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Distinct Roles for the XPB/p52 and XPD/p44 Subcomplexes of TFIIH in Damaged DNA Opening during Nucleotide Excision Repair

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Distinct Roles for the XPB/p52 and XPD/p44 Subcomplexes of TFIIH in Damaged DNA Opening during Nucleotide Excision Repair
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  Molecular Cell  Article Distinct Roles for the XPB/p52 and XPD/p44Subcomplexes of TFIIH in Damaged DNA Opening during Nucleotide Excision Repair Fre´ de´ ric Coin, 1, * Valentyn Oksenych, 1 and Jean-Marc Egly 1, * 1 Institut de Ge´ ne´ tique et de Biologie Mole´ culaire et Cellulaire, CNRS (UMR7104)/INSERM (U596)/ULP, BP 163,67404 Illkirch Cedex, C.U. Strasbourg, France*Correspondence: fredr@igbmc.u-strasbg.fr (F.C.), egly@igbmc.u-strasbg.fr (J.-M.E.) DOI 10.1016/j.molcel.2007.03.009 SUMMARY  Mutations in XPB, an essential subunit of thetranscription/repairfactorTFIIH,leadtonucleo-tide excision repair (NER) defects and xero-derma pigmentosum (XP). The role of XPB inNER and the molecular mechanisms resultinginXParepoorlyunderstood.Here,weshowthatthe p52 subunit of TFIIH interacts with XPB andstimulatesitsATPaseactivity.AmutationfoundamongXP-Bpatients(F99S)weakensthisinter-action and the resulting ATPase stimulation,thereby explaining the defect in the damagedDNA opening. We next found that mutations inthe helicase motifs III (T469A) and VI (Q638A)that inhibit XPB helicase activity preserve theNER function of TFIIH. Our results suggesta mechanism in which the helicase activity of  XPB is not used for the opening and repair of damaged DNA, which is instead only drivenby its ATPase activity, in combination with thehelicase activity of XPD. INTRODUCTION The human transcription/repair factor IIH (TFIIH) consistsof ten subunits. XPB, XPD, p62, p52, p44, p34, and p8/ TTDA form the core complex, while cdk7, MAT1, and cy-clin H form the cdk-activating kinase (CAK) subcomplex,linked to the core via XPD. Hereditary mutations in eitherXPB, XPD, or p8/TTDA yield the xeroderma pigmentosum(XP), XP combined with Cockayne syndrome (XP/CS), ortrichothiodystrophy (TTD) syndromes ( Lehmann, 2003;Giglia-Mari et al., 2004; Oh et al., 2006 ). These diseasesexhibit a broad spectrum of clinical features includingphotosensitivity of the skin due to defects in nucleotideexcision repair (NER) ( Lehmann, 2003 ). NER is part of a cellular defense system that protects genome integrityby removing a wide diversity of helix-distorting DNA le-sions induced by ultraviolet (UV) light and bulky chemicaladducts. The removal of lesions requires their recognitionby the repair factor XPC-HR23b and the subsequent un-winding of the DNA duplex by TFIIH. The single-strandedstructure is then stabilized by XPA and RPA, and the mar-gins of the resulting DNA bubble are recognized by XPGand ERCC1-XPF, thereby generating 3 0 and 5 0 incisionsrelative to the damage ( O’Donnovan et al., 1994; Sijberset al., 1996 ).DNA helicases are motor proteins that can transientlycatalyze the unwinding of the stable duplex DNA mole-cules using NTP hydrolysis as the source of energy. Theyare characterized by seven ‘‘helicase motifs,’’ constitutedof conserved amino acid sequences ( Tuteja and Tuteja,2004 ). It was always hypothesized that XPB and XPDhelicase subunits of TFIIH supply opposite unwinding ca-pacities required for local