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Reconstitution of Yeast Nucleotide Excision Repair with Purified Rad Proteins, Replication Protein A, and Transcription Factor TFIIH

Reconstitution of Yeast Nucleotide Excision Repair with Purified Rad Proteins, Replication... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270. No. 22, Issue of June 2, pp. 12973-12976, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Communication Printed in U.S.A. UV-damaged DNA (2). Rad3 is a single-stranded DNA-depend­ Reconstitution of Yeast ent ATPase, and it also has DNA helicase and DNA·RNA Nucleotide Excision Repair with helicase activities (3-5). Rad3 exhibits a preference for binding UV-damaged DNA that is dependent upon ATP and the degree Purified Rad Proteins, of negative superhelicity in DNA (6). Rad25 also possesses a Replication Protein A, and single-stranded DNA-dependent ATPase and DNA helicase ac­ tivities (7). Radl and RadIO exist as a complex in vivo, and Transcription Factor TFIIH* complex formation between these proteins is essential for their biological action (8). Radl and RadIO together comprise a DNA (Received for publication, April 3, 1995) endonuclease (9, 10), and Rad2 also is a DNA endonuclease Sami N. Guzder, Yvette Habraken, Patrick Sung, (11). Louise Prakash, and Satya Prakashr Another level of complexity in understanding the biological From the Sealy Center for Molecular Science, University roles ofNER genes was introduced from the observation that in of Texas Medical Branch, Galveston, Texas 77555-1061 addition to their requirement in NER, RAD3 and RAD25 are also essential for cell viability (1). Studies of temperature­ Nucleotide excision repair (NER) functions to remove sensitive conditional lethal mutations of these genes have in­ DNA damage caused by ultraviolet light and by other dicated that both are essential for RNA polymerase II tran­ agents that distort the DNA helix. The NER machinery scription (7, 12, 13). Studies with the rad3 and rad25 mutants has been conserved in structure and function from yeast defective in ATPaselDNA helicase activities have indicated to humans, and in humans, defective NER is the under­ lying cause of the cancer-prone disease xeroderma pig­ that whereas Rad3 ATPaselhelicase activity is required for mentosum. Here, we reconstitute the incision reaction NER, the Rad25 ATPaselhelicase is essential for both NER and of NER in Saccharomyces cerevisiae using purified pro­ polymerase II transcription (7, 13, 14). Rad3 and Rad25 pro­ tein factors. The RadI4 protein, the Rad4-Rad23 com­ teins are two of the six subunits of polymerase II transcription plex, the Rad2 nuclease, the RadI·RadiO nuclease, rep­ factor TFIIH, and it has been proposed that the entire TFIIH lication protein A, and the RNA polymerase II functions in NER (15-18). transcription factor TFIIH were purified to near homo­ In addition to the above mentioned protein factors, the sin­ geneity from yeast. We show that these protein factors gle-stranded DNA binding protein RPA has been suggested to are both necessary and sufficient for dual incision of have a role in an early step of NER (19, 20). In a reconstituted DNA damaged by either ultraviolet light or N-acetoxy­ system reported recently, Mu et at. (21) have shown that 2-aminoacetylfluorene. Incision in the reconstituted human RPA is essential for incision of a DNA substrate con­ system requires ATP, which cannot be substituted by taining a cholesterol adduct. adenosine 5'-O-(3-thiotriphosphate), suggesting that the Our goal has been to reconstitute nucleotide excision repair hydrolysis of ATP is indispensable for the incision reac­ in yeast with purified proteins. Because of the amenability of tion. The excision DNA fragments formed as a result of S. cereuisiae to genetic and biochemical analyses, development dual incision are in the 24-27-nucleotide range. of such a system is essential for a detailed understanding of processes that effect different steps of NER, including tran­ scription-coupled DNA repair. Various yeast protein factors Nucleotide excision repair (NER)l represents the most im­ that have been implicated in NER in genetic and biochemical portant cellular mechanism for the removal of DNA damage studies have now been purified to near homogeneity in our induced by ultraviolet light (UV). Genetic studies in the yeast laboratory. Here, we present our results, which indicate that Saccharomyces cerevisiae have identified seven genes, RADl, the incision step of NER can be accomplished by combining the RAD2, RAD3, RAD4, RADIO, RAD14, and RAD25, that are following highly purified protein components: RadI4, Rad4­ essential for NER (1). To begin to define the biological roles of Rad23 complex, Radl-RadiO complex, Rad2, RPA, and TFIIH. these genes, we have