Two-stage dynamic DNA quality check by xeroderma pigmentosum group C protein
2009; Springer Nature; Volume: 28; Issue: 16 Linguagem: Inglês
10.1038/emboj.2009.187
ISSN1460-2075
AutoresUlrike Camenisch, Daniel Träutlein, Flurina C. Clement, Jia Fei, Alfred Leitenstorfer, Elisa Ferrando‐May, Hanspeter Naegeli,
Tópico(s)Evolution and Genetic Dynamics
ResumoArticle16 July 2009free access Two-stage dynamic DNA quality check by xeroderma pigmentosum group C protein Ulrike Camenisch Ulrike Camenisch Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Daniel Träutlein Daniel Träutlein Department of Physics and Center for Applied Photonics, University of Konstanz, Konstanz, Germany Search for more papers by this author Flurina C Clement Flurina C Clement Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Jia Fei Jia Fei Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Alfred Leitenstorfer Alfred Leitenstorfer Department of Physics and Center for Applied Photonics, University of Konstanz, Konstanz, Germany Search for more papers by this author Elisa Ferrando-May Elisa Ferrando-May Bioimaging Center, University of Konstanz, Konstanz, Germany Search for more papers by this author Hanspeter Naegeli Corresponding Author Hanspeter Naegeli Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Ulrike Camenisch Ulrike Camenisch Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Daniel Träutlein Daniel Träutlein Department of Physics and Center for Applied Photonics, University of Konstanz, Konstanz, Germany Search for more papers by this author Flurina C Clement Flurina C Clement Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Jia Fei Jia Fei Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Alfred Leitenstorfer Alfred Leitenstorfer Department of Physics and Center for Applied Photonics, University of Konstanz, Konstanz, Germany Search for more papers by this author Elisa Ferrando-May Elisa Ferrando-May Bioimaging Center, University of Konstanz, Konstanz, Germany Search for more papers by this author Hanspeter Naegeli Corresponding Author Hanspeter Naegeli Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland Search for more papers by this author Author Information Ulrike Camenisch1,‡, Daniel Träutlein2,‡, Flurina C Clement1, Jia Fei1, Alfred Leitenstorfer2, Elisa Ferrando-May3 and Hanspeter Naegeli 1 1Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Zürich, Switzerland 2Department of Physics and Center for Applied Photonics, University of Konstanz, Konstanz, Germany 3Bioimaging Center, University of Konstanz, Konstanz, Germany ‡These authors contributed equally to this work *Corresponding author. Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Winterthurerstrasse 260, Zurich 8057, Switzerland. Tel.: +41 44 635 87 63; Fax: +41 44 635 89 10; E-mail: [email protected] The EMBO Journal (2009)28:2387-2399https://doi.org/10.1038/emboj.2009.187 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Xeroderma pigmentosum group C (XPC) protein initiates the DNA excision repair of helix-distorting base lesions. To understand how this versatile subunit searches for aberrant sites within the vast background of normal genomic DNA, the real-time redistribution of fluorescent fusion constructs was monitored after high-resolution DNA damage induction. Bidirectional truncation analyses disclosed a surprisingly short recognition hotspot, comprising ∼15% of human XPC, that includes two β-hairpin domains with a preference for non-hydrogen-bonded bases in double-stranded DNA. However, to detect damaged sites in living cells, these DNA-attractive domains depend on the partially DNA-repulsive action of an adjacent β-turn extension that promotes the mobility of XPC molecules searching for lesions. The key function of this dynamic interaction surface is shown by a site-directed charge inversion, which results in increased affinity for native DNA, retarded nuclear mobility and diminished repair efficiency. These studies reveal a two-stage discrimination process, whereby XPC protein first deploys a dynamic sensor interface to rapidly interrogate the double helix, thus forming a transient recognition intermediate before the final installation of a