helix opening to form the openDNA intermediates in NER ( Bootsma and Hoeijmakers,1993 ). Indeed, mutations in the ATP binding site of theseproteins inhibit NER in vivo and in vitro ( Guzder et al.,1994; Sung et al., 1988 ), due to a defect in the opening of the damaged DNA structure ( Coin et al., 2006 ). How- ever, these studies used mutants that act by targetingthe ATPase A Walker I motif, but not the other helicasemotifs(fromIItoVI).Thus,questionsremain astowhetherDNA opening during repair requires both XPB and XPDhelicases to open a short sequence of 24/32 nucleotidesencompassing the lesion.Thusfar,investigationsofthemechanisticdefectslead-ing to XP, CS, or TTD have been beneficial in understand-ing the function of XPB, XPD, and p8/TTDA in NER andin transcription ( Evans et al., 1997; Keriel et al., 2002;Dubaele et al., 2003, Coin et al., 2006 ). In this study, we unveiled the role of both the XPB and XPD subunits of TFIIH in NER by analyzing several mutations found in XPpatients or other engineered mutations introduced inhighly conserved domains of the corresponding proteins.WefoundthatthehelicaseactivityofXPBwasnotusedfordamaged DNA opening, which is instead driven by its ATPase activity, in combination with the helicase activityof XPD. Furthermore, we demonstrated that the p52 sub-unit of TFIIH upregulates the ATPase activity of XPBthrough a direct XPB/p52 contact that is impaired inXP-B patients. The TFIIH from these patient is unable toinduce the opening of the DNA around the lesion, due totheincorrectXPB/p52interactionandATPasestimulation. Molecular Cell  26 , 245–256, April 27, 2007 ª 2007 Elsevier Inc.  245  RESULTS The F99S Mutation in XPB Impairs DamagedDNA Opening ToprovideinsightsintotheroleofXPBinNER,weinvesti-gated the DNA repair activity of two TFIIH complexespurified from cell extracts of XP-B patients carrying eitherthe F99S (XP) or the T119P (TTD) mutations ( Oh et al.,2006 ) ( Figure 1 A, left panel). Western blot analysis reveals asimilarsubunitcompositionoftheimmunopurifiedTFIIH/ XPB(WT), TFIIH/XPB(F99S), and TFIIH/XPB(T119P) com-plexes ( Figure 1 A, right panel). Upon addition of TFIIH/ XPB(F99S) to a reconstituted in vitro dual incision assay( Coin et al., 2004 ), a low level (10% activity) of excised damaged oligonucleotides was observed, comparedwith TFIIH/XPB(WT) ( Figure 1B, NER, compare lanes 5and 6 with lanes 3 and 4). TFIIH/XPB(F99S) was more effi-cient in a reconstituted transcription assay ( Gerard et al.,1991 ) (75% activity) than in dual incision (Tx, comparelanes 5 and 6 with lanes 3 and 4). The T119P mutationdid not affect either dual incision or transcription activities(compare lanes 7 and 8 with lanes 3 and 4).GiventheroleofTFIIHinNER,wecarriedoutaperman-ganate footprinting assay measuring the opening of theDNA around the damage ( Evans et al., 1997 ). Addition of  either TFIIH/XPB(WT) or TFIIH/XPB(T119P) to a reactioncontaining XPC-HR23b, in addition to the cisplatinatedDNA fragment, resulted in an increased sensitivity of nu-cleotides at positions T-4, T-5, and, to a lesser extent,T-7 and T-10 indicative of DNA opening ( Figure 1C, com-pare lane 2 with lanes 5 and 9). In contrast, addition of TFIIH/XPB(F99S) did not trigger a detectable opening of the damaged DNA ( Figure 1C, lane 7). However, furtheraddition of the NER factor XPA to TFIIH/XPB(F99S) pro-moted a weak but significant opening of the DNA, com-pared