purified their encoded proteins from yeast The damage-specific incision reaction has a strict dependence and characterized their biochemical activities. This undertak­ on ATP, and our results imply that the two incision nicks are ing has allowed us to infer the involvement of these proteins in made in a highly coordinated fashion. different steps of the incision process, viz. in damage recogni­ tion, in DNA unwinding, and in dual incision of the damaged MATERIALS AND METHODS DNA strand. RadI4 is a zinc metalloprotein with an affinity for UV Irradiation and AAF Treatment of Plasmid DNA-Replicative form M13 mp18 DNA (~90% supercoiled form) was purified from in­ * This work was supported by Grants CA35035 and CA41621 from fected Escherichia coli strain JM101 by two rounds of cesium chloride the National Cancer Institute and Grant DE-FG03-93ER61706 from banding. The DNA, at a concentration of 100 ILg/ml in TE (10 mM the Department of Energy. The costs of publication of this article were Tris-HCI, pH 7.0, 0.2 mM EDTA), was irradiated in 15-ILI droplets for 60 defrayed in part by the payment of page charges. This article must s with a germicidal lamp emitting at 254 nm and a fluence rate of 5 therefore be hereby marked "advertisement" in accordance with 18 2/s J/m to introduce ~2.5 photoproducts/1000 base pairs of DNA. For U.S.C. Section 1734 solely to indicate this fact. treatment with N-acetoxy-2-aminoacetylfluorene (AAF), the plasmid :j:To whom correspondence should be addressed: Sealy Center for DNA, 10 ILg or 30 nmol of nucleotides, was incubated at 37°C for 12 h Molecular Science, University of Texas Medical Branch, 6.104 Medical in the dark with 0.5 nmol ofAAFin 200 ILl of50 mM sodium acetate, pH Research Bldg., 11th & Mechanic St., Galveston, TX 77555-1061. Tel.: 5.8. After extraction with diethyl ether, the DNA was purified by 409-747-8602; Fax: 409-747-8608. ethanol precipitation and redissolved in TE to 100 ILg/ml. 1 The abbreviations used are: NER, nucleotide excision repair; Reconstitution of NER in Vitro--(i) In the incision assay, reaction PAGE, polyacrylamide gel electrophoresis; RPA, replication protein A; mixtures (10 ILl, final volume) were assembled in buffer R (45 mM ATP-yS, adenosine 5'-O-(3-thiotriphosphate); AAF,N-acetoxy-2-amino­ acetylfluorene. K-HEPES, pH 7.9, containing 8 mM MgCI , bovine serum albumin at This is an Open Access article under the CC BY license. 12974 Reconstitution of Yeast Nucleotide Excision Repair 120 JLg/ml , 1.5 mxi dithiothreitol, 2 mxt ATP, a n ATP-regenerating Rad.flRad23 TH ill syste m consisting of 30 mxt creatine phosphate and 200 ng of creatine Radl~ Purffl cation Purification l'urtflcat km kinase, a nd 60 rnxt potassium acetate a nd 20 rnxt KCl th at were du e to addition of the vario us NE R pr otein factors) a nd contained 100 ng of TFIIH, 50 ng of RPA , 8 ng of Rad 1-Rad10 complex , 10 ng of Rad 2 Clarified Extract Clarified Extract C larified Extract protein, 20 ng of Rad4 -Rad2 3 complex, a nd 10 ng of Rad1 4 protein. Th e Rad1-Rad1 0 comp lex for use in these studies was form ed by in cubating Ammonium Ammonium Hie -Rex 70 equimola r a mounts of purified Rad1 a nd RadIO pr oteins for 24 h on ice sulfate preci pitation sulfate prec ipitation in 20 mxt Tri s-HCl, pH 7.5, containing 1 mxi dithiothreitol a nd 500 I I ug/m l bovin e se rum albumin to give a final concentration of 1 JLM of th e DEAE Scphaccl Q Scpb•n ose Q SC p h<lTOSC protein complex. Th e combina tion of NE R factors were incubated at I I I 25 °C for 5 min before the undam aged DNA or dam aged DNA , 100 ng SP Scpharose SP Scpharosc Hydroxyapatite each, was added in 1 JLI. After incubation a t 30 °C for 10 min , SDS and protein ase K were added to 0.5% a nd 200 ug/m l, res pectively, followed I I Nickel Agarosc Hydroxyapatite Mono Q by a 5-min incubation at 37 °C to dep roteini ze th e reaction mixtures. Sa mples wer e run in 0.8% agarose ge ls in TAE buffer (20 mM Tris I I acetate, pH 7.4, 0.5 mxr EDTA). Th e gels were treate d with eth idiu m :'-10110 S Hydroxyapatite Mono S bromide (l ug/rnl in H"Q) to stain DNA, soa ked in a large volume of I I water to reduce background staining, a nd th en photogr aphed th rough a ~ 1 ()n () Q xtono S red filter. (ii ) For the excision assay, reacti on mixtures (50 JLI, final volume) were assemb led in buffer R an d contained 500 ng of TFIIH, 250 B c D ng of RPA, 40 ng of Rad1 -Rad10 complex , 50 ng of Rad2, 100 ng of Rad 4-Rad 23 complex, a nd 50 ng of Rad 14. Th e combination of NER 97 ­ ~ ~ pr otei ns was incubated at 25 °C for 5 min before 1 JLg of undam aged or 68 - 116 - UV-da maged DNA was added in 10 JLl of TE. Th e complete reaction 200 - -Rad25 mixtures were incub ated at 30 °C for 60 min a nd th en depro tein ized by 97 - 45 - 116 ­ - Rad3 ext ract ion wit h a n equa l