more static repair-initiating complex. Introduction Nucleotide excision repair (NER) is a fundamental protective system that promotes genome stability by eliminating a wide range of DNA lesions (Gillet and Schärer, 2006). In addition to (6-4) photoproducts and cyclobutane pyrimidine dimers (CPDs) caused by ultraviolet (UV) light, the NER pathway removes DNA adducts generated by electrophilic chemicals as well as intrastrand DNA cross-links, DNA-protein cross-links and a subset of oxidative lesions (Huang et al, 1994; Kuraoka et al, 2000; Reardon and Sancar, 2006). The NER system operates through the cleavage of damaged strands on either side of injured sites, thus releasing defective bases as the component of oligomeric DNA fragments (Evans et al, 1997). Subsequently, the excised oligonucleotides are replaced by repair patch synthesis before DNA integrity is restored by ligation. Hereditary defects in this NER process cause devastating syndromes such as xeroderma pigmentosum (XP), a recessive disorder presenting with photosensitivity, a >1000-fold increased risk of skin cancer and, occasionally, internal tumours and neurological complications (Cleaver, 2005; Andressoo et al, 2006; Friedberg et al, 2006). XP patients are classified into seven repair-deficient complementation groups designated XP-A through XP-G (Cleaver et al, 1999; Lehmann, 2003). In the NER pathway, the initial detection of DNA damage occurs by two alternative mechanisms. One subpathway, referred to as transcription-coupled repair, takes place when the transcription machinery is blocked by obstructing lesions in the transcribed strand (Hanawalt and Spivak, 2008). The second subpathway, known as global genome repair (GGR), is triggered by the binding of a versatile recognition complex, composed of XPC, Rad23B and centrin 2, to damaged DNA anywhere in the genome (Sugasawa et al, 1998; Nishi et al, 2005). XPC protein, which is the actual damage sensor of this initiator complex, displays a general preference for DNA substrates that contain helix-destabilizing lesions including (6-4) photoproducts (Batty et al, 2000; Sugasawa et al, 2001). In the particular case of CPDs, this recognition function depends on an auxiliary protein discovered by virtue of its characteristic UV-damaged DNA-binding (UV-DDB) activity (Nichols et al, 2000; Fitch et al, 2003). The affinity of this accessory factor for UV-irradiated substrates is conferred by a DNA-binding subunit (DDB2) mutated in XP-E cells (Scrima et al, 2008). To achieve its outstanding substrate versatility, XPC protein interacts with an array of normal nucleic acid residues surrounding the lesion in a way that no direct contacts are made with the damaged bases themselves (Buterin et al, 2005; Trego and Turchi, 2006; Maillard et al, 2007). This exceptional binding strategy has been confirmed by structural analyses of Rad4 protein, a yeast orthologue that shares ∼40% similarity with the human XPC sequence. In co-crystals, Rad4 protein associates with DNA through a large transglutaminase-homology domain (TGD) flanked by the three β-hairpin domains BHD1, BHD2 and BHD3 (Supplementary Figure 1; Min and Pavletich, 2007). In view of the position of these structural elements relative to the accompanying model substrate, a recognition mechanism has been proposed in which BHD3 would 'sample the DNA's conformational space to detect a lesion' (Min and Pavletich, 2007). These earlier studies describing the features of an ultimately stable XPC/Rad4–DNA complex explain its ability to serve as a molecular platform for the recruitment of transcription factor IIH (TFIIH) or other downstream NER players (Yokoi et al, 2000; Uchida et al, 2002). However, one of the most challenging issues in the DNA repair field is the question of how a versatile sensor-like XPC/Rad4 examines the Watson–Crick double helix and faces the task of actually finding base lesions among a large excess of native DNA in a typical mammalian genome (Schärer, 2007; Sugasawa and Hanaoka, 2007). To address this long-standing question, we exploited fluorescence-based imaging techniques (Houtsmuller et al, 1999; Houtsmuller and Vermeulen, 2001; Politi et al, 2005) to