated in the absence or presence of full-length Ku70/80 (20 nM), XRCC4-Lig4 (LX; 0.2 μM), XLF (1 μM), and the indicated wild-type or mutant APLF (0.2 μM), and then employed in EMSA. Download figure Download PowerPoint It is worth noting that we employed a short DNA duplex probe of 19 bp in the above experiments, which is similar to the footprint of a single Ku heterodimer, to prevent DNA binding by XRCC4-Lig4 and so specifically address the role of protein–protein interactions on XRCC4-Lig4 assembly into Ku-DNA complexes (Kysela et al, 2003; Lu et al, 2007). However, similar results were observed with a longer duplex probe (60 bp) that allows DNA binding by multiple Ku heterodimers and direct DNA binding by XRCC4-Lig4 (Figure 5C). As reported previously (Nick McElhinny et al, 2000), XRCC4-Lig4 was able to super-shift Ku complexes in the absence of A
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