volum e of buffered ph en ol. Th e DNA was - -TFBI 97 - 68 - , precipitated by etha nol a nd red issolve d in 10 JLl of TE , a nd 5 JLI of 31- 68- which was treated wit h 7.5 units of calf thymus terminal tra nsfera se <Boehringer Ma nnheim l wit h 5 JLCi of [,,-""Pldideoxy ATP (Ame rsha rn 2 1- _ Corp.; 5000 Ci/rnrnol) for 60 min a t 37 °C in a final volume of20 JLI of th e 45 - 45 - buffer su pplied by th e vendor . Th e labeling mixtures wer e deprotein­ 14 - _ - p38 M 1 2 3 ized by trea tment with 0.5% SDS a nd 300 JLg/ml pr otein ase K for 10 min M 1 2 3 at 37 °C, followed by the precip itation of t he DNA. Th e pellets were dissolved in 4 JLl of bu ffer and a nalyze d on 15% seque ncing gels. The FIG. 1. Protein factors for in vitro reconstitution of NER. A, gels wer e dried a nd exposed to x-ray film s (Koda k Bio Ma x MR ) to pu rifi cation schemes for Rad1 4, Rad 4-Rad 23 complex, a nd TFIIH. B-D, reveal th e excision DNA fra gments. SDS-PAGE of pu rified NER factors. B, RadlO (1 JLg in lane t ), Rad1 4 (1 JLg in lane 2), and RPA (2 JLg in lane 3) consisti ng of the 69-, 36-, a nd RESULTS 13-kD a subuni ts each marked by an asterisk wer e ru n in a 12% den a­ turing polyacrylamide ge l a nd stained with Cooma ssie Blue. M, molec­ Purification of NER Factors-RadIO protein was purified ular size standards . C, 1 JLg each of Rad l tlane l ), Rad 2 (lane 2 ), and th e - l ,OOO-fold to near homo gen eity (Fig. lB , lan e 1) from yeast complex of Rad4 (upper band marked by asterisk in lane 3) and Rad23 strain CMY135 harboring the overproducing plasmid pSU C8 (lower band mark ed by asterisk in lane 3) were run in a 7% denaturing polyacrylam ide gel a nd stai ned with Cooma ssi e Blue. M, molecular size as described (22). Th e RAD14 gene contains a n intron and standa rds. D, TFIIH (500 ng total protein ) was run in an 8% den aturing encodes a protein with a pr edicted size of 43 kDa, and it polyacrylamide gel a nd silver-stained with a kit pu rchased from Bio­ exhibits a size of 48 kDa in SDS-PAGE (23). Rad14 protein has Rad . Th e va rious subu nits of TFIIH a re indicated . Th e identi ty of been purified - l ,OOO-fold to near homogen eity (Fig. lB , lan e 2 ) subunits was verified by immunoblotting usin g affinity-purified anti­ from yeast strain YRPll harboring th e overproducing plasmid Rad25, a nti- Rad3, anti-TFB1, a nd a nti-SS Ll a ntibodies. pR14.l5 containing RAD14 under th e control of the yeast alco­ ity-purified anti-Rad23 antibodies (25), we have verified that hol dehydrogenase I (ADC1) promoter, with th e purification scheme outlined in Fig. lA . Radl protein was purified - 2,000­ this 57-kDa species was indeed Rad23 protein . Because in wild fold to near homogen eity (Fig. l C, lan e 1) from yeast stra in type yeast Rad23 is more abunda nt th an Rad4 (25), only over­ CMY135 harboring th e overproducing plasmid pRR168 as de­ production of Rad4 was necessary to purify the Rad4-Rad2 3 complex (Fig. l C, lan e 3). TFIIH was purified > lO,OOO-fold to scribed previously (9). Rad2 protein wa s purified - 3,000-fold to near homogen eity (Fig. l C, lan e 2) from yeast strain LY2 har­ near homogen eity (Fig. lD) from yeas t strain YPH!I'FB1.6HIS boring th e overpro ducing plasmid pR2.26 as described (11). To that contains a 6-histidine tag in th e TFBI subunit of TFIIH overprodu ce th e RAD4-en coded protein in yeast for purifica­ (26) as outlined in Fig. lA . Du ring purification, the elution of TFIIH from various chromatographic matrices was monitored tion, th e pr otein coding frame of RAD4 was fused to the yeas t AD C1 promoter to yield plasmid pR4.l, whi ch wa s introduced by immunoblotting usin g affinity-purified anti bodies specific for th e Rad 3, Rad2 5, TFBl, and SSLI subunits (23). The TFIIH into yea st stra in YRPl 1. Th e scheme pr esented in Fig. lA was used to purify Rad4 protein - 4,000-fold to near homogen eity pr eparation used in this study conta ined six subunits, Rad25, Rad 3, TFBl, SSLl , p55, and p38 (Fig. iz». RPA, consisting of (Fig. l C, lan e 3) from YRPll(pR4.l). Th e elution of Rad4 pro ­ tein from various chroma togra phic matrices was monitored by three subunits of 69, 36, and 13 kDa, was purified to near hom ogeneity (Fig. lB , lane 3) from yeast strain LP27 49-9B immunoblotting usin g affinity- purified polyclonal antibodies raised against an inso luble form of Rad4 protein expressed in using th e procedure of Brill and Stillman (27). ATP-dependent In cision of UV-damaged DNA-To examine insect cells with th e use of baculovirus (data not shown). Rad4 protein has a pred icted molecular ma ss of 87 kDa (24), but it wheth er th e purified yeast factors Rad14, Rad4-Rad2 3 com­ plex, Radl-RadlO complex, Rad2, RPA , and TFIIH are suffi­ migrates in SDS-polyac ryla mide gels with a relative molecular mass of 116 kDa. Through out th e purification of Rad4, we cient for reconstituting NER in vitro, th ey wer e incubated at 30 °C in buffer containing ATP with plasmid DNA previously observed a precise co-elution of a protein with a relative mo- . lecular mass of 57 kDa, which is th e same as that described for irradiated with UV light to introduce photoproducts into the DNA. Reaction mixtures were deproteini zed and ana lyzed by the RAD23 gene produc t (25) that plays an important role in the proficien cy of NER <1 , 25). By immunoblotting using a ffin- agarose gel electrophoresis, followed by sta ining with ethidium Reconstitution of Yeast Nucleotide Excision Repa ir 12975 -A,AF .AAF A DNA • UV + UV A DNA .