visualize the mobility of XPC protein at work in the chromatin context of living cells. Our results point to a two-stage discrimination process, in which the rapid DNA quality check driven by a dynamic sensor of non-hydrogen-bonded bases precedes the final engagement of BHD3 with lesion sites. Results Instantaneous recognition of DNA lesions in human cells Damage-induced changes of molecular dynamics in the nuclear compartment have been followed by C-terminal conjugation of the human XPC polypeptide with green-fluorescent protein (GFP). The time-dependent relocation of this fusion product was tested by transfection of repair-deficient XP-C fibroblasts that lack functional XPC because of a mutation leading to premature termination at codon 718 (Chavanne et al, 2000). Individual nuclei containing low levels of XPC-GFP (similar to the XPC expression in wild-type fibroblasts) were identified on the basis of their overall fluorescence (Supplementary Figure 2). To induce lesions, the nuclei were subjected to near-infrared irradiation using a pulsed multiphoton laser, thereby generating spatially confined and clearly detectable patterns of DNA damage with minimal collateral effects (Meldrum et al, 2003). The resulting laser tracks contained (6-4) photoproducts (Figure 1A) and CPDs (Figure 1B), representing the major UV lesions processed by the NER system. As expected, wild-type XPC-GFP was rapidly concentrated at nuclear sites containing such photolesions (Figure 1A and B). As earlier studies showed that the UV-induced accumulation of XPC is stimulated by DDB2 protein (Fitch et al, 2003; Moser et al, 2005), we applied the same procedure to XP-E cells, in which an R273H mutation generates a DDB2 product that is inactive in DNA binding and fails to be expressed to detectable levels (Nichols et al, 2000; Itoh et al, 2001). In this XP-E background, XPC-GFP is nevertheless effectively relocated to UV-irradiated tracks (Figure 1C), consistent with the known ability of XPC protein to detect (6-4) photoproducts in the absence of UV-DDB activity (Batty et al, 2000; Kusumoto et al, 2001). Figure 1.Instantaneous recognition of DNA damage by XPC protein in living cells. (A) High-resolution patterns of DNA damage and XPC-GFP accumulation. XP-C fibroblasts expressing low levels of XPC-GFP were laser treated to generate ∼5000 UV lesions along each linear irradiation track. The cells were fixed after 6 min and (6-4) photoproducts were detected by immunochemical staining using the red dye Alexa 546. B/W, black-and-white images illustrating the pattern of UV lesions (upper panel) and the accumulation of XPC-GFP (lower panel). Merged, superimposed images in which the relocation of XPC-GFP matches the pattern of DNA damage. Hoechst, DNA staining visualizing the nuclei. (B) Co-localization of XPC-GFP and CPDs. (C) Efficient relocation of XPC-GFP to UV irradiation tracks in XP-E cells devoid of UV-DDB activity. (D) Real-time kinetics of DNA damage recognition. A single 10-μm line of UV photoproducts was generated across each nucleus of XP-C cells. The accumulation of XPC-GFP at different time points is plotted as a percentage of the average fluorescence before irradiation (n=7). Error bars, standard errors of the mean. Download figure Download PowerPoint To determine the kinetics of protein redistribution, DNA photoproducts were formed along a single 10-μm line crossing the nucleus of XP-C cells. Maximal accumulation of XPC protein was detected after treatment with a near-infrared radiation of 300–360 GW ·cm−2 (Supplementary Figure 3). Subsequently, DNA damage was induced with 314 GW cm−2 to generate ∼5000 UV lesions in each cell or, on the average, 1 UV lesion in ∼1.6 × 106 base pairs (see Materials and methods). Under these conditions, the local fluorescence in irradiated areas increased nearly instantaneously leading to a clearly distinguishable relocation of XPC fusion protein already 3 s after irradiation (Supplementary Movie 1). With progressive accumulation of wild-type XPC, a half-maximal increase in local fluorescence intensity was reached after ∼40 s (Figure 1D). A plateau level of fluorescence in the irradiation tracks, reflecting a steady-state