Two-stage dynamic DNA quality check by xeroderma pigmentosum group C protein

Ulrike Camenisch '.4, Daniel Trautlein2.4, Flurina C Clement', Jia Fei', Alfred Leitenstorfer2, Elisa Ferrando-M ay3

and Hanspeter Naegeli"* 'Institute of Pharmaco logy and Tox ico logy, University of ZUrich­Vetsuisse, ZUrich, Switzerland, ' Department of Physics and Center for App lied Photonics, University of Konstanz, Konstanz, Germany and 3Bioi maging Center, University of Konstanz, Konstanz, Germany

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 p-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 p-turn extension that promotes the mobility of XPC molecules searching for lesions. The key function of this dynamic interaction surface is shown by a site­directed charge invers ion, which results in increased affinity for native DNA, retarded nuclear mobility and diminished repair efficiency. These studies reveal a two­stage 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 sta tic repair-initiating complex.

Subject Categories: geno me stability & dynamiCS Keywords: DNA repair; genome stability; protein dynamics

Introduction

Nucleotide excision repair (NER) is a fundamental protective system that promotes genome stability by eliminating a wide range of DNA lesions (G illet and Scharer, 2006). In add ition to (6-4) photoproducts and cyclobutane pyrimidine dimers (CPOs) caused by ultraviolet (UV) light, the NER pathway removes DNA adducts genera ted by electrophilic chemica ls

' Corresponding author. Insti tute of Pharma co logy and Toxicology, University of Zurich-Vetsu isse, Winterthurerstrasse 260, Zurich 8057, Switzerland. Tel.: + 41 446358763 ; Fax: -I 41 4463589 10; E-mai l: naegelih@vetpharm.uzh.ch 4These authors contribu ted equally to thi s work

as well as intrastrand DNA cross-links, DNA-protein cross­links and a subset of oxidative lesions (Huang et ai, 1994; Kuraoka et ai, 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 co mponent of oligomeriC DNA fragments (Evans et ai, 1997) . Subsequently, the excised oligonucl eotides are re­placed by repair patch synthesis before DNA integrity is restored by liga tion. Hereditary defects in this NER process cause devastating syndro mes such as xeroderma p igmento­su m (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 ai, 2006; Friedberg et ai, 2006). XP patients are classified into seven repair-deficient co mplementation groups designated XP-A through XP-G (Cleaver et ai, 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 repa ir (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 ai, 2005) . XPC protein, which is the actual damage sensor of this initiator complex, displays a general preference for DNA substrates that conta in helix-des tabiliz ing lesions including (6-4) photoproducts (Batty et al, 2000; Sugasawa et ai, 2001). In the particular case of CPOs, this recognition function depends on an auxiliary protein discovered by virtue of its characterist ic UV-da maged DNA-binding (UV-DDS) activity (Nichols et a i, 2000; Fitch et ai, 2003) . The affinity of thi s accessory factor for UV-irradiated substrates is conferred by a DNA-binding subunit (DDB2) mutated in XP-E cells (Scrima et ai, 2008).

To achi eve its outstanding substra te versatility, XPC pro­tein interacts with an array of normal nucleic acid residues surrounding the lesion in a way that no direct co ntacts are made with the damaged bases themselves (Buterin et ai, 2005; Trego and Turchi , 2006; Maillard et ai, 2007) . This exceptional binding stra tegy has been co nfirm ed by stru ctural analyses of Rad4 protei n, a yeast ort hologue that shares -40% similarity with the human XPC sequence. In co­crystals, Rad4 protein associates with DNA through a large transglutami nase-homology domain erGO) fl anked by th e three ~- hairpin domains BHDl, 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 recogni tion 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 tile features of an ultimately stable XPC/Rad4- DNA co mplex explain its ability

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to serve as a molecular platform for the recruitment of transcription factor lIH (TFlIH) or other downstream NER players (Yokoi et ai, 2000; Uchida el ai, 2002). However, one of th e most chall enging issues in th e DNA repa ir fi eld is the ques tion of how a versatile sensor-like XPC/Rad4 exa mines 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 (Scharer, 2007; Sugasawa and Hanaoka, 2007). To address thi s long-standing question , we exploited flu orescence-based imaging techniques (Houtsmuller et ai, 1999; Houtsmuller and Vermeulen, 2001; Politi el ai, 2005) to vi suali ze 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 th e fina l engagement of BHD3 with lesion sites .

Results

Instantaneous recognition of DNA lesions in human cells Damage-induced changes of mol ecular dynamics in the nu­clear compartment have been followed by C-terminal con­jugation of the human XPC polypeptide with green­fluorescent protein (GFP). The time-depend ent reloca ti on of thi s fusion product was tested by transfection of repair­deficient XP-C fibrobl as ts th at la ck fun ctional XPC beca use of a mutation leading to premature termination at codon 718 (Chavanne et ai, 2000). Individual nuclei containing low levels of XPC-GFP (similar to the XPC expression in wild­type fibroblasts) were identifi ed on the basis of their overall fluorescence (Suppl ementary Fi gure 2). To induce lesions, th e nuclei were subjected to nea r-infrared irradiation using a pulsed multiphoton laser, thereby generating spatially confined and clearly detecta bl e pJtterns of DNA damJge with minimal colla teral effects (Meldrum et ai, 2003).

A BIW B

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The resulting laser tracks co ntained (6-4) photoproducts (Figure 1A) and CPOs (Figure 1 B), representing the major UV lesions processed by the NER system. As expected, wild­type XPC-GFP was rapidly co ncentrated at nuclear sites containing such photolesions (Figure 1A and 8). As earlier studies showed that the UV-induced accumulat ion of XPC is stimulated by DDB2 protein (Fitch et ai, 2003 ; Moser et ai, 2005) , we app lied the same procedure to XP-E cells, in which an R273H mutat ion generates a DDB2 product that is inactive in DNA binding and fails to be expressed to detectab le levels (Nichols et ai, 2000; Itoh et ai, 2001). In this XP-E back­ground , XPC-GFP is nevertheless effectively relocated to UV­irradiated tracks (Figure 1C) , consistent with the known abi lity of XPC protein to detect (6-4) photoproducts in the absence of UV-DDB activity (Batty et ai, 2000; Kusumoto el ai, 2001).