--I. i r----11 I R1 R1 Factor omitted Bl None 81 None 110 R2 R14 A4 DB RPA Factor omined Bl None 81 None /10 R2 R14 R4J23 UH RPA 123 ~ -oc -oc _ _ __ _ _ _ _ 0- 4 _ -sc -sc - - - ---- - - 3 4 8 9 10 BONA -w +IN · AAF + AAF B ONA r--11 i r---1 1 NER Factors -+-++++++++ _+e+++++++ + NER Factors Nucleotide ATP ATP ATP ATP ADP dATP 'Y$ e l P GfP UTP Nucleotide ATP ATP ATP ATP • ADP dATP "1'5 crs GYP UTP -oc -oc - - - - -sc - - - -- ---- -sc --- -- - - - - 2 3 4 5 6 7 8 9 10 11 2 3 4 5 6 7 8 9 10 11 F IG. 2. ATP·dependent in cis ion of UV-damaged DNA. A, recon­ FIG. 3. ATP·dependent incision of DNA containin g AAF ad­ sti tution of the incision reaction using purified protein facto rs. Th e ducts. A, the NER factors incise AAF-cont ai ning DNA. The und amaged DNA used was either not treated (- UV , lan es 1 and 2) or trea ted with DNA (lan e 2) OJ' damaged DNA tla ne 4) was incubated with the Radl ­ UV (+UV , lan es 3- 10). The full complement of NE R factors <Rad l ­ Rad lO complex, Rad2, Rad4·Rad23 complex, Radl4, TF IIH , and RPA at RadI O complex, Rad2, Rad4-Rad 23 complex, Radl4, TFIIH, and RPA) 30 °C for 10 min in buffer containing ATP. The reaction mixtur es in was incubated with und am aged DNA (lane 2) and UV-damaged DNA lanes 5- 10 conta ined AAF·damaged DNA and ATP , but one of the NER (lane 4) in buffer containing ATP at 30 °C for 10 min . One of the factors was omitted; Rad l -RadlO complex was absent in lane 5, Rad2 afore ment ioned NE R factors was absent in th e reaction mixtu res in was absent in lan e 6, Rad14 was absent in lane 7, Rad4-Rad 23 complex lan es 5- 10 th at contai ned UV-damaged DNA and ATP; Radl-Radl0 was absent in lan e 8, TF IIH was absent in la ne 9, and RPA was absent complex was omitted in lan e 5, Rad2 was omitted in lan e 6, Rad1 4 was in lane 10. Bl , DNA incubated in buffer with out any of the NER factors itted in lan e 7, Rad4-Rad 23 complex was omitted in la ne 8, TF IIH om ilanes I an d 3). B, incision of AAF-cont aining DNA requi res ATP. The was omitted in lan e 9, an d RPA was omitted in lan e 10, as indica ted at full complement of NER factors (lane 2 and lanes 4 - 11) was incu bated th e top of the figu re. Bl , DNA incubated in buffer without any of the with either undam aged DNA (- AAF, lane 2 ) or with AAF-containing NER factors (lanes 1 an d 3). B, incision of UV-da mage d DNA requires DNA (+AAF, la nes 4 -11) in the presence of ATP (lan es 2 and 4 ), ADP ATP. Th e full complement of NER factors (lane 2 an d lanes 4-11) was (lane 6), dATP tlane 7), ATP yS ilane 8), CTP (lan e 9), GTP ilane 10), incubated wit h eit her und am aged DNA (- UV, lane 2) or with UV­ UTP (lan e 11), or without any nucleotid e tlane 5) at 30 °C for' 10 min. In dam aged DNA (+UV , lan es 4· 11) in the pre sence of ATP (lanes 2 and 4), lanes 6 and 8, creatine kina se was omitted to inacti vate the ATP­ ADP (lan e 6), dATP (lan e 7), ATPyS (lane 8), CTP (lane 9), GTP (lane rege nerating sys tem. No NER factors wer e added in lanes I and 3. S C, 10), UTP (lane 11), or without any nucleotide (lane 5) at 30 °C for 10 supercoiled form; ac, open circular form. min. In lanes 6 and 8, creatine kinase was omitted to inactivate the ATP-regenerating system. No NER factors were added in lan es 1 and 3 . 2B) . Interestingly, th e non-hydro lyzable ATP ana log ATPyS is SC, supercoiled form; ac, open circula r form . also inactive (Fig. 2B , lan e 8), suggesting that ATP hydrolysis is in fact required for the incision reaction. Furth ermore, Mg + br omide to exa mine the fate of the DNA substrate. Fig. 2A is also required for incision (data not shown). shows th at whe n a ll the purified NER factors were present, In cision ofDNA Damaged by AAF-The NER machinery has greate r than 80% of the UV-da maged superco iled plasmid DNA specificity for a variety of DNA lesions. We examined whether was converte d to the open circular form (lane 4), indicating th at our pu rified NER protein s would also incise DNA dam aged by incision of th e dam aged DNA had occurred. By contrast, plas­ N-acetoxy-2-aminoacetylflu oren e. As shown in Fig. 3A, plas­ mid DNA that had not been subjecte d to UV treatme nt was not mid DNA containing the AAF adduct was incised when all th e acte d on by th e same protein factors (Fig . 