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• W2080726979 abstract "Article18 October 2007free access Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA Oleg V Tsodikov Oleg V Tsodikov Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USAPresent address: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA Search for more papers by this author Dmitri Ivanov Dmitri Ivanov Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Barbara Orelli Barbara Orelli Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Lidija Staresincic Lidija Staresincic Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Ilana Shoshani Ilana Shoshani Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Robert Oberman Robert Oberman Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA Search for more papers by this author Orlando D Schärer Orlando D Schärer Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Department of Chemistry, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Gerhard Wagner Gerhard Wagner Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tom Ellenberger Corresponding Author Tom Ellenberger Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA Search for more papers by this author Oleg V Tsodikov Oleg V Tsodikov Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USAPresent address: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA Search for more papers by this author Dmitri Ivanov Dmitri Ivanov Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Barbara Orelli Barbara Orelli Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Lidija Staresincic Lidija Staresincic Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Ilana Shoshani Ilana Shoshani Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Robert Oberman Robert Oberman Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA Search for more papers by this author Orlando D Schärer Orlando D Schärer Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA Department of Chemistry, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Gerhard Wagner Gerhard Wagner Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tom Ellenberger Corresponding Author Tom Ellenberger Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA Search for more papers by this author Author Information Oleg V Tsodikov1,‡, Dmitri Ivanov1,‡, Barbara Orelli2, Lidija Staresincic2, Ilana Shoshani2, Robert Oberman4, Orlando D Schärer2,3, Gerhard Wagner1 and Tom Ellenberger 4 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA 2Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA 3Department of Chemistry, Stony Brook University, Stony Brook, NY, USA 4Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA ‡These authors contributed equally to this work *Corresponding author. Department of Biochemistry and Molecular Biophysics, Washington University, Washington University School of Medicine, 660 S Euclid, Campus Box 8231, St Louis, MO 63110, USA. Tel.:+1 314 747 8893; Fax: +1 314 632 4432; E-mail: [email protected] The EMBO Journal (2007)26:4768-4776https://doi.org/10.1038/sj.emboj.7601894 Present address: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein–protein and protein–DNA interactions within NER complexes. We have investigated an essential protein–protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo. Introduction The repair of chemical insults to DNA caused by UV light and other mutagens is essential for coping successfully with the intrinsic reactivity of DNA and preserving genetic information. Inherited diseases resulting in the failure to correct spontaneous or environmentally induced damage are typically associated with genomic instability and a predisposition to various cancers (Friedberg et al, 2005). Contrarily, DNA repair is undesirable when DNA-damaging agents are used for chemotherapy of cancer and other diseases. In these settings, the ability to modulate the DNA repair activities of cells targeted for destruction is a desirable goal (Ding et al, 2006). The nucleotide excision repair (NER) pathway is essential for normal genomic maintenance, removing bulky chemical adducts from DNA that are otherwise mutagenic or would pose lethal blocks to replication. NER involves over 30 proteins that recognize damaged sites in DNA and excise an oligonucleotide containing the damage (de Laat et al, 1999; Gillet and Schärer, 2006). Following excision of the damaged DNA segment, the resulting gap is filled by templated DNA synthesis and ligase seals the nick to complete the repair. Cell biological and biochemical studies have shown that NER operates