To determine the kinet ics of protein redi stribution, DNA photoproducts were fonn ed along a single lO-~lm line cross­ing the nucleus of XP-C cells. Max imal accumulation of XPC protein was detected after treatment with a near-infrared radiation of 300-360 GW . cm- 2 (Supplementary Figure 3). Subsequent ly, 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 x lOG base pairs (see Materials and method s). Under th ese conditi ons, th e loca l fluoresce nce in irradiated areas increased nearly instantaneously leading to a clearly distinguishable reloca ti on of XPC fusion protein already 3 s after irradiation (Supplementary Movie 1). With progressive accumulation of wild-type XPC, a half-maximal increase in loca l fluorescence intensity WJS reached after ~40 s (Figure 1D) . A plateau level of fluorescence in the irrad iati on tracks, refl ecting a steady-s tate situa tion with constant turnover, was detected after ~ 300 s.

Concordance of relocation and DNA-binding activity Besides the truncating XPC mutation, the XP-C fibrob las ts used in this study (GM16093) are characterized by a

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Figure 1 Instantaneous recognition of DNA damage by XPC protein in li ving ce ll s. (A) High-resolution patterns of DNA damage and XPC-GFP accumu lation . XP-C fibrob lasts expressing low levels of XPC-GFP were laser treated to generate - 5000 UV lesions along each linear irradiation track. The cells were fi xed after 61ll in and (6-4) photoproducts were de tectecl by imJ11unochemica l 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 wh ich the relocation of XPC-GFP matches the pattern of DNA damage. Hoechst, DNA staining visualizing the nuclei. (B) Co-localization of XPC-GFP and CP Os. (el Efficient reloca tion of XPC-GFP to UV irradiation tracks in XP-E ce lls devoid of UV-DDB act ivity. (0) Rea l-time kineti cs of DNA damage recogn ition. A single lO-pm line of UV photoproducts was generated across each nucl eus of XP-C cell s. The acc umulati on of XPC-GFP at different time poi nts is plotted as a percentage of the average flu orescence before irrad iati on (n = 7) . Error bars, standard errors of the mea n.

comparably low level of DDB2 protein (Supp lementary Figure 4). This reduced DDB2 express ion suggested that the CM l6093 fibrob las ts may prov ide a cellu lar co ntext in wh ich. in contrast to an earlier report (Yasuda et al. 2007). the damage recognition defect of XPC mutants becomes evident without preceding DDB2 down-regu lation. This view was confirmed by tes ting the nuclear dynamics of a repair-deficient W690S mutant with minimal DNA-binding affi ni ty (Bunick et ai, 2006; Maillard et al. 2007; Hoogstraten et al. 2008). In co njunction with the CFP fusion partner. this pathogen ic mutant is expressed in similar amounts as the wi ld-type control and also localizes to the nuclei. However, in th e XP-C fibroblas ts of thi s st udy. th e single W690S mut ati on causes > fiv e-fold redu cti on in th e reloca ti on to UV-damaged areas (Figure 2A; Suppl ementary Mov ie 2). These findin gs were confirm ed when another techn iq ue was used to infli ct geno toxic stress. th at is by UV-C irradiation (254 nm wavelength) th rough the pores of polycarbonate filte rs (Mone el 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). Oligo nucleotide-binding assays with XPC protein expressed

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in in sect ce ll s confi rmed that thi s W690S muta ti o n and th e corresponding alanine substitution (W690A) abrogate the interac tion with DNA (Figure 2B) .

The same analys is was ex t.end ed to furth er repair-defi cient XPC mutants targeting conserved aromatic residues (Maillard et al. 2007). A nearly complete loss of DNA b inding is co nferred by the F733A mutat ion . whereas the W531A and W542A substitutions are associated with more moderate defects (Figure 2B). When tes ted in CM 16093 fibroblasts as CFP fusions. the damage-dependent red ist ribution of these different mutants correlates closely with the respective DNA-binding properties . In fact , the W690S. W690A and F733A derivatives display a poor ab ility to concentrate at damaged sites . In contras t. the residua l DNA-binding activi ty of W531A and W542A leads to an intermediary level of accumulation in areas co ntaining UV photoproducts (Figure 2C). From this tight correspondence between DNA binding and nuclear red istribution . we concluded that the rapid relocation of XPC protei n to UV lesion sites reflects the intrinsic capacity of this sensor subu nit to detect DNA damage through direct interactions with the nucleic acid substrate.

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Figure 2 Dependence on intrinsic DNA-binding activity. (A) Represen tative image (in colour and black-and-white) showi ng the low residual accumulati on of the W690S mutant 6 min after irradiation . DNA lesions were counterstain ed by antibodies agains t CPOs. (8) DNA-binding acti vity determined by direct pu ll down. Wild-type (wI) XPC or mutants were expressed in S{9 cells as fu sion constructs with maltose-binding protein (MBP) . Ce ll Iysates con taining si mi lar amou nts of XPC protein (Mai llard e/ al . 2007) were incubated with a single-stranded l3S-mer oli gonucleo tide. Subsequent ly. racliolabell ed DNA molecules captured by XPC protein were separated from the free probes using anti -MBP antibodies linked to magnetic beads, and the rad ioactivity in each fract ion was Quan tified in a sci ntillation counter. DNA binding is represented as the percentage of radioactivit y immobilized by wt XPC protein after deduction of a background va lue determined with empty beads (n = 3). Error ba rs. standard dev iati on. (e) Co rrelation between DNA binding and the kineti cs of XPC accumulati on in XP-C cells (n = 7) . See legend to Figure 10 for details.

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Role of the transglutaminase-like domain As the transglutaminase- like region maps to the N-termi nal part of human XPC (Figure 3A), we generated N-terminal truncations (XPCI1 8-9~ O' XPC~27-9' I O and XPC607- 9>1O) to test how the TGD sequences contribute to DNA damage recogn i­tion in living cells. The posi tions US and 607 were selected for these truncations to allow for comparisons with an ear lier in vitro study monitoring the DNA-, Rad23B- and TFlIH­binding act ivity of XPC fragments (Uchida et ai, 2002). Another truncate (XPCI _~~5) was included as a negative cont ro l that lacks the ent ire C-termi nal half. The functionality of these constructs, co njugated to GFP at their C-terminus, was compared in a host-cell reactiva tion assay that has been developed to measure the cellular GGR activity (Carreau et ai, 1995). Briefl y, XP-C fibrob lasts were transfected with a dual luciferase reporter system along with an expression vector codi ng for full -length or truncated XPC fu sions. The reporter

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plasmid, which carries a Photinus luciferase gene, wa s da­maged by exposure to UV-C light and supplement ed with an undamaged vector tha t expresses the Renilla luciferase. GGR effic iency was assessed a ft er IS- h incubati ons by determ ini ng Photinus luciferase activit y in cell lysa tes, followed by normali za tion against the Renilla control.