situation with constant turnover, was detected after ∼300 s. Concordance of relocation and DNA-binding activity Besides the truncating XPC mutation, the XP-C fibroblasts used in this study (GM16093) are characterized by a comparably low level of DDB2 protein (Supplementary Figure 4). This reduced DDB2 expression suggested that the GM16093 fibroblasts may provide a cellular context in which, in contrast to an earlier report (Yasuda et al, 2007), the damage recognition defect of XPC mutants becomes evident without preceding DDB2 down-regulation. This view was confirmed by testing the nuclear dynamics of a repair-deficient W690S mutant with minimal DNA-binding affinity (Bunick et al, 2006; Maillard et al, 2007; Hoogstraten et al, 2008). In conjunction with the GFP fusion partner, this pathogenic mutant is expressed in similar amounts as the wild-type control and also localizes to the nuclei. However, in the XP-C fibroblasts of this study, the single W690S mutation causes >five-fold reduction in the relocation to UV-damaged areas (Figure 2A; Supplementary Movie 2). These findings were confirmed when another technique was used to inflict genotoxic stress, that is by UV-C irradiation (254 nm wavelength) through the pores of polycarbonate filters (Moné et al, 2004). In fact, compared with wild-type XPC, the W690S mutant exhibits only a marginal tendency to accumulate in UV-C radiation-induced foci (data not shown). Oligonucleotide-binding assays with XPC protein expressed in insect cells confirmed that this W690S mutation and the corresponding alanine substitution (W690A) abrogate the interaction with DNA (Figure 2B). Figure 2.Dependence on intrinsic DNA-binding activity. (A) Representative image (in colour and black-and-white) showing the low residual accumulation of the W690S mutant 6 min after irradiation. DNA lesions were counterstained by antibodies against CPDs. (B) DNA-binding activity determined by direct pull down. Wild-type (wt) XPC or mutants were expressed in Sf9 cells as fusion constructs with maltose-binding protein (MBP). Cell lysates containing similar amounts of XPC protein (Maillard et al, 2007) were incubated with a single-stranded 135-mer oligonucleotide. Subsequently, radiolabelled DNA molecules captured by XPC protein were separated from the free probes using anti-MBP antibodies linked to magnetic beads, and the radioactivity in each fraction was quantified in a scintillation counter. DNA binding is represented as the percentage of radioactivity immobilized by wt XPC protein after deduction of a background value determined with empty beads (n=3). Error bars, standard deviation. (C) Correlation between DNA binding and the kinetics of XPC accumulation in XP-C cells (n=7). See legend to Figure 1D for details. Download figure Download PowerPoint The same analysis was extended to further repair-deficient XPC mutants targeting conserved aromatic residues (Maillard et al, 2007). A nearly complete loss of DNA binding is conferred by the F733A mutation, whereas the W531A and W542A substitutions are associated with more moderate defects (Figure 2B). When tested in GM16093 fibroblasts as GFP fusions, the damage-dependent redistribution of these different mutants correlates closely with the respective DNA-binding properties. In fact, the W690S, W690A and F733A derivatives display a poor ability to concentrate at damaged sites. In contrast, the residual DNA-binding activity of W531A and W542A leads to an intermediary level of accumulation in areas containing UV photoproducts (Figure 2C). From this tight correspondence between DNA binding and nuclear redistribution, we concluded that the rapid relocation of XPC protein to UV lesion sites reflects the intrinsic capacity of this sensor subunit to detect DNA damage through direct interactions with the nucleic acid substrate. Role of the transglutaminase-like domain As the transglutaminase-like region maps to the N-terminal part of human XPC (Figure 3A), we generated N-terminal truncations (XPC118−940, XPC427−940 and XPC607−940) to test how the TGD sequences contribute to DNA damage recognition in living cells. The positions 118 and 607 were selected for these truncations