by the sequential assembly of protein factors at the sites of DNA damage, rather than through the action of a preformed repairosome (Houtsmuller et al, 1999; Volker et al, 2001). The recruitment of NER factors into protein–DNA ensembles is guided at each step by numerous protein–protein interactions (Araujo et al, 2001), imparting specificity to the recognition and verification of damaged sites. Damage recognition in NER culminates in the incision of DNA 5′ and 3′ to the lesion site by ERCC1-XPF and XPG, respectively, to release a 24–32 nucleotide segment containing the damage (de Laat et al, 1999; Gillet and Schärer, 2006). For the NER pathway, DNA cleavage by ERCC1-XPF requires physical interaction with XPA, a scaffold protein that interacts with DNA and several repair proteins, including RPA, TFIIH and the ERCC1 subunit of ERCC1-XPF (Li et al, 1994, 1995; Park and Sancar, 1994; Saijo et al, 1996). Although XPA was originally described as a DNA damage-specific sensor or verification protein, recent work suggests that XPA instead recognizes the DNA structural intermediates arising during processing by NER (Jones and Wood, 1993; Camenisch et al, 2006). XPA recruits ERCC1-XPF to NER complexes (Volker et al, 2001), positioning the XPF nuclease domain at the 5′ side of the damage site (Enzlin and Schärer, 2002). ERCC1-XPF has other roles in DNA metabolism outside of NER, notably in interstrand crosslink repair and homologous recombination (Hoy et al, 1985; Niedernhofer et al, 2001). The importance of these additional, NER-independent functions of ERCC1-XPF is underscored by the pronounced sensitivity to crosslinking agents caused by mutations of ERCC1 or XPF in mice and humans (McWhir et al, 1993; Weeda et al, 1997; Niedernhofer et al, 2006). However, the exact biochemical role(s) of ERCC1-XPF in crosslink repair remain to be discovered. Li et al (1994, 1995) identified residues 59–114 in XPA as the site of interaction with ERCC1, and showed that deletion of three conserved glycines (Gly72, Gly73, Gly74) abrogates the XPA–ERCC1 interaction as well as the ability of the XPA protein to confer UV resistance to XP-A cells. Furthermore, the expression of a truncated protein comprising residues 59–114 of XPA renders cells sensitive to UV light and cisplatin (Rosenberg et al, 2001), suggesting that this region is sufficient to disrupt the interaction of the native XPA protein with ERCC1-XPF. Conversely, it can be inferred from several previous studies that residues 92–119 of ERCC1 are necessary for the interaction with XPA (Li et al, 1994; Sijbers et al, 1996; Gaillard and Wood, 2001). Following these seminal studies, understanding of the biochemical and structural basis for XPA's interaction with ERCC1 has not advanced, although more is known about the individual proteins. XPA contains a well-defined central domain (residues 98–219; Figure 1A), although the remainder of the protein including the ERCC1 interaction domain appears to be poorly structured (Buchko et al, 1998, 2001; Ikegami et al, 1998; Iakoucheva et al, 2001). Residues 92–119 of ERCC1 fall within the central domain of ERCC1 (Tsodikov et al, 2005) that is structurally homologous to the nuclease domain of the archaeal XPF-like proteins Aeropyrum pernix Mus81/XPF (Newman et al, 2005) and Pyrococcus furiosus Hef (Nishino et al, 2003). A V-shaped groove on the surface of ERCC1 corresponds to the nuclease active site of XPF (Enzlin and Schärer, 2002; Nishino et al, 2003; Newman et al, 2005), except that ERCC1's groove lacks the catalytic residues of a nuclease active site and is instead populated with basic and aromatic residues (Gaillard and Wood, 2001). We previously showed that the central domain of ERCC1 binds to single-stranded DNA in vitro (Tsodikov et al, 2005), and proposed the V-shaped groove as the DNA-binding site. Figure 1.XPA domain organization and structure of the ERCC1-binding peptide. (A) The ERCC1-binding region of XPA (residues 67–77) is located between the central domain (Zn2+-binding and DNA-binding subdomains; residues 98–219) and an N-terminal region (residues 1–58) that is dispensable for functional complementation of NER in whole-cell extracts from XP-A mutant cells (Miyamoto et al, 1992) and a TFIIH-binding region (Park et al, 1995). (B) 15N HSQC spectrum of 15N-labeled XPA59−93 in complex with unlabeled ERCC1, and in the unbound state (inset). The spectrum of the unbound XPA59−93 (inset) is characteristic of an unfolded peptide. The appearance of new well-dispersed NMR peaks in the XPA spectrum upon addition of