The full -length protein (XPC I_940) and an XPC I18- 9>IO derivative, isolated by functiona l complementa tion (Legerski and Peterson, ] 992), were profici ent in correcting the repa ir defect of XP-C cells (Figure 3B), thus showing that gene reactivat ion is determined by the ability of the GGR pathway to excise offendi ng UV lesions. However, this repair activity could not be rescued by XPC427- 940 and XPCG07- 940 (Figure 3B), implying that the N-terminal part of XPC protein is essen ti al for the GGR reaction. All tes ted fragments were detect ed in transfected fibrob lasts in similar amounts as the full -length co ntrol or the funct ional XPC11 8- 940 derivative

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Figure 3 Mapping of the damage sensor domain to the C-terminal part of human XPC. (A) Scheme illustrat ing the position of the TCD sequences relati ve to the N-tenninal XPC truncates. (B) CCR activity determined by host-cell reactivation assay (n = 5; error bars, standard deviation). (e) Immunoblot ana lys is of XP-C cells transfected with expression vectors codi ng for the indicated fu sions. The protein level was probed using anti -CFP antibodies. C, endogenous CAPDH control. (D) Representative image showing that an XPC fragment lacking the C­terminus (XPC I 495) fa ils to accllmulate in laser-damaged areas. The XP-C fibrohla sts were fixed 6 min after irradiation . Bj W, black-and-white images showing that the tracks of DNA damage (upper panel) do not induce an accumu lation of truncated XPC fusions (lower panels). (E) Represen tative images (in colour and black and wh ite) showing that XPC427 9>\0 and XPC607 <)40 accumulate in damaged areas of XP-C fibrob lasts. The distribution of fluorescent fusion products was monito red 6m in after irrad iation. (F) Loca l increase of fluorescence resulting from the damage-induced red istri bu tion of full -length XPC or XPC607- 9,IO' A 10-llmline of UV photoproducts was generated across each nucleus and the resulting accumulation of fusion prote ins (after a 6-min incuba ti on) is plotted as a percentage of th e average fluorescence before irradiation (n = 7). Error bars, standard errors of the means. (G) Representative image illustrating that XPC607- 9'IO accumulates in foci generated by UV-C irradiation (lOOJ m 2) through the pores of po lycarbonate filters. The XP-C cell s were fi xed 15 min after treatment Jnd CPOs were detected by immunochemical stain ing. The posit ion of XPC6D7- 9,1O foci is indicated by the arrows.

(Figure 3C), indicating that their repa ir defici ency does not result from reduced expression or enhanced degradation.

Next, all GGR-deficient trunca tes were tested for th eir damage recognition proficiency in XP-C fibrobl as ts. Neither XPC I- 495 (Figure 3D) nor XPC I_718 (Supplementary Figure 4) were red istributed 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 XPC'1 27-9~0 retains the ability to concentrate in laser-in'a­diated areas (Figure 3E). Even mo re surprising was the observation that the smaller fragment XPC607- ? ,1O read ily accumulates at sites containing UV photolesions (Figure 3E). The quantifi ca tion of defin ed lO-~lm tracks showed that XPC607- 9'IO is only -30% less efficient than full -length XPC in relocating to damaged sites (Figure 3F) . Thus, a large N-term inal 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 sensi ng process itself. This conclusion is confirmed by the accumulation of XPC607 940 in UV-C foci generated by irra­diati on th rough the pores of polyca rbonat e filters (Figure 3G) .

Differential contribution of p-hairpin domains According to the Rad4 crysta l, three consecutive ~- hairpin

domains (BHD1, BHD2 and BHD3) mediate the interaction with damaged DNA (see Supplementary Figure I) . In the homologous XPC sequence, these structural elements range from residue 637 (start of BHDI) 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 XPC I_741 (comprising BHDl and BHD2) and XPC I_831 , which includes all three BHDs (Figure 4A). Again, the trunca tion position 741 was chosen to allow for comparisons with an earli er in vitro study (Uchida et ai, 2002) . The constructs were conjugated to GFP at their C-terminus and tested for th eir ability to initi ate the GGR reaction. [n the case of XPC 1 741, the repair funct ion is reduced to a background level observed with empty GFP vector (Figure 4B). However, the repo rter gene was reacti ­vated to -40% of control in the presence of XPC1 831, indicating that despite its C- terminal truncation, this large fragment reta ins 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 essen­tia lly the same GGR activity was induced by XPC I_766, that is by adding only 25 amino acids to XPC I_7'11 (Figure 4B). A comparison with the Rad4 orthologue indicates that these 25 amino acids (residues 742- 766) be long to an N-terminal extension of BHD3, which fold s into a ~- turn structure (see Figure 4A).

The UV-induced relocation of trunca ted XPC derivatives was tested in XP-C fibrobla sts ex pressing sim il ar low levels of each GFP construct (Supplementary Figure 5) . Consistent with its distinctive functionality in the GGR assay, we ob­served that XPC 1 766 accumulates more effectively than XPC I_7'11 to the lO-~lm tracks of photolesions generated by laser irradiation (Figure 4C). An unequivocal pattern of XPC I- 766 accumulation along the radiat ion tracks was also recorded in XP-E fibrobl as ts, that is ill the absence of UV-DDB activity (Figure 40). 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 show ing that th e dam age-s pecific accumulation of XPC trun­cates as well as the effect of the ~- turn structure takes place in the absence of DDB2 protein . A clear difference between XPC I- 766 and XPC I_7'1 1 was reprodu ced when foci of flu or­escence were monitored after UV-C irradiation through the pores of polycarbonate filters (Figure 4F) . Taken together, this efficien t redistribution of XPC I- 766, irrespective of the cell type or technique used to infli ct DNA damage, establishes for th e fi rst time th at most of BII D3 is not required for the initi al damage-sensing process.

The p-turn structure enhances XPC dynamics The GGR and re location assays of Figure 4 revealed a striking difference between XPC I_741 and XPC I- 766 because of the 25-amino-acid ~-turn extension. To analyse the function of this ~-turn structure, we compared the nuclear mobility of differ­ent truncates using flu orescence recovery after pho tobl each­ing (FRAP; Houtsmuller and Vermeulen, 2001). [n cells that express similarly low levels of GFP fu sion constructs, a nuclea r area of 4 ~lm 2 was bleached and, subsequently, protein movements were tested by recording the recovery of local fluorescence, which is dependent on the ab ility of the GFP fusions to move rapidly within the nuclea r compartment.