2A, lane 2). Thus, the purified protein factors were pr esent (lane 4) bu t not whe n any combination of NER protein s carries out an incision reaction of th e fact ors was omitte d from th e reaction mixture (lanes that is highl y specific for UV dam age. Importantly, omission of 5- 10). Th e incision of AAF-modified DNA also has a specific any of the comp onents Radl-RadlO complex, Rad 2, Radl4, requirement for ATP or dATP (Fig. 38). ATP yS did not promote Rad4-Rad 23 complex , TFIIH, or RPA from the reaction mixture incision (Fig. 38), again suggesting a requirement for ATP abolishe d the formation of the open circular form (Fig. 2A, hydrolysis in the repair of AAF-dam aged DNA. lan es 5-10), indicating that incision of UV-dam aged DNA re­ S ize of Excision DNA Fragments- NE R in human s has been qui res all of th ese pu rifi ed pr otein factors . It is of particular shown to occur by way of du al incision of th e DNA strand that interest that nicking of the UV-damaged DNA did not occur contains th e lesion, resulting in th e release of a DNA fragment whe n eithe r the Radl-RadlO endonuclease or th e Rad2 endo­ 27- 29 nucl eotides in length (28). To detect the excision DNA nucl ease was omitte d from th e reaction mixture (Fig. 2A, lanes fragment, we incubated plasmid DNA dam aged by UV light in 5 and 6). Th is obse rvation strongly suggests th at both th e the reconstituted re pair syste m. Following phenol extraction Radl-Radl0 complex and the Rad 2 protein , in addition to bein g to remove repai r pr otein s, the DNA was purified and treated th e endonucleolyt ic comp onents, have a pivotal role in th e with calf th ymus terminal transferase in the presence of 32P proper assembly of the incision enzyme compl ex at the damage [a- ldid eoxy ATP to label any excision DNA fragments that site, thus ensuring that th e two incision nicks are mad e in a might have been generate d. As shown in Fig. 4, a series of DNA coordinated fashion. fragments rangin g in size from 25 to 28 nucleotid es was de­ Because Rad 3 and Rad2 5 protein s both possess an ATP tected in the reaction mixture th at conta ined UV-da maged (dATP)-dependent helicase activity th at is required for NER (7, DNA as substrate (lane 5) but not in th e cont rol that contained 13, 14), it was of considerabl e importance to determine whether undam aged DNA (lane 2). Importantly, th e DNA fragments the incision of dam aged DNA in our reconstituted sys te m re­ were not produced if ATP was absent (Fig. 4, lane 6) or whe n qui res ATP . We found th at in the abse nce of ATP , the inci sion TFIIH was omitted from the repair reaction (Fig. 4, lan e 4 ). of UV-da maged DNA does not occur (Fig. 2B , compare lanes 4 Because the :J2P-labeling protocol added one nucl eotide to the and 5). Whereas dATP is as effective as ATP in pr omoting th e excision DNA fra gment s, the actual size ra nge of these frag­ incision reaction, ADP , CTP, GTP, and UTP are inactive (Fig. ments is 24-27 nucleotid es. 12976 Reconstitution of Yeast Nucleotide Excision Repair - Uv + UV DNA th at th e en donucleolytic sciss ions at th e dam age site occur in a ,------, AlP + + nated manner, as well as to minimize gratuitous nicking coordi FaclorOmitted BI none 81 lUi none none of DNA. In summary, the followin g major conclusion s emerge from this work. First, th e damage recognition factor Radl4, the Rad4-Rad2 3 complex, the Radl-Radl0 endonuclease, th e Rad2 endonuclease, RPA, and TFIIH are all essential for the incision step ofNER. Second , becau se of th e high degr ee of purity of th e protein fact ors used, we infer that th e combination of th ese protein s is sufficient for th e incision reaction. Thi rd , th e inci­ sion reaction occurs only in the pr esence of ATP , and our - 30 results strongly sugges t a requirement of ATP hydrolysis in this reaction. Finally, our obse rvation that th e size of the excision fragme nt produced by th e yeas t incision enzyme com­ -25 plex resembles that in human s indicates that th e yeast and human NER machineri es act in a highly similar manner. Ackn owledgm ent s-We are very grateful to A. Sancar and colleagues -20 for communicat ion of th eir results prior to public ati on and to W. J . Feaver and R. D. Kornberg for yea st stra in YPH!I'FB1.6HIS, for th e E. coli plasmid th at expresses 6-histidine tagged SSLl, and for a sa mple of ank S. Wilson for help­ purified 6-histidine tagged SSLI protein . We th ful discussions and A. McCullough for anti-SSLI antibodies. 2 3 4 5 6 REFERENCES FIG. 4. Size of the excision DNA fragments. UV-irra diated (lanes 1. Prak ash , S., Sung, P., a nd Pr ak ash , L. (1993) Annu. Rev. Genet. 