ERCC192−214 (shown in the larger spectrum) indicates that a portion of the XPA peptide adopts a defined conformation in complex with ERCC1. Download figure Download PowerPoint These observations prompted us to investigate the structural and functional basis for the interaction of XPA and ERCC1, and its role in recruiting the XPF-ERCC1 endonuclease to sites of NER. Here, we describe the structure of a short peptide motif from XPA in complex with the central domain of ERCC1. We show that this XPA peptide specifically inhibits the NER reaction in vitro, creating an opportunity for structure-based design of NER inhibitors targeting this protein–protein interaction. Point mutations in the corresponding region in XPA abolish NER activity in vitro, underscoring the importance of the XPA–ERCC1 interactions for NER. In addition to providing insights into how protein–protein interactions mediate progression through the NER pathways, our studies will provide a blueprint to develop small molecules that intercept the interaction between XPA and ERCC1. Such molecules should be valuable for studying the biochemical functions of ERCC1-XPF in NER and other repair pathways including DNA interstrand crosslink repair and homologous recombination by selectively inhibiting NER. Results Induced fit of the XPA peptide upon interaction with ERCC1 Previous reports have suggested that the ERCC1-interacting region of XPA (Figure 1A) is unfolded in solution, based on NMR studies and its sensitivity to proteolytic cleavage (Buchko et al, 2001; Iakoucheva et al, 2001). To investigate the structure of the XPA ligand, we collected HSQC NMR spectra of an 15N-labeled XPA59−93 peptide alone and in complex with unlabeled central domain of ERCC1 (the ERCC192−214 protein; Figure 1B). In the absence of ERCC1, the resonance signals for XPA cluster in a narrow range of chemical shifts (Figure 1B, inset) that is characteristic of an unstructured polypeptide with poor spectral dispersion. In the complex with ERCC1, a subset of XPA backbone amides become well dispersed and the peaks are broader. These changes are indicative of a well-structured region within the bound XPA peptide. Only a few resonance peaks are markedly perturbed when XPA59−93 is bound to ERCC1, and among these, three glycine residues (assigned as Gly72, Gly73 and Gly74) are strongly perturbed in the complex. In order to overcome the peak broadening observed in NMR spectra of the XPA59−93 peptide at concentrations above 0.1 mM, we sought to identify shorter XPA peptide ligands for ERCC1. A minimal, 14-residue XPA67−80 peptide (described below) was identified by examining a series of overlapping XPA fragments from the region previously shown to interact with ERCC1 (Li et al, 1994). The structure of XPA in complex with ERCC1 A synthetic XPA67−80 peptide with amino-acid sequence KIIDTGGGFILEEE forms a stable complex with ERCC196−214 that can be purified by gel filtration chromatography. Like full-length XPA protein, the XPA59−93 and the XPA67−80 peptides behave similarly and efficiently co-purify with ERCC1, suggesting that XPA67−80 contains all significant binding determinants. We confirmed that XPA and ERCC1 form a stoichiometric 1:1 complex by estimating the amount of each subunit in the purified complex using an Edman degradation reaction, and by analytical centrifugation of the complex. Equilibrium sedimentation data for the complex (Supplementary Figure 1C) were best fit to the expected masses for a 1:1 complex of XPA59−93 and ERCC192−214 (Mw=(19.4±1.2) kDa) and unbound ERCC192−214 (Mw=(15.0±1.0) kDa). We confirmed that ERCC196−214 binds stoichiometrically to the XPA67−80 peptide with a KD of 0.78 μM (Supplementary Figure 2). A structure of the XPA67−80–ERCC196−214 complex (Figure 2A) was determined by a combination of NMR-derived distance restraints and X-ray diffraction data extending to 4 Å resolution (Table 1 in the Supplementary data and Materials and methods) as described below. Figure 2.Structure of the XPA–ERCC1 complex. (A) The XPA67−80 peptide (orange) is bound to a V-shaped groove of the central domain of ERCC196−214 (green). An orthogonal view of the bound XPA peptide (left side) is shown in comparison to the peptide in complex with ERCC1 (right-hand side). (B) The XPA-binding site on the surface of ERCC1 (colored red) was identified by resonance perturbations larger than 0.2 ppm that are indicative of direct interactions with XPA. Download figure Download PowerPoint Identification of the ERCC1-binding site in complex with XPA The binding site for XPA