The control experiment of Figure SA shows how, in the absence of a fusion partner, the GFP moiety moves freely inside the cells. Instead, the nucl ear mobility of full -length XPC-GFP is restrained by its larger size and propensity to undergo macromolecular interactions, as reported earlier (Hoogstraten el ai, 2008). Surprisingly, in a direct comparison between XPC I_74I , XPC I_766 and XPC I- 831, a larger size correlated with increased nuclear mobility (Figure 5B) . The FRAP curves obtained with these different truncates were used to ca lcu late effec tive diffu sion coeffi cients (Doff; Supplementary Tab le I). It was un expected to find that. in undamaged cells, XPC I_766 (containing BHDl, BHD2 and the ~-turn structure) and XPC I- 831 (containing all three BHDs) move more rapidly inside the nucleus (Dorr= 0.44 and 0.49 j.1m2 S- I, respectively) than the shorter polypeptide XPC I_74 1 lacking the ~- turn (Dcrr = 0.34j.1m2 S- I) . We con­cluded that these C-termina l truncations disclose the exis­tence of a dynamic interface, residing within the ~-turn

structure, which enhances the const itutive nuclear mobility of XPC protein in the absence of genotoxic stress.

Subsequently, the FRAP approach was used to assess the corres ponding responses to UV-C irradiation. [n accord with its poo r accumulation along DNA damage tracks (Figure 4C) , the mobility of XPC I_74 1 is on ly margina lly affected by the induction of photo les ions (Figure 5C) . [n contrast, the diffu­sion rates of XPC I- 766 (Figure 5D) and XPC I- 831 (Figure 5E) , whil:h accuJllulat e in UV lesion tracks, are sign ifican tly reduced (the respective Dcrr values are listed in Supplementary Table I) . In the case of XPC I_83 I, the induc­tion of DNA damage had a two-fold effect. First, UV lesions decreased the initial rate of protein diffusion exactly as observed with XPC I_76G . Second, similar to the response of full -length XPC (Hoogstraten el ai, 2008), th e overall fluor­escence recovery is less co mp lete on UV irradiation (Figure 5E), indicat ing that a fra ction of XPC I- 83 1 is immo­bi lized in a damage-s pecific mann er. In summary, th ese

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Figu re 4 BHD3 is not required for DNA damage detect ion. (A) Scheme illustra ting the location of BHD and ~-turn sequences relative to the C­terminal XPC truncates of this study. (B) GGR ac ti vity determined by host-ce ll reactivation assay in XP-C fibroblasts (n = 5; error bars, standard deviation). (e) Representative images (ta ken 6 min after irradiation) comparing the accumulation of XPC' _7GG and XPC' _74 ' at damaged sites. In the black-and-white representation, the li near irradia tion tracks are surrounded by a dashed rectangle. (D) Representati ve image illustrating the accumulation of XPC' _7GG along UV radiation tracks generated in XP-E fib roblasts devoid of UV-DDB act ivity. (E) The local increase in fl uorescence, because of damage- induced red istribution s of XPC truncates, was measu red in XP-C and XP-E cells and ploUed as the percen tages of wt control as outlined in Figure 10 (11 = 5; error bars, standard errors of the mean). (F) XPC' _7(,6 is also more effi cient than XPC '_7<1 ' in accuillu lating in DNA damage foci generated by UV-C irradiation through th e pores o1'polycarbonate fi lt ers (see Figure 3G for detail s). XPC' _7('(' [top) and XPC' _74' fo ci (bollom) arc indicated by the arrows.

protein mobility studies show that BHD3 induces the forma­tion of a stable nucleoprotein complex once the lesion has been de tected .

Antagonistic composition of the dynamic sensor domain The truncation studi es of Figures 4 and 5 suggested that residues 607-766 may be suffici ent to find lesion si tes in the ge nome. Thi s hypo th es is wa s co nfirmed by exp ress ing short protein fragment s in XP-C fibrob las ts (Figure 6A) . In th e case

of XPC607- 766 (consisting of BHD1/BHD2 and the p-turn stru ctu re). a clea r pa ttern of damage-induced accumulation was detected immed iately after laser irrad iation (Figlll'e 6B). In co ntras t, XPCGQ7_74, (lacking the p-turn) fai led to accumu­late in the tracks of UV lesions. XPC607- 741 was unab le to relocate to damaged areas regardless of whether the GFP moiety was placed at the C- (Figlll'e 6C) or at the N- terminus (data not shown) . These res ults support the conclusion that XPC607- 766 di sp lays a minimal senso r surface with damage recognit ion activi ty in liv ing human cells.

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TIme(s) Time (s)

0 1.2 E 1.2 Immobile fraction Retarded diffusion

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0.2 • 1- 766(+ UV) 0.2 • 1- 831 (+ UV)

0 0 -5 0 5 10 15 20 25 30 35 - 5 0 5 10 15 20 25 30 35 40

TIme(s) Time (s)

Figure 5 Identifica tio n of a dynamic co re and two-stage damage recognition. (A) Principle of FRAP ana lysis. An area of 4 ~m2 in the nuclei of XP-C fi broblasts ex pressing a particular GFP construct is bleached with a 488-nm wavelength lase r. Th e kineti cs and ex tent of flu orescence 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. Th e difference between XPC , ~66 and XPC ,_83 , is not signifi cant. (e) The nucl ear mobility of XPC , 741 remains unaffected by UV-C irradiation at a dose of lOJ m- 2 (n = 12) . (0) The initial diffusion of XPC ,- 766 is reduced by UV light (IOJ m- 2• n = 12). reflecting transient molecu lar interactions during stage 1 of the damage recognition process. (E) A fra ction of XPC ,_83 , is stably immobi lized after UV irradiation (lOJ cm 2. n = 12). refl ecting stage 2 of the damage recognition process.

The fragments XPC607 74 1. X PCG07 766 and X PC607 83 1 have been isolated to assess their DNA-binding properties using 13s-mer DNA substrates. All three fragments were expressed and purified as so luble polypeptides without any signs of aggregation or precipitation that would be indicative of defective protein folding (Figure 60). We compared their binding with three different DNA conformations: homodu­plexes. heteroduplexes with three contiguous base mis­matches or single-s tranded oligo nucleotides of the same length . Although XPC607 74 1 (containing BHDI and BHD2) is un ab le to find DNA lesions in living ce ll s. thi s fra gmenl displays a preference for unpaired bases embedded in double­stranded DNA. In fact. XPC607- 741 binds with higher affinity to heteroduplex DNA rela tive to homoduplexes or single­stranded oligonucleotides (Fi gure 6E).