27, 33- 70 3-6) and uni rr adi at ed (lanes 1 and 2) plasmid DNAs were incubated 2. Guzder, S. N., Sung, P., Pr akash . L., and Pra kash , S. (1993) Proc. Na tl. Acad . with the full complement of NER factors (lanes 2, 5, and 6) in th e Sci. U. S. A. 90, 5433- 5437 3. Su ng, P., Pra kash , L., Weber, S., and Prak ash , S. (1987) Proc. Na tl. A cad . Sci. presence of ATP ttanes 2 and 5) or in its absence (lane 6). Th e reacti on U. S. A. 84, 6045-6049 lane 4 contained ATP but lacked TFIIH. The numbers to th e mixtu re in 4. Sung, P., Pr ak ash , L., Matson, S. W., a nd Pr akas h, S. (1987) Proc. Na tl. Acad . right of the autora diogra m indicate th e positi ons in nuc1eotides of th e Sci. U. S. A. 84, 8951- 8955 DNA marker s used. Bl , DNA incubat ed in buffer without any of th e 5. Bailly, V., Sung, P., Prakash, L., and Prak ash , S. (1991) Proc. Na tl. Acad . Sci. NER factors (lanes 1 and 3). U. S. A. 88, 9712-9716 6. Sung, P., Watkins, J . F., Pra kash, L., and Pr akash, S. (1994) J. Bioi. Chem. 269, 8303-8308 DISCUSSION 7. Guzder, S., Sung, P., Bailly, V., Prakash , L., a nd Prakash, S. (1994) Na ture 369, 578 - 581 In this work we have achi eved th e reconstitution of a syste m 8. Bailly, V., Sommer s, C. H., Sung, P., Pr ak ash , L., a nd Prakash , S. (1992 ) Proc. th at mediates du al inci sion of DNA damaged eithe r by UV or N atl. A cad . Sci. U. S. A. 89, 8273- 8277 9. Sung, P., Reynolds, P., Prakash, L., and Prak ash , S. (1993)J. Bivl. Chern. 268, AAF, by combining the following essentially homogeneous 2639 1-26399 S. cerevisiae factors: Radl-Radl0 complex, Rad2, Rad4-Rad23 10. Tomki nson, A. E., Bard well, A. J ., Bardwell, L., Tap pe, N. J ., and Fri edberg, E. complex, Radl4, RPA, and TFIIH consisting of Rad3, Rad25, C. (1993 ) Nature 362, 860-862 11. Habraken , Y., Su ng, P., Prak ash, L., a nd Pr a kash, S. (1993 ) Na ture 366, TFBl, SSLl, p55, and p38 subunits . Sinc e all these purified 365-368 protein factors are indispen sable for damage-specific inci sion , 12. Guzder , S. N., Qiu, H., Sommer s, C. H., Sung, P., Prakash , L., and Pr ak ash , S. (1994) Na ture 367, 91-94 th ey represent th e minimum set of factors for accomplishing 13. Qiu, H., Park, E., Pra kash, L., a nd Pra kash, S. (1993) Gelles & Dev. 7, th is reaction. 2161-2171 14. Sung, P., Higgins, D., Prakash, L., and Prakash, S. (1988) EMBO J. 7, In our reconstituted system, inci sion of dam aged DNA shows 3263-3269 a strict dependenc e on ATP or dATP, whose hydrolysis is re­ 15. Feaver , W. J ., Svejst rup , J . Q., Bard well, L., Bardwell, A. J ., Bura towski, S., quired for th e repair reaction because the non-hydrolyzable Gulyas, K D., Donah ue, T. F., Fri edberg, E. C., a nd Korn berg, R. D. (1993) Cell 75, 1379 - 1387 ana log ATP yS does not promote th e inci sion of damaged DNA. 16. Wan g, Z., Svejstrup, J . Q., Feaver , W. J ., Wu, X., Kornb erg, R D., a nd Th e requi rement for ATP and its hydrolysis in th e incision ste p Friedb erg, E. C. (1994) Na ture 368, 74- 76 of NER is consistent with the results from genetic studies 17. Drapkin, R , Reardon, J . T., Ansa ri, A., Huan g, J . C., Zawel, L., Ahn, K , Sa ncar, A., an d Reinberg, D. (1994) Na ture 368, 769-772 conducted with mutan t varia nts of Rad3 and Rad25 protein s 18. van Vuu ren , A. J ., Verm eulen, W., Ma, L., Weeda, G., Appeldoorn , E., J aspers, that are defective in ATP hydrolysis (7, 13, 14). We suggest that N. G. J ., va n del' Eb, A. J ., Bootsma , D., Hoeijm ak er s, J . H. J ., Humbert , S., Schaeffer, L., and Egly, J .-M. (1994) EMBO J . 13, 1645-1653 at the expense of ATP hydrolysis, th e combined helicase func­ 19. Coverley, D., Kenn y, M. K , Lan e, D. P., a nd Wood, R D. (1992) N ucleic Acid s tion of Rad 3 and Rad25 create s a single-stranded region at the Res. 20, 3873-3880 dam age site for du al incisi on by th e Radl-Radl0 and Rad2 20. Clugston, C. K , McLaughlin, K , Kenny, M. K , and Brown , R. (1992) Cancer Res. 52, 6375-6379 endonucleases . 21. Mu, D., Pa rk, C.-H., Matsunaga, T., Hsu , D. S., Reardon, J. T., a nd Sa ncar, A. It is intriguing that nickin g of th e damaged DNA does not (1995 ) J . BioI. Chem . 270, 2415- 2418 22. Sung, P., Prak ash , L., a nd Pr ak ash , S. (1992 ) Nature 355, 743- 745 occur if either th e Radl-Radl0 endonuclease or the Rad2 en­ 23. Guzder, S. N., Bailly, V., Sung , P., Prakash, L., and Pra kash, S. (1995)J. BioI. donu clease is present alone with th e remainder of th e incision Chern. 270 , 8385-8388 NER components. Thi s finding st rongly suggests th at in addi­ 24. Gietz, R D., a nd Prakash , S. (1988 ) Gene (A rnst.) 74, 535-54 1 25. Wat kin s, J . F., Sung, P., Prakash, L., and Prakash, S. (1993) Mol. Cell. BioI. tion to providing th e endonucleolytic activities for dual incision , 13, 7757- 7765 th e Radl-Radl0 protein comp lex and th e Rad2 protein are also 26. Svejst rup, J . Q., Feav er , W. J ., LaP oint e, J ., and Korn berg, R. D. (1994 )J . BioI. Chern. 