on the surface of ERCC1 (Figure 2B) was identified using two-dimensional HSQC experiments. The spectrum of unliganded 15N-labeled ERCC192−214 (blue, Figure 3) showed significant differences from that of the complex with unlabeled XPA67−80 (red, Figure 3). However, complexes of ERCC196−214 with either XPA67−80 or XPA59−93 were identical, suggesting that the shorter XPA peptide makes all of the significant binding contacts. The 15N HSQC spectrum of the ERCC1–XPA complex is consistent with a slow-exchange regime, implying a dissociation equilibrium constant below 1 μM for the complex. The XPA-binding site on the ERCC1 central domain was identified using the backbone assignments for ERCC196−214 alone and in complex with XPA (see Materials and methods). A comparison of the 15N HSQC spectra for ERCC1 in the presence and absence of XPA reveals that, with only one exception, the most prominent changes in chemical shifts involve a cluster of residues within a V-shaped groove of the ERCC1 central domain (Figures 2B and 3). Figure 3.XPA67−80 binds in a shallow groove of ERCC1. (A) A comparison of the two-dimensional HSQC spectra for 15N-labeled ERCC192−214 in the presence and absence of an unlabeled XPA67−80 peptide. The 15N HSQC spectra reveal significant chemical shift changes for some ERCC1 residues in the absence (blue) or presence (red) of unlabeled XPA67−80. (B) Combined average chemical shift perturbations are calculated as Δχave=(((Δχ1H)2+(Δχ15N/5)2)/2)1/2 for each backbone amide of ERCC1 and shown as a histogram. Download figure Download PowerPoint The bound XPA peptide fits snugly into the V-shaped groove of ERCC1 (Figure 2) that we previously speculated could be a binding site for single-stranded DNA (Tsodikov et al, 2005). Three consecutive glycines (Gly72, Gly 73, Gly74) of the XPA peptide insert into the groove, making a U-turn with close steric complementary to the binding site. These are the same three conserved glycines previously reported to be essential for the interaction of XPA with ERCC1 and required for the functional complementation of XP-A cells (Li et al, 1994, 1995). A total of 1039 Å2 of accessible surface area from XPA peptide is buried in the complex with ERCC1, accounting for 61% of the solvent accessible surface area of XPA residues 67–77, which are in close proximity to the binding site. The XPA ligand derives many interactions from the core sequence motif (shown in boldface; KIIDTGGGFILEEE) of the XPA67−80 peptide. The side chains of Phe75, Leu77 and Thr71 of XPA are clustered together at the mouth of the V-shaped groove (Figure 2A) where Phe75 stacks against Asn110 of ERCC1, and the Ile76 side chain packs against the aliphatic portion of ERCC1 side chains Arg144 and Leu148. The binding groove in ERCC1 is capped by XPA Leu77. The glycine-rich loop of XPA67−80 extends far into the groove of ERCC1 where main chain atoms of these XPA residues stack against the side chains of Tyr145 and Tyr152 from ERCC1 (Figure 2A). The main chain amides of these glycines could participate in hydrogen-bonding interactions with the ERCC1-binding site, although these interactions cannot be directly observed from our NMR experiments nor can they be reliably confirmed by low-resolution (4 Å) X-ray diffraction data (Table 1 in the Supplementary data). Based on the proximity of atoms modeled in the complex, we infer that the carbonyl oxygen of Gly74 may bond with the main chain amide of Ser142 from ERCC1. The orientations of the Tyr145 and Tyr152 side chains from ERCC1 would permit their hydroxyl groups to make hydrogen-bonding interactions with the backbone carbonyls of Thr71 and Gly73, respectively. The side chain of XPA Asp70 could participate in electrostatic interactions with the side chain His149 of ERCC1. It is notable that a solvent-exposed salt bridge between the side chains of Asp129 and Arg156 of ERCC1 (PDB code 2A1I; Tsodikov et al, 2005) becomes almost completely buried when XPA is bound. Phe75 of XPA is completely buried within the ERCC1-binding site (Figure 2A). We tested whether an alanine substitution at this position interferes with binding to 15N-labeled ERCC1 by measuring chemical shifts in the 15N HSQC spectra in the presence of the mutant peptide designated XPA67−80 F75A. Addition of the mutant peptide failed to perturb the chemical shifts of ERCC1 seen upon addition of wild-type XPA67−80 (data not shown), indicating that the mutant peptide does not bind to ERCC1. The TGGGFI-binding motif of the XPA ligand and the corresponding residues of the ERCC1-binding site are strictly