A simi lar preference for hetero- over homoduplexes is retained by XPC607- 766 . which includes both BHDl / BHD2 and the ~- turn structure (Figure 6F). thus supporting the notion that this minimal senso r is active in living cells by sea rching for destabilized base pairs. A side-by-side com­parison of dose-dependent DNA-binding ac tivities with XPCG07- 74 1 and XPCG07- 76G showed that the ~- turn structure leads to a substantial reduction in nucleic acid binding

(Figure 6F) . In particul ar. we found that the aSSOCiatIOn constant representing the interaction with homoduplex DNA decreases nearly lO-fold from 2.7 x 109 M- ' for XPC607_ 74 1 to 2.8 X 108 M I for XPC607- 766' This drop in binding to the native double helix implies that the enhanced nuclea r mobility conferred by amino acids 742-766 (Figure sB) results from an antagonistic DNA-repulsive effect.

Finally. to tes t the contribution of BHD3, the same 13s-mer substrates were used to monitor the DNA-binding propert ies of a longer fragm ent (XPC607 83 1) comprising all three BHDs. Figure 6G shows that th is larger fragment has the character­istics of a single-s tranded DNA-binding protein, indicating that BHD3 itself confers a pronounced selectivity for single­stranded conformations. The characteristic DNA-binding pro­file of thi s larger fragment X P C 607- 83 1 corresponds roughly to that detected when identical reactions were carried out with full -length XPC protein (Supp lementary Figure 6).

Design of an XPC mutant with retarded nuclear mobility We postulated that part of the DNA-repulsive action med iated by the ~- turn structure (Figure 6F) arises from negatively charged side chains that clash with the phosphates of the nucleic acid backbone. Thi s hypothesis predicts that it should

2393

2394

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Figure 6 Antagonist ic composition of the minimal damage senso r. (A) Immunoblot analys is of XP-C fibroblasts after transfection with vectors coding for the indicated XPC-GFP sequences. The expression was probed using anti -GFP antibodies. NTC, non-transfected cell s; GFP, cells transfected with the GFP sequence alone; G, GAPDH co ntrol. (B) Representative image illustrating that fragment XPC('n7 7('(' readily accumutates in damaged areas containing DNA photolesions. The distribution of fluorescent fu sion products was monitored Imin after laser irradiation. B/W, black-and-white image. (e) XPC607- 7'" is unable to recognize UV les ions in li ving cells. Fibroblasts were subjected to fi xa tion 1 min after irradiation and (6-4) photoproducts were detected by immunochemi ca l staining. B/ W, black-a nd-white im ages showing that UV lesions (upper panel) did not lead to accumulation of the fusion protein (lower panel). (0) Gel electrophoretic ana lysis of purifi ed XPC fragments expressed as glutathione-S-transferase (GST) fusions in E. coli or with a histidine (His) tag in S(9 cells. (E) DNA binding of XPC607_74 1 determined by oligonucleotide capture. The indica ted concentrations of XPC-GST fragments were incubated with radiolabe lled 135-mer oligonucleo tides (3-misrnatch heteroduplexes, homoduplexes and single strands) . Thereafter, DNA molecules immobilized by XPC fragments were sepa rated from the free oligo nucleot ides using glutathionc-Sepharose beads. followed by th e quantifica tion of radi oacti vity associated with the beads. DNA binding is represented as the percentage of total input radioacti vity captured by XPC fragmen ts after deduction of a background value determined with empty beads (n = 6; error bars, standard deviation). (F) DNA-binding profi le of th e minim al damage sensor (XPC607 -766) determined as described in the legend to Figure 6E. (G) Con tribution of BHD3 . The DNA-bind ing profi le of XPC607 83 1 was determined as outlined in the legend to Figure 6E, except that pu ll downs were performed with Ni-NTA agarose beads.

be possibl e to mitigate this DNA-repellent effect by replacing negatively charged am ino acids with positively charged ana­logues. We identi fied a glutamat e moi ety at positi on 755 of the human ~- turn motif that is conserved among higher eukaryotes (Figure 7 A) and inverted the charge of this particular side chain by substitution with lysine.

The consequence of this engineered charge inversion was first tes ted by co mpal'ing th e interac ti on with nat ive double­stranded DNA in biochemical assays. For that purpose, the lys ine subst itution was introduced into XPCG07- 76G, thus generating a mutated fragm ent of 160 amino acids (E755K607- 766) that , simila r to its wild-type counterpart (XPC607- 766), is amenab le to ex pression and pu rificat ion as a soluble polypeptide. DNA homoduplexes of 135 base pairs were used to determine the DNA-binding capacity of this mutated fragment in relation to the wild-type control. As illustrated in the comparison of Figure 7B, the E75SK muta­tion was able to partially reverse the drop in DNA binding

resulting from the presence of the ~- turn structure in XPC607- 766 ' Binding sa turation studies with homoduplex DNA indicates that the associat ion co nstant increased from 2.8 x 108 M- I for XPC607- 766 containing the wild-type se­quence (determined in the earlier section) to 7.4 x 108 M- 1

for the E7SSK607- 766 derivative, which carries the single charge inversion.

These fi ndings led us to generate a mutant GFP fu s ion co nstru ct to co nfirm that the effect of the ~-turn structure in enhancing the XPC dynamics, observed with truncated deri ­vatives (Figure 5B), is retained in the full -length protein context. Unlike other repa ir-defective XPC mutants (W531A, W542A, W690A, W690S and F733A), all of which display a higher nuclear mobility than the wi ld-type control (I-1oogstraten et ai, 2008 and data not shown), the novel E7SSI< mutant is characterized by a strikingly reduced nucle­ar mob ility (Fi gure 7C) acco mpani ed by a signifi ca nt GGR defect (Figure 70) . Collectively, these effects induced by a

A

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.:l:- *" .:f:> i:J'?-~~':) ~'OC8 ~'OC8 «t Figure 7 Analysis of the dynamic interface by site-directed mutagenesis. (A) Identification of a conserved glutama te (arrow) in the p-turn motif of higher eukaryotes . Th is residue is not conserved in the Rad4 sequence, suggesting that the yeast orlhologue may have diffe rent dynami c propert ies. (B) A single E7SSK mutation reduces the DNA-repellent effect of the p-tUI'll structure. The associat ion of XPC('1I7 7''' , XPC(,t17 7(,(, and E7SSK('07 7(,(, with homoduplex DNA was compared at a polypeptide concentration of ISO nM, as outlined in the legend to Figure 6E. DNA binding is represented as the percentage of total input radioacti vity captured by XPC fragments (n = 6; error bars, standard devia tion). A control reaction was carried out with empty beads. (e) FRAP analys is showing that, in undamaged cells, the nuclear mobi lity of the full -length E7SSK mutant is retarded relati ve to the wt control (n = 12; error bars, standard error of the mean) . (D) !-Ios t·cell react iva tion assay showi ng that the E7SSK mutation co nfers a signi fica nt GGR defect. All results were corrected for the background activity in XP·C ce lls transfected with the GFP vector (n = 5; error bars, standard dev iation) .

single site-d irected mu tat ion co nfirm that th e dynamic prop­erties of its minimal sensor surface, co nferred by the ~-turn

struclure, are crilica l for the abi lity of human XPC protein to act as a sensor of DNA damage.