269 , 28044 - 28048 required for th e proper assembly of th e NER ensemble at th e 27. Brill, S. J ., an d Stillman, B. (1989) N ature 342, 92- 95 dam age site . Th e involvement of the two endonucleolytic com­ 28. Hua ng, J. C., Svoboda , D. L., Reardon, J . T., a nd Sa nca r, A. (1992) Proc. Na tl. ponents in as sembling the NER complex may serve to ensure Acad. Sci. U. S. A. 89, 3664 - 3668 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Reconstitution of Yeast Nucleotide Excision Repair with Purified Rad Proteins, Replication Protein A, and Transcription Factor TFIIH

Guzder, Sami N.; Habraken, Yvette; Sung, Patrick; Prakash, Louise; Prakash, Satya
Journal of Biological Chemistry – Jun 1, 1995
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0021-9258
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10.1074/jbc.270.22.12973
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270. No. 22, Issue of June 2, pp. 12973-12976, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Communication Printed in U.S.A. UV-damaged DNA (2). Rad3 is a single-stranded DNA-depend­ Reconstitution of Yeast ent ATPase, and it also has DNA helicase and DNA·RNA Nucleotide Excision Repair with helicase activities (3-5). Rad3 exhibits a preference for binding UV-damaged DNA that is dependent upon ATP and the degree Purified Rad Proteins, of negative superhelicity in DNA (6). Rad25 also possesses a Replication Protein A, and single-stranded DNA-dependent ATPase and DNA helicase ac­ tivities (7). Radl and RadIO exist as a complex in vivo, and Transcription Factor TFIIH* complex formation between these proteins is essential for their biological action (8). Radl and RadIO together comprise a DNA (Received for publication, April 3, 1995) endonuclease (9, 10), and Rad2 also is a DNA endonuclease Sami N. Guzder, Yvette Habraken, Patrick Sung, (11). Louise Prakash, and Satya Prakashr Another level of complexity in understanding the biological From the Sealy Center for Molecular Science, University roles ofNER genes was introduced from the observation that in of Texas Medical Branch, Galveston, Texas 77555-1061 addition to their requirement in NER, RAD3 and RAD25 are also essential for cell viability (1). Studies of temperature­ Nucleotide excision repair (NER) functions to remove sensitive conditional lethal mutations of these genes have in­ DNA damage caused by ultraviolet light and by other dicated that both are essential for RNA polymerase II tran­ agents that distort the DNA helix. The NER machinery scription (7, 12, 13). Studies with the rad3 and rad25 mutants has been conserved in structure and function from yeast defective in ATPaselDNA helicase activities have indicated to humans, and in humans, defective NER is the under­ lying cause of the cancer-prone disease xeroderma pig­ that whereas Rad3 ATPaselhelicase activity is required for mentosum. Here, we reconstitute the incision reaction NER, the Rad25 ATPaselhelicase is essential for both NER and of NER in Saccharomyces cerevisiae using purified pro­ polymerase II transcription (7, 13, 14). Rad3 and Rad25 pro­ tein factors. The RadI4 protein, the Rad4-Rad23 com­ teins are two of the six subunits of polymerase II transcription plex, the Rad2 nuclease, the RadI·RadiO nuclease, rep­ factor TFIIH, and it has been proposed that the entire TFIIH lication protein A, and the RNA polymerase II functions in NER (15-18). transcription factor TFIIH were purified to near homo­ In addition to the above mentioned protein factors, the sin­ geneity from yeast. We show that these protein factors gle-stranded DNA binding protein RPA has been suggested to are both necessary and sufficient for dual incision of have a role in an early step of NER (19, 20). In a reconstituted DNA damaged by either ultraviolet light or N-acetoxy­ system reported recently, Mu et at. (21) have shown that 2-aminoacetylfluorene. Incision in the reconstituted human RPA is essential for incision of a DNA substrate con­ system requires ATP, which cannot be substituted by taining a cholesterol adduct. adenosine 5'-O-(3-thiotriphosphate), suggesting that the Our goal has been to reconstitute nucleotide excision repair hydrolysis of ATP is indispensable for the incision reac­ in yeast with purified proteins. Because of the amenability of tion. The excision DNA fragments formed as a result of S. cereuisiae to genetic and biochemical analyses, development dual incision are in the 24-27-nucleotide range. of such a system is essential for a detailed understanding of processes that effect different steps of NER, including tran­ scription-coupled DNA repair. Various yeast protein factors