conserved in higher eukaryotes. In lower eukaryotes, the corresponding sequences of both proteins have diverged from this consensus, perhaps indicating the coevolution of these two proteins and their functions. The XPA peptide inhibits NER in mammalian cell extracts The direct interaction of XPA67−80 peptide with the ERCC1-binding pocket raised the possibility that this peptide might specifically interfere with the recruitment of the ERCC1-XPF nuclease into the NER reaction pathway. We investigated the effect of XPA67−80 and the mutant XPA67−80 F75A peptide on the dual incision of a DNA lesion during NER in cell-free extracts. A plasmid containing a single site-specific 1,3-cisplatin intrastrand crosslink was incubated with HeLa cell-free extract in the presence of increasing concentrations of XPA peptide (Shivji et al, 1999). In the absence of XPA peptide, the characteristic NER excision products of 28–33 nucleotides containing the lesion were evident (Figure 4A, lane 1). Increasing concentrations of XPA67−80 interfered with excision of the oligonucleotide, and complete inhibition was achieved at a concentration of XPA peptide in the low micromolar range (Figure 4A, lanes 2–6). In contrast, the addition of XPA67−80 F75A did not affect NER activity at concentrations up to 92 μM, the maximum concentration tested (Figure 4A, lanes 7–11). Figure 4.The XPA67−80 peptide is an effective inhibitor of NER activity. (A) XPA67−80 inhibits the in vitro NER reaction, whereas the mutant XPA67−80 F75A peptide has no effect. HeLa cell extracts were incubated with a plasmid containing a 1,3-intrastrand cisplatin adduct in the presence of increasing concentrations of either XPA67−80 or XPA67−80 F75A (lane 1, no XPA; lanes 2 and 7, 46 nM XPA peptide; lanes 3 and 8, 460 nM; lanes 4 and 9, 4.6 μM; lanes 5 and 10, 46 μM; lanes 6 and 11, 92 μM). Products were visualized by a fill-in reaction following annealing to an oligonucleotide complementary to the excision product with a 4-nt overhang (Shivji et al, 1999). The marker DNA ladder is labeled LMW DNA ladder. (B) XPA67−80 and XPA67−80 F75A do not affect the intrinsic nuclease activity of ERCC1-XPF. The stem12-loop22 substrate (6.6 nM) was incubated with different concentrations of ERCC1-XPF (lanes 2, 4 and 6: 6.7 nM ERCC1-XPF; lanes 3, 5 and 7: 26.8 nM) and 0.4 mM MnCl2 in the presence of no peptide (lanes 1–3), 92 μM XPA67−80 (lanes 4 and 5), and 92 μM XPA67−80 F75A (lanes 6 and 7). The DNA substrate and the cleavage products are indicated. Download figure Download PowerPoint The XPA peptide might inhibit NER activity in vitro by directly interfering with the endonuclease activity of ERCC1-XPF, instead of blocking its interaction with XPA. To account for the former possibility, we tested the effects of XPA peptides on the DNA incision reaction catalyzed by purified ERCC1-XPF using stem-loop DNA substrate (de Laat et al, 1998). ERCC1-XPF efficiently cleaves on the 5′ side of the loop and the XPA peptide has no effect on this activity (Figure 4B) even at a concentration (92 μM) that completely abolishes NER activity (Figure 4A). We conclude that the inhibitory effect of XPA67−80 on the NER reaction results from disrupting the interaction of ERCC1 with XPA, an essential protein–protein interaction for the dual incision of DNA by the NER pathway. Mutations in the ERCC1-binding epitope of XPA abolish NER The specificity of inhibition of NER by XPA67−80 suggested that mutations of single residues such as F75 might diminish the NER activity of the XPA protein. We generated mutant XPA proteins containing an F75A mutation and ΔG73 single and ΔG73/ΔG74 double deletion, and compared their activities to that of the wild-type XPA protein. The ability of the XPA proteins to mediate NER activity was tested by incubating a plasmid containing a 1,3-cisplatin intrastrand crosslink with a cell-free extract generated from XPA-deficient cells supplemented with purified wild-type or mutant XPA protein (Shivji et al, 1999). Addition of wild-type XPA protein to this mixture resulted in robust NER activity, as evidenced by formation of the characteristic excision products of 24–32 nts in length (Figure 5A, lanes 1–2). By contrast, no NER activity was observed following addition of the F75A or G73Δ/G74Δ mutants, while the G73Δ single deletion mutant displayed marginal NER activity. Figure 5.Mutation of the ERCC1-binding epitope of XPA abolishes NER but not DNA-binding activity. (A) XP-A (XP2OS) cell extracts were incubated with a plasmid containing a 1,3-intrastrand cisplatin adduct