Discussion

We elucidated the mechanism by which XPC protein scruti ­nizes DNA quality in li ving cells. The most outstanding finding is th e identifica ti on of a two-s tage di scrimi nati on process triggered by a dyna mic sensor interface that detects DNA damage without the involve ment of a pro minent DNA­binding doma in (BHD3). wh ich was thought to represen t the primary lesion recognition module on the basis of the Rad4 crys tal structure (Min and Pavletich, 2007). The newly iden­tified sensor interface serves to rap idly scree n th e double helix for the presence of unpaired bases, thus localizi ng damaged target sites th at are amenable to the subsequent installat ion of an ulti mate repair-initiat ing co mplex.

Dynamic molecular dialogue with the DNA double helix Accord ing to the aforement ioned Rad4 structure, the TGD region cooperates with BHDI to associa te with a po rtion of double-s tranded DNA fl anking the lesio n (see Suppl ementary Figure 1) . However, we observed that a large N-terminal segment (6S % of the human sequence including most TGD sequences) has a stimulatory ro le, but is not directly requi red for the relocation of XPC protein to focus on DNA lesions (Figure 3). [n the absence of this TGD segment , a stro ng interaction with the normal duplex is nevertheless med iated

by the earlier described (Uchida et ai, 2002) minimal DNA­binding fragment XPC607- 741 , which co nsists of BHDI and BHD2 (Figure 6E) . Instead, a longer fragment covering all three BHDs displays a co mparably low affi nil y for the norma l duplex (Figure 6G), indica ting that the double-stranded DNA­binding acti vity of BHDl/BHD2 is opposed by the neighbour­ing BHD3 sequence. The furth er dissection of this critical XPC region revealed that a short ~-turn extension of BHD3 is suffici ent to mediate in par t such an antagonis tic effect (Figure 6F).

Several observat ions in li ving cells support the not ion that the add ition of this ~-turn extension co nveys a true gain of function rather than causing the destabilizat ion of adjacent struct ural elements in the respective XPC constructs. First, XPC I - 76(, and XPC 1_R3 1 display a residual GGR function that is missing in the case of XPC I _ 74I, which lacks the ~- turn

structure (Figure 48). The fac t that XPCI- 766 and XPC I_831 exert a similarly low co mplementing act ivity is likely because of the absenc'e of a t least so me co mponents of the TFII H­recruiting doma in in th eir C- terminal region (Uchida el ai, 2002). Second, a side-by-side comparison of the same C­terminal trunca tes shows that the enhanced nucl ear mobility co nferred by the ~- turn structure (Figure SB) co rrela tes with a morc efficient relocati on 1.0 UV lesions (Figure 4E). Thi rd, the nucl ea r mobility of XPC I- 766, but not XPC I _ 7•I I , is retarded by UV damage (Figure SC and D) , confirming that the fo rmer detects DNA lesions more effecti vely. Fourth , in living cells, the dam age-induced accumul ati on of an earli er defined mini­mal DNA-binding fragment (XPC607- 741) is stri ctly dependent on the presence of the ~- turn structure (Figure 6B). Finally,

2395

2396

the critical role of this dynamic ~-turn subdomain is sup­ported by a site-d irec ted E755K substitution tha t reverts in part its DNA-repell ent ac tion . The in creased a ffinity of thi s novel mutant for the native double helix results in decreased nuclear mobility and markedly reduced repair activity (Figure 7). According to the Rad4 structure, the critical position 755 maps to an amino-acid sequence that is in close contact with th e DNA substrate (Min and Pavletich, 2007). Thus, our find ings indicate that the ~-turn structure displays both DNA-attractive and DNA-repulsive forces that dictate the dynamic interplay with duplex DNA such that , in the full genome context , this subdomain facilitates damage recognition by providing sufficient mobility to the XPC molecules searching for les ions.

Identification of a transient recognition intermediate On binding to damaged substrates, XPC protein indu ces loca l DNA melting and kinking (Evans et ai, 1997; Janicijevic et ai , 2003; Mocquet et ai, 2007). A structural basis for these rearrangements is again provided by the Rad4 crystal , in which the ~-hairpin of BHD3 is inserted through the DNA dupl ex, ca using two base pa irs to enti rely flip out of th e double helix (see Supplementary Figure 1) . In view of these feat ures of the Rad4-DNA co mpl ex, it was unexpected to find that most of BHD3 including the protruding ~-hairpin is actually not necessary to sense DNA damage in living cells. In fact, an XPC fragment that conta ins the ~- turn structure, but is devoid of the remaining BHD3 sequence because of a truncat ion at position 766 (XPC I 766), accumulates in UV foci with remarkabl e efficiency (- 60% of the full -length control ; Figure 4E), but without forming stable nucleoprotein com­plexes (Figure 50) . Similar to the W690S mutant, this trun­cated XPC I - 766 derivative is even able to induce GGR activity (Figure 4B), although to moderate levels that are not suffi ­cient to complement the repair defect of XP-C cells. A damage-specific accumulation of XPC I _ 766 was also detected in DDB2-deficient XP-E fibrobla sts (Fi gure 40 and E) a nd V79 hamster cells (data not shown), thus excluding that the BHD3-independent relocation occurs in an indirect manner by association with UV-DDB. Finally, the conclusion that XPC protein form s a transient damage recognition intermediate withou t the in volvement of BHD3 is supported by th e finding that a small fragment (XPC607- 766) consisting only of BHDl/ BHD2 and the ~- turn structure (together - 15 % of the human XPC seq uence) s till function s as a cellular DNA damage sensor (Figure 6B). This minimal sensor surface displays a binding preference for duplexes containing non-hydrogen­bonded bases, a generic feature of damaged DNA, and hence functions as a mol ecular ca liper of thermodynami c base-pair s tability.