with the full opening obtained with either TFIIH/ XPB(WT) or (T119P) ( Figure 1C, compare lane 8 to lanes6 and 10). This defect in DNA opening parallels and ex-plains the low removal of damaged oligonucleotidesseen in Figure 1B.To dissect the molecular mechanism of the NER defectobserved with the F99S mutation, we purified from bacu-lovirus-infected insect cells a recombinant TFIIH complex(IIH6) containing the six subunits of the core TFIIH (XPB,XPD, p62, p52, p44, and p34). The following experimentswere performed only with the core TFIIH, since the CAKcomplex did not play any role in our in vitro NER assay( Coin et al., 2006 ). Similarly to the endogenous TFIIH/  XPB(F99S), the recombinant IIH6/XPB(F99S) showed alower repair activity, compared with either IIH6/XPB(WT)or (T119P) ( Figure 1D, compare lane 5 with lanes 1 and7). Interestingly, the addition of the NER-specific TFIIHsubunit p8/TTDA toIIH6/XPB(F99S) didnot stimulate inci-sion, compared with the increase in the removal of dam-aged oligonucleotides observed with either the IIH6/ XPB(WT) or IIH6/XPB(T119P) complexes ( Figure 1D,compare lane 6 with lanes 2 and 8). Next, we observedthat addition of p8/TTDA and XPA to IIH6/XPB(F99S) didnot trigger optimal opening of the damaged DNA in a per-manganate footprinting assay, compared with either IIH6/ XPB(WT) or IIH6/XPB(T119P) ( Figure 1E, compare lanes10–12 with lanes 4–6 and 13–15). As a control, mutationin the XPB ATPase A Walker I motif totally abolished theIIH6/XPB(K346R) repair activity ( Figure 1D, lane 3), dueto an inhibition of the damaged DNA opening ( Figure 1E,lane 7) and regardless of the presence of p8/TTDA ( Fig-ure 1D, lane 4, and Figure 1E, lanes 8 and 9). Finally, the recruitment of both TFIIH and XPA to thelesion was tested in vivo following local UV irradiation of wild-type MRC5 and XPCS2BA (bearing the F99S muta-tion) nuclei ( Volker et al., 2001 ). Fluorescence signals of  XPB colocalized with cyclobutane pyrimidine dimer (CPD)spots both in wild-type MRC5 and XPCS2BA cells ( Fig-ures2 A–2D),indicatingthatTFIIH/XPB(F99S)translocatestothe sites of DNA photolesions. Incontrast, XPA was notrecruited to the lesions in XPCS2BA, compared to MRC5cells ( Figures 2E–2H). At this point, we concluded that therepairdefectharboredbyTFIIH/XPB(F99S) isattheopen-ing step, following the binding of TFIIH to the damagedDNA. p52 Stimulates the ATPase Activity of XPB Having observed that the F99S mutation does not impairthe helicase activity of the recombinant XPB protein (datanotshown),wefocusedontheATPaseactivityofXPB.WeobservedthatthecoreIIH6/XPB(F99S)complexdisplayeda lower ATPase activity (30% activity) than those of IIH6/ XPB(WT) and IIH6/XPB(T119P) ( Figure 3 A). Enigmatically,the free XPB(F99S) polypeptide exhibited a catalytic ATPaseactivitysimilartothoseofXPB(WT)orXPB(T119P)( Figure 3B). These observations prompted us to examineif XPB-interacting subunits in TFIIH could modulate its ATPase activity. Addition of increasing amounts of p52, apartner of XPB in TFIIH ( Jawhari et al., 2002 ), to a fixed amountofpurifiedXPBsignificantlystimulateditsATPaseactivity ( Figure 3C, lanes 2–4). To the contrary, addition of either p44 or p8/TTDA, two subunits of TFIIH that do notinteractwithXPB,hadnoeffectontheATPase( Figure3C,lanes 5, 6, 8, and 9).WenextinvestigatedifXPB(F99S)andXPB(T119P)weredetrimental for the XPB/p52 interaction. Equal amountsof recombinant