to allow for comparisons with an earlier in vitro study monitoring the DNA-, Rad23B- and TFIIH-binding activity of XPC fragments (Uchida et al, 2002). Another truncate (XPC1−495) was included as a negative control that lacks the entire C-terminal half. The functionality of these constructs, conjugated to GFP at their C-terminus, was compared in a host-cell reactivation assay that has been developed to measure the cellular GGR activity (Carreau et al, 1995). Briefly, XP-C fibroblasts were transfected with a dual luciferase reporter system along with an expression vector coding for full-length or truncated XPC fusions. The reporter plasmid, which carries a Photinus luciferase gene, was damaged by exposure to UV-C light and supplemented with an undamaged vector that expresses the Renilla luciferase. GGR efficiency was assessed after 18-h incubations by determining Photinus luciferase activity in cell lysates, followed by normalization against the Renilla control. Figure 3.Mapping of the damage sensor domain to the C-terminal part of human XPC. (A) Scheme illustrating the position of the TGD sequences relative to the N-terminal XPC truncates. (B) GGR activity determined by host-cell reactivation assay (n=5; error bars, standard deviation). (C) Immunoblot analysis of XP-C cells transfected with expression vectors coding for the indicated fusions. The protein level was probed using anti-GFP antibodies. G, endogenous GAPDH control. (D) Representative image showing that an XPC fragment lacking the C-terminus (XPC1−495) fails to accumulate in laser-damaged areas. The XP-C fibroblasts were fixed 6 min after irradiation. B/W, black-and-white images showing that the tracks of DNA damage (upper panel) do not induce an accumulation of truncated XPC fusions (lower panels). (E) Representative images (in colour and black and white) showing that XPC427−940 and XPC607−940 accumulate in damaged areas of XP-C fibroblasts. The distribution of fluorescent fusion products was monitored 6 min after irradiation. (F) Local increase of fluorescence resulting from the damage-induced redistribution of full-length XPC or XPC607−940. A 10-μm line of UV photoproducts was generated across each nucleus and the resulting accumulation of fusion proteins (after a 6-min incubation) is plotted as a percentage of the average fluorescence before irradiation (n=7). Error bars, standard errors of the means. (G) Representative image illustrating that XPC607−940 accumulates in foci generated by UV-C irradiation (100 J m−2) through the pores of polycarbonate filters. The XP-C cells were fixed 15 min after treatment and CPDs were detected by immunochemical staining. The position of XPC607−940 foci is indicated by the arrows. Download figure Download PowerPoint The full-length protein (XPC1−940) and an XPC118−940 derivative, isolated by functional complementation (Legerski and Peterson, 1992), were proficient in correcting the repair defect of XP-C cells (Figure 3B), thus showing that gene reactivation is determined by the ability of the GGR pathway to excise offending UV lesions. However, this repair activity could not be rescued by XPC427−940 and XPC607−940 (Figure 3B), implying that the N-terminal part of XPC protein is essential for the GGR reaction. All tested fragments were detected in transfected fibroblasts in similar amounts as the full-length control or the functional XPC118−940 derivative (Figure 3C), indicating that their repair deficiency does not result from reduced expression or enhanced degradation. Next, all GGR-deficient truncates were tested for their damage recognition proficiency in XP-C fibroblasts. Neither XPC1−495 (Figure 3D) nor XPC1−718 (Supplementary Figure 4) were redistributed to sites of photoproduct formation in the irradiated nuclei of living cells, confirming that the C-terminal half of XPC protein is necessary for lesion recognition. However, unlike these C-terminal truncations, fragment XPC427−940 retains the ability to concentrate in laser-irradiated areas (Figure 3E). Even more surprising was the observation that the smaller fragment XPC607−940 readily accumulates at sites containing UV photolesions (Figure 3E). The quantification