A two-stage quality-control inspection Although the BHD3 segment (residues 767-831) a nd its ~- ha irpi n are not required to attract XPC protein to les ion s ites, thi s additiona l domain favours the subsequent forma­tion of stable nucleoprotein complexes, reSUlting in an im­mobile fraction of XPC protein in response to DNA damage (Figure 5E) . The biochem ica l ana lys is of purified fragm e nts shows that, unlike the BHD1/B I-lD2/~-turn minimal sensor, which displays a preference for duplexes with unpaired bases, BHD3 confers an exq uis ite selectivity for single­s tranded DNA co nforma ti ons (Figure 6G). [n conjunction

with th e ea rli er mention ed Rad4 stru cture, th ese findin gs indica te that BHD3 does not participate in the early and tra nsie nt recognition intermediate, but, ins tead, facilitates the subsequent stabiliza tion of a repa ir-initiat ing complex using its Si ngle-s tranded DNA-binding activity to encircle the undamaged strand across les ion sites.

To co nclud e, thi s is th e first report providing evidence for a two-stage discrimination mechanism by w hich XPC protein carries out its versatile recognition function (Figure 8). This two-stage process obviates th e diffi culty of probing every genomic base pair for its susceptibility to undergo a BHD3-mediated ~- hairpin insertion. Instead, the energetica lly less demanding search conducted by the dynamic BHDl/BHD2/ ~- turn interface is likely to precede more extensive BHD3-dependent s truct ural adjustments. This initia l search leads to the detection of non-hydrogen-bonded residues tha t are more prone than native base pa irs to be flipp ed out of the double helix and , hence, become an interaction partner for the single-s tranded DNA-binding ac tivi ty of BHD3. A crit ical step of this two-stage quality-control process is the transition from an ini tially labile sensor intermediate to the more stable ultimate recognition complex. Two co nstitutive interaction partners of XPC protein , Rad23B and centrin 2 , are thought to exert an accessory function not only by inhibiting XPC degradation, but also by st imulating its DNA-binding activity (Ng et ai, 2003; Xie et ai, 2004; Nishi et ai, 2005). Such an a uxiliary role is supported for Rad23B by the observation that XPC607- 94 0, a fragment that fails to associate with Rad23B (Uchida et ai, 2002), has a reduced DNA damage recognition capacity in living cells (Figure 3F). In addition, the two-step discrimination process identifi ed in this study raises the possibility that Rad23B, centrin 2 or other binding partners may facilitate the installat ion of an ultimate XPC-DNA com­plex by lowering the energetic cost of critical nucleoprotein rea rrangements required for th e fin al ~-hairpin insertion.

B

I I I I I I I I I

Uttimate 1 recognition

complex

Figure 8 Two-stage detection of DNA lesions by XPC protein. Model depicting the switch from a dynamic damage sensor inter­mediate to the ultimate recognition complex. CA) This study iden­tifi es a minimal senso r interface that rapidly scrutini zes base-pa ir integrit y. This initial search, carried out by BHDl/BI-tD2 in con­junction with the ~ - turn structure, results in the formation of a labile nucleoprotein intermediate. (B) The single-s tranded DNA­binding activity of BHD3 promotes the subsequent transition to a stable recognition complex by capturing ex truded nucleo tides in the undamaged strand.

Materials and methods

XPC constructs The human XPC complemen tary DNA was cloned into pEGFP-N3 (Clontech) using the restric ti ons enzy mes Kpnl and XmaI. The same enzymes were used to generate the truncated XPC fragments. Primers for the insert ion of restrictions sites and s ite-directed mutagenesis (QuickChange, Stratagene) are li sted in the Supple­mentary Tab le II. All clones were sequenced (Microsynth) to exclude accidenta l mutations.

Cell culture Simian viru s 40-transformed human XP-C fibroblasts (GM 16093J and untrans fonn ed XP-E fibrobla sts (GM02415), derived from patients XPI4BR and XP2RO, respectively, were purchased from the Coriell Institute for Medical Research (Camden, New Jersey, USA). The XP-C cells carry a homozygous C--.T transi ti on at position 2152 of the XPC sequence (Chavanne et ai, 2000). The GM02415 cell s carry a G--.A trans ition in the DDB2 sequence generating an inactive R273H mutant that is not expressed to detectable levels (Nichols et ai, 2000; Itoh et ai , 2001 ). These fibrobla sts , as we ll as V79 hamster cells deficient in UV-DDB activity (Tang et ai, 2000) were cultured in Dulbecco's modifi ed Eagle's medium (DMEM; Gibco), supplemented with 10% fetal ca lf serum (FCS), penicillin G (100 units mr') and streptomycin (100 ,Ig mrl). The cells were maintained at 37°C in a humidified incubator containing 5% CO2 •

Transfections One day before transfection, 60() 000 cells were seeded into 6-well plates conta ining glass cover slips. At a confluence of 90-95 %, the cells were transfected with l,rg XPC-pEGFP-N3 (or truncated constructs) using 4,t1 FuGENE 1-10 reagent (Roche) and incubated for another 18 h. Expression of XPC polypeptides was monitored by western blotting (Maillard et ai, 2007).

High-resolution DNA damage induction The growth medium was replaced by phenol red-free DMEM (Gibco) supplemented with 10% FCS and 25mM HEPES (pH 7.2). Single cells were irradiated with a femtosecond fibre laser (Trautlein el ai, 2008) coupled to a confocal microscope (LSM Pascal, Zeiss) tha t generates pulses of 775 nm (duration 230 fs, repetition rate 107 MHz). The peak power density a t the focal plane was 350 GW cm 2 and the pixel dwell time was 44.2 ms. Nuclei were irradiated along a single track or two intersecting lines . The area of each irradiation track was < 10 ,un2 and its volume < 20 ,UTI" .

By multiphoton excitation, three photons of low energy (775 nm wavelength) cause DNA les ions normally produced by the absorption of a s ingle photon of higher energy (equivalent to 258 nm wavelength). Irrad iation in the near-infrared range induces CPOs, (6-4) photoproducts and oxidative les ions (Lan el al, 2004; Dinant et ai, 2007). In a n earli er report (Meldrum et al, 2003), it has been calculated that three-photon irrad iation with a peak power density of 350GWcm- 2 generates ~ 7000 UV lesions in each treated ce ll. Taking in to accoun t our sli gh tl y modifi ed parameters, we calcul a ted that in thi s study, the same power density produced ~ 5000 UV les ions along each linear 10-,lm track.