XPB(WT), XPB(F99S), and XPB(T119P),immobilized on agarose beads, were incubated with p52-expressing extracts. Following extensive washing, weobserved in our experimental conditions that XPB(F99S)interacts much less with p52 than do XPB(WT) orXPB(T119P) ( Figure 3D, compare lanes 8 and 9 and 5and 6 with lanes 2 and 3). When tested in an ATPaseassay, p52 weakly stimulated XPB(F99S), compared withXPB(WT) ( Figure 3E, compare lanes 5–7 with lanes 2–4). Altogether, these results demonstrate first that p52 regu-lates XPB ATPase activity and second that a mutationfound in XP-B/CS patients weakens the interaction be-tween the regulatory subunit p52 and XPB, leading to alowstimulationoftheATPaseactivityandareducedopen-ing of DNA around the damage. 246  Molecular Cell  26 , 245–256, April 27, 2007 ª 2007 Elsevier Inc. Molecular Cell Role of XPB and XPD in DNA Repair  Figure 1. The F99S Mutation Impairs Damaged DNA Opening (A)(Left)SchematicrepresentationofXPB.Thedarkgrayboxesindicatethehelicasedomains.Thelightgrayboxindicatestheconserved N-terminaldomain. Mutations found in XP-B patients (F99S and T119P) and mutation in the ATPase A Walker I motif (K346R) are depicted. (Right) Two estab-lished clones derived from the XPCS2BA cell line (mutation F99S) and expressing either the F99S (XP) or T119P (TTD) XPB ( Riou et al., 1999 ) wereused together with the MRC5 control cell line for TFIIH purification. TFIIH/XPB(WT), TFIIH/XPB(F99S), and TFIIH/XPB(T119P) were immunoprecipi-tated with antibody toward p44, a subunit of the core TFIIH, from whole-cell extracts and eluted with a competitor peptide ( Coin et al., 1999 ). Thesamples were resolved by SDS-PAGE and western blotted (WB) with anti-TFIIH antibodies. The subunits of TFIIH are indicated.(B) Fifty and one hundred nanograms of TFIIH/XPB(WT) (lanes 3 and 4), TFIIH/XPB(F99S) (lanes 5 and 6), or TFIIH/XPB(T119P) (lanes 7 and 8) weretested in a dual incision assay (NER) containing the recombinant XPC-HR23b, XPA, RPA, XPG, ERCC1-XPF factors and a closed-circular plasmidcontainingasingle1,3-intrastrand d(GpTpG)cisplatin-DNAcrosslink(Pt-DNA) asatemplate( Fritetal.,2002 )orinareconstitutedtranscriptionassay(Tx) composed of recombinant TFIIB, TFIIF, TBP, TFIIE factors, the purified RNA polymerase II, and the adenovirus major late promoter template( Gerard et al., 1991 ). Sizes of the incision products or transcripts are indicated.(C)TFIIH(100ng)wasincubated witharadiolabeled linearDNAfragmentfromthePt-DNAplasmid and40ngofXPC-HR32b.XPA(25ng)wasaddedwhen indicated. Lane 1, Pt-DNA with BSA only. Residues are numbered with the central thymine of the crosslinked GTG sequence designated T0. Arrows indicate KMnO4-sensitive sites.Adducted strandresiduestothe 3 0 and5 0 endsofT0aredenoted bypositiveandnegativeintegers(+N,  N).(D)TherecombinantIIH6/XPB(WT),IIH6/XPB(K346R)(mutatedintheATPaseAWalkerIsite),IIH6/XPB(F99S),andIIH6/XPB(T119P)lackingCAKandp8/TTDA were produced in baculovirus-infected insect cells ( Tirode et al., 1999 ). TFIIH (100 ng) was tested in dual incision in the presence of 3 ng of  recombinant p8/TTDA when indicated (lanes 2, 4, 6, and 8).