of defined 10-μm tracks showed that XPC607−940 is only ∼30% less efficient than full-length XPC in relocating to damaged sites (Figure 3F). Thus, a large N-terminal part of human XPC (65% of the full-length protein including its TGD regions) stimulates DNA damage recognition, but is not absolutely required for the sensing process itself. This conclusion is confirmed by the accumulation of XPC607−940 in UV-C foci generated by irradiation through the pores of polycarbonate filters (Figure 3G). Differential contribution of -hairpin domains According to the Rad4 crystal, three consecutive β-hairpin domains (BHD1, BHD2 and BHD3) mediate the interaction with damaged DNA (see Supplementary Figure 1). In the homologous XPC sequence, these structural elements range from residue 637 (start of BHD1) to residue 831 (end of BHD3). To examine how each of these domains contributes to DNA damage recognition in living cells, we generated the C-terminal truncations XPC1−741 (comprising BHD1 and BHD2) and XPC1−831, which includes all three BHDs (Figure 4A). Again, the truncation position 741 was chosen to allow for comparisons with an earlier in vitro study (Uchida et al, 2002). The constructs were conjugated to GFP at their C-terminus and tested for their ability to initiate the GGR reaction. In the case of XPC1−741, the repair function is reduced to a background level observed with empty GFP vector (Figure 4B). However, the reporter gene was reactivated to ∼40% of control in the presence of XPC1−831, indicating that despite its C-terminal truncation, this large fragment retains in part the ability to recruit NER factors to lesion sites. Although attempting to delineate the borders of a minimal sensor domain, we surprisingly found that essentially the same GGR activity was induced by XPC1−766, that is by adding only 25 amino acids to XPC1−741 (Figure 4B). A comparison with the Rad4 orthologue indicates that these 25 amino acids (residues 742–766) belong to an N-terminal extension of BHD3, which folds into a β-turn structure (see Figure 4A). Figure 4.BHD3 is not required for DNA damage detection. (A) Scheme illustrating the location of BHD and β-turn sequences relative to the C-terminal XPC truncates of this study. (B) GGR activity determined by host-cell reactivation assay in XP-C fibroblasts (n=5; error bars, standard deviation). (C) Representative images (taken 6 min after irradiation) comparing the accumulation of XPC1−766 and XPC1−741 at damaged sites. In the black-and-white representation, the linear irradiation tracks are surrounded by a dashed rectangle. (D) Representative image illustrating the accumulation of XPC1−766 along UV radiation tracks generated in XP-E fibroblasts devoid of UV-DDB activity. (E) The local increase in fluorescence, because of damage-induced redistributions of XPC truncates, was measured in XP-C and XP-E cells and plotted as the percentages of wt control as outlined in Figure 1D (n=5; error bars, standard errors of the mean). (F) XPC1−766 is also more efficient than XPC1−741 in accumulating in DNA damage foci generated by UV-C irradiation through the pores of polycarbonate filters (see Figure 3G for details). XPC1−766 (top) and XPC1−741 foci (bottom) are indicated by the arrows. Download figure Download PowerPoint The UV-induced relocation of truncated XPC derivatives was tested in XP-C fibroblasts expressing similar low levels of each GFP construct (Supplementary Figure 5). Consistent with its distinctive functionality in the GGR assay, we observed that XPC1−766 accumulates more effectively than XPC1−741 to the 10-μm tracks of photolesions generated by laser irradiation (Figure 4C). An unequivocal pattern of XPC1−766 accumulation along the radiation tracks was also recorded in XP-E fibroblasts, that is in the absence of UV-DDB activity (Figure 4D). A quantitative comparison in both XP-C and XP-E cells highlights the increase in damage recognition when the truncation was introduced at residue 766 as compared with the truncation at position 741 (Figure 4E), thus showing that the damage-specific accumulation of XPC truncates as well as the effect of the β-turn structure takes place in the absence of DDB2 protein. A