in the presence of wild-type XPA (XPA-WT) or mutant XPA proteins (XPA-F75A, XPA-G73Δ or XPA-G73Δ/G74Δ). The reaction products were visualized by a fill-in reaction after annealing the excision product to an complementary oligonucleotide with a 4-nt overhang (Shivji et al, 1999). Different XPA concentrations of 200 nM (lanes 1, 3, 5 and 7) and 800 nM (lanes 2, 4, 6 and 8) were tested. The position of a 25 mer of the LMW DNA ladder is indicated. (B) A 5′-labeled DNA three-way junction (1 nM) was incubated with wild-type and mutant XPA proteins for 30 min at room temperature, then the XPA-bound (xd) and free DNA (d) oligonucleotides were separated on an 8% native polyacrylamide gel. The reaction products generated with different concentrations of XPA are shown: 0 (lane 1), 4 nM (lanes 2, 7, 13, 17), 10 nM (lanes 3, 8, 13, 18), 25 nM (lanes 4, 9, 14, 19), 60 nM (lanes 5, 10, 15, 20) and 150 nM (lanes 6, 11, 16, 21). Download figure Download PowerPoint To test if these XPA mutations only affected binding to ERCC1, we also compared the DNA-binding activities of wild-type and mutant XPA proteins. We investigated the binding of wild-type and mutant XPA to a DNA three-way junction, representing a high-affinity target for XPA in band-shift assays (Missura et al, 2001). The wild-type, F75A, G73Δ and G73Δ/G74Δ XPA proteins all bound with similar affinity to a three-way junction (Figure 5B), indicating that the mutant proteins are fully proficient in DNA binding and unlikely to be misfolded or otherwise inactive. These results show that single point mutations in XPA can result in a defect in NER activity by weakening the interaction between ERCC1 and XPA. Due to the highly cooperative nature of NER (Moggs et al, 1996), other NER functions and interactions may be disrupted as a result of blocking the recruitment of XPF–ERCC1. XPA competes with single-stranded DNA for binding to ERCC1 Because XPA binds in the groove on ERCC1 (Figure 2) that was previously implicated in DNA-binding activity (Tsodikov et al, 2005), we directly tested whether or not XPA competes with single-stranded DNA for binding to ERCC1. DNA-binding activity was measured by monitoring fluorescence anisotropy, using single-stranded 40-mer oligonucleotide labeled on the 5′ end with 6-carboxyfluorescein. The XPA67−80 peptide does not detectably bind to DNA (not shown), although it does compete with DNA for binding to ERCC1 (Supplementary Figure 3). This result confirms that the DNA-binding site on ERCC1's central domain overlaps with the XPA-binding site. The EC50 for binding of XPA67−80 is in the micromolar range, but quenching of the fluorescent probe by high concentrations of XPA precluded an accurate measurement of the binding constant. We previously reported an equilibrium-binding constant of 1.5 μM for DNA binding to the central domain of ERCC1 (Tsodikov et al, 2005). By fitting the XPA competition titration data to a competitive binding model (Equation 9 in the Supplementary data), we obtain the estimated binding constant of Kd=(540±280) nM for the XPA–ERCC1 complex. This result agrees well with the affinity determined directly for this interaction (Supplementary Figure 2). Thus, XPA binds to the central domain of ERCC1 with approximately three-fold higher affinity than single-stranded DNA. Discussion The removal of bulky and helix-distorting DNA lesions by the NER pathway requires the coordinated assembly of a large multiprotein complex (Houtsmuller et al, 1999; Volker et al, 2001) that exposes the damaged DNA strand and excises an oligonucleotide containing the lesion (Gillet and Schärer, 2006). We have investigated one of the essential protein–protein interactions in this pathway. The specific interaction of XPA with ERCC1 is responsible for recruitment of the ERCC1-XPF nuclease to the DNA repair complex (Li et al, 1994). Our structural studies have defined the XPA ligand as a TGGGFI sequence motif that inserts into a pocket of the central domain of ERCC1 (Figure 2). It was previously shown that deletion of the GGG triplet within this motif abolishes the interaction of XPA with ERCC1 (Li et al, 1995). These glycines insert deep into the ERCC1-binding site and are likely to make hydrogen-bonding interactions using main chain atoms (Fi" @default.
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• W2080726979 date "2007-10-18" @default.
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• W2080726979 title "Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA" @default.
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