(E) A KMnO4 assay was performed as described in Figure 1C with 100 ng of recombinant IIH6 complex incubated with a radiolabeled linear DNA fragment from the Pt-DNA plasmid and XPC-HR32b. XPA (25 ng) and p8/TTDA (6 ng) were added when indicated. Lane 1, Pt-DNA with BSA only.Lane 2, positive control with TFIIH purified from HeLa. Molecular Cell  26 , 245–256, April 27, 2007 ª 2007 Elsevier Inc.  247 Molecular Cell Role of XPB and XPD in DNA Repair  To map the region of p52 that is involved in the stimula-tion of XPB ATPase activity, we designed the p52(1–304)and the p52(305–462) truncated polypeptides ( Figure 4 A),knowing that p52 interacts with XPB through two distinctdomainscomprising the residues1–135and304–381( Ja-whari et al., 2002 ). Equal amounts of purified recombinantp52(WT), p52(1–304), and p52(305–462) were incubatedwith fixed amount of purified recombinant XPB(WT) in an ATPase assay. Both p52(WT) and p52(305–462) stimu-lated XPB ATPase activity ( Figure 4B, lanes 3–5 and 9–11,respectively), while addition of p52(1–305) did not showany significant effect (lanes 6–8). We also noticed thatp52(1–358), a truncated p52 polypeptide mimicking amutation found in yeast ( Jawhari et al., 2002 ), efficiently stimulated the XPB ATPase (data not shown and Jawhariet al. [2002] ). Altogether, our data indicate that the XPB ATPasestimulation dependsonthesecondXPB-interact-ing domain in p52, delimited by residues 305 and 358. Mutations in Helicase Domains of XPB PreserveTFIIH Repair Activity  We next explored the combined action, if any, of both the ATPase and helicase activities of XPB in NER. Since thehelicase activity of XPB depends on the integrity of sevenconserved motifs ( Weeda et al., 1990 ), we designed two recombinant XPB proteins. The first T469A mutation islocated in the helicase motif III, which is involved in theunwinding of the DNA. Such mutation in the domain IIIhasbeenreportedtoimpairthehelicaseactivityofseveralSF2 helicase family members ( Pause and Sonenberg,1992; Papanikou et al., 2004 ), including XPB ( Lin et al., 2005 ). The second Q638A mutation is located in the heli-case motif VI, involved in the interaction with the single-stranded DNA ( Tuteja and Tuteja, 2004 ) ( Figure 5 A), and was shown to be detrimental for XPB helicase activity( Lin et al., 2005 ). We found that both recombinant XPB(T469A) and (Q638A) displayed a very low 3 0 –5 0 heli-case activity, compared with XPB(WT) ( Figure 5B, upperpanel, compare lanes 5 and 6 and 8 and 9 with lanes 2and 3), while neither T469A nor Q638A mutations inter-fered with XPB ATPase activity ( Figure 5B, lower panel).Remarkably, IIH6/XPB(T469A) and IIH6/XPB(Q638A) re-moved damaged DNA as efficiently as did IIH6/XPB(WT)in a dual incision assay ( Figure 5C, compare lanes 7–9and 10–12 with lanes 1–3), while their ability to allow RNA synthesis was decreased when added to a reconstitutedtranscription system (50% and 20% activity, respectively)( Figure 5D, compare lanes 7–9 and 10–12 with lanes 1–3).In contrast, IIH6/XPB(K346R), deficient in the ATPaseactivity of XPB, was inactive both in DNA repair and tran-scription ( Figures 5C and 5D, lanes 4–6). In a permanga-nate assay, addition of either TFIIH/XPB(WT) or TFIIH/ XPB(T469A) to a reaction containing XPC-HR23b andp8/TTDAresultedinaDNAopeningaroundthelesion,de-pendent on the addition of ATP ( Figure 5E, lanes 3–5 and9–11). By contrast, a mutation in the ATPase A Walkermotif I (TFIIH/XPB[K346R]) inhibited the DNA-damagedopening (lanes 6–8) ( Coin et al., 2006 ). ToassesstheimportanceofthehelicaseactivityofXPBduring NER in vivo, a host cell reactivation assay was per-formed ( Carreau et al., 1995 ). A reporter construct (pLuc), carryingaluciferasegene,wasdamagedbyUVirradiationand transfected in the repair-deficient CHO27-1 cells,mutated in the XPB ( Ma et al., 1994 ), together with an Figure 2. Recruitment of TFIIH and XPA at Sites of UV Damage XPCS2BA(F99S)andwild-typeMRC5(labeledwithbluebeads)cellswereplatedonthesameslide.CellswereUVirradiatedwith70J/m 2 througha3 m mporefilterandfixed30minlater.Immunofluorescentlabelingwasperformedusingarabbitpolyclonalanti-XPB(A),amousemonoclonalanti-CPD(B and F) or a rabbit polyclonal anti-XPA (E). Nuclei were counterstained with DAPI (C and G), and slides were merged (D and H). 248  Molecular Cell  26 , 245–256, April 27, 2007 ª 2007 Elsevier Inc. Molecular Cell Role of XPB and XPD in DNA Repair  undamaged control vector coding for  b -galactosidase andan expression vector coding for the human XPB proteinsof interest. Expression of the UV-irradiated reporter genewassuppressedinCHO27-1,duetotheirrepairdefect( Fig-ure 5F, compare lane 2 with lane 3). Cotransfection of XPB(WT)cDNApartiallyrestoredluciferasegeneexpression(lanes 3 and 4), while cotransfection of XPB(fs740) cDNA containing a mutation that abolishes NER ( Coin et al.,2004 ) did not ( Figure 5F, lane 6). The recovery of luciferase activity is incomplete, probably due to species-specificdifferences between human and hamster XPB. Cotrans-fectionofXPB(T469A)cDNAallowedanincreaseinthelu-ciferase expression that reaches the level observed withXPB(WT) (lanes 4 and 5), demonstrating that the T469A mutation spares TFIIH repair activity in vivo. Altogether,weshowthat,whilethehelicaseactivityofXPBisdispens-able for effective NER, its ATPase activity is required. Mutations Impairing XPD Helicase Activity Thwartthe Repair Activity of TFIIH We next addressed if XPD, the other helicase of TFIIH,mightbecontributingtotheopeningofthedamagedDNA.We designed recombinant IIH6 complexes with XPD con-taining either the R658H, R683W, or R722W mutationsfound within XP/TTD patients or the K48R mutation lo-cated in the ATPase A Walker I motif ( Figure 6 A). As theyprevent the interaction of XPD with p44, the R683W andR722W mutations impair the helicase activity of XPD,while R658H conveys to its partial inhibition ( Dubaeleet al., 2003 ). The K48R mutation inhibits both ATPaseand helicase activities of XPD ( Tirode et al., 1999 ). Using the permanganate footprinting assay, we showed thatdamaged DNA opening was impeded in the absence of the XPD ATPase activity ( Figure 6B, lanes 5 and 6). Simi-larly, R683W and R722W hindered damaged DNA open-ing, even in the presence of p8/TTDA ( Figure 6B, lanes7, 8, 11, and 12). In contrast, R658H is sensitive to theadditionofp8/TTDA,andweobservedalimitedbutsignif-icantopening of theDNAaround the lesionwiththe corre-sponding mutated complex ( Figure 6B, lane 10). Interest-ingly, the rate of dual incision activity obtained with theIIH6/XPD(R658H) complex (50% activity) ( Figure 6C,compare lanes 6–8 with lanes 3–5) parallels the level of DNA opening. Finally, the presence of the XPB(T469A)subunit within IIH6/XPD(R658H) resulted in the IIH6/ XPD(R658H)/XPB(T469A) complex exhibiting a dual inci-sionactivitysimilartothatofIIH6/XPD(R658H),regardlessof the presence of p8/TTDA ( Figure 6C, compare lanes 9–11 with lanes 6–8). In conclusion, our results reveal thatDNA opening in NER depends on the ATPase, but noton the helicase, activity of XPB in combination with thehelicase activity of XPD. DISCUSSION