Image analysis Fluorescence meas urements were carried out through a x 40 oil immersion objective lens with a numerical aperture of 1.4 (EC-Plan­Neo-Fluar, Ze iss) using an Ar I source (488 nm). The selec ted parameters, including laser power and magnification factor, were kept constant throughout a ll experiments. To monitor the distribu ­tion of fluorescent fu sions, a t least 60 images were taken for up to 10 min after irradiation and ana lysed using the ImageJ software (http://rsb.info.nih.gov /ij). An initi al-control image was taken immediately before damage induction. Signals were corrected for bleaching (http://www.embl-heidelberg.de/eamnet/html/body_ bleach_correction .html) and cell movements (http://bigwww. epfl. ch/th evenaz/s tackreg). For eve ry time point, the average flu o rescence intensiti es were measured in the a rea of accumulation and, as a background reference, in a neighbouring area of ident ica l s ize. Finally, the background-corrected values were normalized to the mean intens ity of the same nuclear region before irrad iation.

Induction of UV foci After removal of the culture medium, the cell s were rinsed w ith phosphate-buffe red sa line (PBS) , covered by a polycarbonate filter (Millipore) with 5-pm pores and irrad iated using a UV-C source (254 nm, 100J m- 2

). Subsequently, the filter was removed and the cells were returned to complete OM EM for ISmin at 37"C before para formaldehyde fixation.

Immunocytochemistry All wash steps and incubations were performed in PBS. At the indica ted times afte r irrad ia ti on, cell s were washed and fix ed for IS min at room tempera ture using 4% (v/v) paraformaldehyde. The cells were then permeabilized twice with 0.1 % (v/v) TWEEN 20 for 10 min and DNA was denatured with 0.07 M NaOI-l for 8 min. Subsequently, the samples were washed fiv e times with 0.1 % TWEEN 20 and incubated (30 min at 37°C) with 20% FCS to inhibit unspecifi c binding. The samples were incubated (I h at 37°C in 5% FCS) with primary a ntibodies (MBL Internationa l Corporation) directed against CPOs (TDM-2, dilution 1:3000) or (6-4) photo­products (64M-2, dilution 1:1000). Next, the samples were washed with 0.1 % TWEEN 20, blocked twice for 10 min with 20% FCS and trea ted with Alexa Fluor 546 dye-conjugated secondary antibodies (Invitrogen, dilution 1:400) for 30 min a t 37°C. After washing with 0.1 % TWEEN 20, the nuclei were stained for 10 min with I-Ioechst dye 332S8 (200 ng mrl). Finally, the samples were washed three times and a nalysed using an oil immersion objective.

GGR assay Triplicate samples of XP-C fibroblasts, at a con flu ence of 90-95%, were transfected in a 6-well plate. The total amount of plasmid DNA (I,lg) included 0.45,rg pGL3 (UV irradiated at 1000J m - 2

, coding for Photinus luciferase), 0.05 ,Ig phRL-TK (unirradiated, coding for Renilla luciferase) and 0.5,rg of XPC-pEGFP expression vector. After 4 h, the transfection mixture was replaced by complete culture medium. After another 18 h, the cells were disrupted in 500,r1 Passive Lysis Buffer (Promega) . The Iysa tes were cleared by centrifugation and the ratio of Photinus and Renilla luciferase activity was determined in a Dynex microtiter luminometer using the Dual -Luciferase assay system (Promega).

FRAP analysis Protein mobility was ana lysed at high time resolution using a Leica TCS SPS confocal microscope equipped with an Ar + laser (488 nm, not inducing DNA lesions) and a x 60 oil immersion lens (numerical aperture of 1.4). The assays were performed in a controlled environment at 37°C and CO2 supply of 5% . A region of interest (ROO covering 4 pm2 was photobleached for 2.3 sat 100% laser intensity. Fluorescence recovery within the ROI was monitored 200 times using lIS-ms intervals followed by 30 frames at 250-ms a nd 10 fra mes at 500 ms. Simultaneously, a reference ROI of the same s ize was measured for each time point to correct for overall bleaching. All data were normalized to the prebleach intensity and the effective diffusion model (Sprague el ai, 2004) was used to es tilllJt e diffu sion coeffic ien ts (see Suppl ementa ry Table 1].

DNA-binding assays Full-length MBP-XPC fusions were expressed in Sj9 cells (Maillard et ai, 2007). Insect ce ll Iysates (5 - 20,r1) were incubated with 32p_ labelled 13S-mer oligonucleotides (4 nM) in 200 pI buffer A (25 mM Tris- HCI , pH 7.5, 0.3 M NaCI , 10% glycerol, 0.01 % Triton X-IOO, 0.2S mM phenylmethane sulfonyl fluorid e and 1 mM EDTAJ. After I h at 4°C, the reaction mixtures were supplemented with monoclonal an tibodies against MBP linked to paramagnetic beads (0 .2 mg, New England BioLabs). After another 2 h a t 4°C, the beads were washed four times with 200 ,t1 buffer A and the oligonucleo­tides associated with pa ramagnetic beads were quantifi ed by liquid scintill at ion counting. All values were corrected for the background radioactivity reS Ulting from unspecific binding to empty beads. The amount of immobilized XPC protein was con trolled by denaturing gel electrophoresis.

GST-XPC607_74 1> GST-XPC607_ 766 and GST-K7S5E607 _766 (ex­pressed in Escherichia coli) as well as His-XPC607_831 (expressed in S{9 cell s) were purified as described (Uchida et ai , 2002). The indicated concentrations of XPC fragments were incubated with radiolabelled 13S-mer oligonucleotides (4nM) in 200,r1 buffer B (2S mM Tris- HC\, pH 7.5, O.IS M NaCI, 10% glycero l, 0.01 % Triton X- IOO , 0. 25 mM ph enyl methane sulfonyl flu oride and ImM EDTA).

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2398

After 1 h at 4°C, the reaction mixtures were supplemented with glutath ione-Sepharose (10 ,tl , Ame rsham) or Ni-NTA agarose beads (10 ,II, Qiagen) . After anot her 1 h at 4°C, the beads were was hed twice with 200 ,tl buffer B and the immobili zed oligonucleotides were quantified by liq uid scintill a ti on count ing. All va lues we re corrected for the background rad ioactivity resulting frolll unspecific binding to empty beads. lb es tima te binding constants, the data frolll sa turation experiments (50- 250 nM pro tein) were subjected to Scatchard a nalysis by p lotting the ratio of bound a nd free XPC fragments as a function of the fraction of bound protein (Husain and Sancar, 1987). The double-s tranded homoduplex or hetero­duplex probes were obta ined by hybridi zat ion of complementary 135-mers in SO mM Tris- HCI (pH 7.4). 10 mM MgCl, and 1 mM dithiothreito l. Equa l amounts of each oligo nucleot ide were hea ted a t 95°C for 10 min followed by slow cooling (3 h at 25°C).

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