clear difference between XPC1−766 and XPC1−741 was reproduced when foci of fluorescence were monitored after UV-C irradiation through the pores of polycarbonate filters (Figure 4F). Taken together, this efficient redistribution of XPC1−766, irrespective of the cell type or technique used to inflict DNA damage, establishes for the first time that most of BHD3 is not required for the initial damage-sensing process. The -turn structure enhances XPC dynamics The GGR and relocation assays of Figure 4 revealed a striking difference between XPC1−741 and XPC1−766 because of the 25-amino-acid β-turn extension. To analyse the function of this β-turn structure, we compared the nuclear mobility of different truncates using fluorescence recovery after photobleaching (FRAP; Houtsmuller and Vermeulen, 2001). In cells that express similarly low levels of GFP fusion constructs, a nuclear area of 4 μm2 was bleached and, subsequently, protein movements were tested by recording the recovery of local fluorescence, which is dependent on the ability of the GFP fusions to move rapidly within the nuclear compartment. The control experiment of Figure 5A shows how, in the absence of a fusion partner, the GFP moiety moves freely inside the cells. Instead, the nuclear mobility of full-length XPC-GFP is restrained by its larger size and propensity to undergo macromolecular interactions, as reported earlier (Hoogstraten et al, 2008). Surprisingly, in a direct comparison between XPC1−741, XPC1−766 and XPC1−831, a larger size correlated with increased nuclear mobility (Figure 5B). The FRAP curves obtained with these different truncates were used to calculate effective diffusion coefficients (Deff; Supplementary Table I). It was unexpected to find that, in undamaged cells, XPC1−766 (containing BHD1, BHD2 and the β-turn structure) and XPC1−831 (containing all three BHDs) move more rapidly inside the nucleus (Deff=0.44 and 0.49 μm2 s–1, respectively) than the shorter polypeptide XPC1−741 lacking the β-turn (Deff=0.34 μm2 s–1). We concluded that these C-terminal truncations disclose the existence of a dynamic interface, residing within the β-turn structure, which enhances the constitutive nuclear mobility of XPC protein in the absence of genotoxic stress. Figure 5.Identification of a dynamic core and two-stage damage recognition. (A) Principle of FRAP analysis. An area of 4 μm2 in the nuclei of XP-C fibroblasts expressing a particular GFP construct is bleached with a 488-nm wavelength laser. The kinetics and extent of fluorescence recovery (shown for GFP and XPC-GFP) depends on diffusion rate, molecular interactions as well as the fraction of immobile molecules. (B) Recovery plots of XPC truncates normalized to prebleach intensity (n=12). Error bars, standard errors of the mean. The difference between XPC1−766 and XPC1−831 is not significant. (C) The nuclear mobility of XPC1−741 remains unaffected by UV-C irradiation at a dose of 10 J m−2 (n=12). (D) The initial diffusion of XPC1−766 is reduced by UV light (10 J m−2, n=12), reflecting transient molecular interactions during stage 1 of the damage recognition process. (E) A fraction of XPC1−831 is stably immobilized after UV irradiation (10 J cm−2, n=12), reflecting stage 2 of the damage recognition process. Download figure Download PowerPoint Subsequently, the FRAP approach was used to assess the corresponding responses to UV-C irradiation. In accord with its poor accumulation along DNA damage tracks (Figure 4C), the mobility of XPC1−741 is only marginally affected by the induction of photolesions (Figure 5C). In contrast, the diffusion rates of XPC1−766 (Figure 5D) and XPC1−831 (Figure 5E), which accumulate in UV lesion tracks, are significantly reduced (the respective Deff values are listed in Supplementary Table I). In the case of XPC1−831, the induction of DNA damage had a two-fold effect. First, UV lesions decreased the initial rate of protein diffusion exactly as observed with XPC1−766. Second, similar to the response of full-length XPC (Hoogstraten et al, 2008), the overall fluorescence recovery is less complete on UV irradiation (Figure 5E), i
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