p52, a New Regulatory Subunit in TFIIH BydissectingtherepairdefectinducedbytheF99Smuta-tion found in XP-B patients, we have shed light on the im-portanceofthepartnershipbetweenXPBandp52inNER.WedemonstratedthattheF99SmutationinXPBweakensthe interaction with p52 and the resulting stimulation of its ATPaseactivity,therebyinhibitingtheopeningofthedam-agedDNAandtheremovalofthelesion.Givenitsrole,p52can be considered as a regulatory subunit of the ATPaseactivity of XPB within TFIIH. Recently, it was shown thatp8/TTDA participates in the regulation of the ATPase ac-tivity of XPB within the TFIIH complex, even though thesetwo subunits do not interact. However, p8/TTDA interactswith p52 ( Coin et al., 2006 ), and it is likely that the free p8/  TTDA,whichwasshowntoshuttlebetweenthecytoplasmand nucleus and to associate with TFIIH when NER-spe-cific DNA lesions are produced ( Giglia-Mari et al., 2006 ), would regulate or stabilize the XPB/p52 interaction withinthe TFIIH complex. Thus, the binding of p8/TTDA to TFIIHandtheresultingstimulationoftheXPBATPaseactivitybyp52 might constitute a crucial NER checkpoint, decidingwhether or not a lesion will be removed. In the light of the 3D structure of an archea XPB homolog ( Fan et al.,2006 ), it was proposed that ATP hydrolysis by XPB drivesa large conformational change inducing a reorientationof a moiety of XPB and its wrapping around the DNA. Accordingly, it is likely that p52 together with p8/TTDA regulates thisconformational changethroughthe stimula-tion of the ATPase activity of XPB. Is XPB a Conventional Helicase in NER? TheremovaloflesionsdependsontheopeningoftheDNA around the damaged site. Natural mutations in either theXPB or the XPD proteins can disable DNA opening ( Evanset al., 1997 ). A remaining question is this: do both DNA helicase activities function during the NER reaction? Mu-tations in the ATP binding site of XPB and XPD totallyimpede the formation of the open DNA structure in NER( Sung et al., 1988; Guzder et al., 1994; Coin et al., 2006 ). However, such observations indicate that the hydrolysisofATPbyXPBisessentialforthefunctionofTFIIHinrepairbut do not demonstrate that the helicase activity of XPB isrequired for NER. It raises the possibility that the ATPaseactivity is not only a provider of energy for the helicaseaction but also displays another independent and distinctfunction. This hypothesis is strengthened by the fact thatthestimulationoftheATPhydrolysisbytheXPB/p52part-nership does not increase XPB helicase activity (data notshown). Furthermore, TFIIH-bearing mutations in the heli-case motifs III or VI of XPB are still functional in NER. Thissupports the idea that XPB doesn’t act as a conventionalhelicase in NER, a role that is devoted to XPD, the otherhelicase of TFIIH. Indeed, we demonstrated that muta-tionsweakeningthecontactofXPDwithitsp44regulatorysubunit ( Coin et al., 1998; Dubaele et al., 2003 ) impair damaged DNA opening. In this context, we favor a modelinwhichthewrappingofXPBaroundtheDNAwillallowfora local melting of the double-stranded DNA around thelesion that would favor the correct anchoring of the XPDhelicase. XPB would therefore play the role of a wedge,using ATP to keep the two strands of the DNA around Molecular Cell  26 , 245–256, April 27, 2007 ª 2007 Elsevier Inc.  249 Molecular Cell Role of XPB and XPD in DNA Repair
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