Nucleotide excision repair (NER)l represents the most im­ that have been implicated in NER in genetic and biochemical portant cellular mechanism for the removal of DNA damage studies have now been purified to near homogeneity in our induced by ultraviolet light (UV). Genetic studies in the yeast laboratory. Here, we present our results, which indicate that Saccharomyces cerevisiae have identified seven genes, RADl, the incision step of NER can be accomplished by combining the RAD2, RAD3, RAD4, RADIO, RAD14, and RAD25, that are following highly purified protein components: RadI4, Rad4­ essential for NER (1). To begin to define the biological roles of Rad23 complex, Radl-RadiO complex, Rad2, RPA, and TFIIH. these genes, we have purified their encoded proteins from yeast The damage-specific incision reaction has a strict dependence and characterized their biochemical activities. This undertak­ on ATP, and our results imply that the two incision nicks are ing has allowed us to infer the involvement of these proteins in made in a highly coordinated fashion. different steps of the incision process, viz. in damage recogni­ tion, in DNA unwinding, and in dual incision of the damaged MATERIALS AND METHODS DNA strand. RadI4 is a zinc metalloprotein with an affinity for UV Irradiation and AAF Treatment of Plasmid DNA-Replicative form M13 mp18 DNA (~90% supercoiled form) was purified from in­ * This work was supported by Grants CA35035 and CA41621 from fected Escherichia coli strain JM101 by two rounds of cesium chloride the National Cancer Institute and Grant DE-FG03-93ER61706 from banding. The DNA, at a concentration of 100 ILg/ml in TE (10 mM the Department of Energy. The costs of publication of this article were Tris-HCI, pH 7.0, 0.2 mM EDTA), was irradiated in 15-ILI droplets for 60 defrayed in part by the payment of page charges. This article must s with a germicidal lamp emitting at 254 nm and a fluence rate of 5 therefore be hereby marked "advertisement" in accordance with 18 2/s J/m to introduce ~2.5 photoproducts/1000 base pairs of DNA. For U.S.C. Section 1734 solely to indicate this fact. treatment with N-acetoxy-2-aminoacetylfluorene (AAF), the plasmid :j:To whom correspondence should be addressed: Sealy Center for DNA, 10 ILg or 30 nmol of nucleotides, was incubated at 37°C for 12 h Molecular Science, University of Texas Medical Branch, 6.104 Medical in the dark with 0.5 nmol ofAAFin 200 ILl of50 mM sodium acetate, pH Research Bldg., 11th & Mechanic St., Galveston, TX 77555-1061. Tel.: 5.8. After extraction with diethyl ether, the DNA was purified by 409-747-8602; Fax: 409-747-8608. ethanol precipitation and redissolved in TE to 100 ILg/ml. 1 The abbreviations used are: NER, nucleotide excision repair; Reconstitution of NER in Vitro--(i) In the incision assay, reaction PAGE, polyacrylamide gel electrophoresis; RPA, replication protein A; mixtures (10 ILl, final volume) were assembled in buffer R (45 mM ATP-yS, adenosine 5'-O-(3-thiotriphosphate); AAF,N-acetoxy-2-amino­ acetylfluorene. K-HEPES, pH 7.9, containing 8 mM MgCI , bovine serum albumin at This is an Open Access article under the CC BY license. 12974 Reconstitution of Yeast Nucleotide Excision Repair 120 JLg/ml , 1.5 mxi dithiothreitol, 2 mxt ATP, a n ATP-regenerating Rad.flRad23 TH ill syste m consisting of 30 mxt creatine phosphate and 200 ng of creatine Radl~ Purffl cation Purification l'urtflcat km kinase, a nd 60 rnxt potassium acetate a nd 20 rnxt KCl th at were du e to addition of the vario us NE R pr otein factors) a nd contained 100 ng of TFIIH, 50 ng of RPA , 8 ng of Rad 1-Rad10 complex , 10 ng of Rad 2 Clarified Extract Clarified Extract C larified Extract protein, 20 ng of Rad4 -Rad2 3 complex, a nd 10 ng of Rad1 4 protein. Th e Rad1-Rad1 0 comp lex for use in these studies was form ed by in cubating Ammonium Ammonium Hie -Rex 70 equimola r a mounts of purified Rad1 a nd RadIO pr oteins for 24 h on ice sulfate preci pitation sulfate prec ipitation in 20 mxt Tri s-HCl, pH 7.5, containing 1 mxi dithiothreitol a nd 500 I I ug/m l bovin e se rum albumin to give a final concentration of 1 JLM of th e DEAE Scphaccl Q Scpb•n ose Q SC p h<lTOSC protein complex. Th e combina tion of NE R factors were incubated at I I I 25 °C for 5 min before the undam aged DNA or dam aged DNA , 100 ng SP Scpharose SP Scpharosc Hydroxyapatite each, was added in 1 JLI. After incubation a t 30 °C for 10 min , SDS and protein ase K were added to 0.5% a nd 200 ug/m l, res pectively, followed I I Nickel Agarosc Hydroxyapatite Mono Q by a 5-min incubation at 37 °C to dep roteini ze th e reaction mixtures. Sa mples wer e run in 0.8% agarose ge ls in TAE buffer (20 mM Tris I I acetate, pH 7.4, 0.5 mxr EDTA). Th e gels were treate d with eth idiu m :'-10110 S Hydroxyapatite Mono S bromide (l ug/rnl in H"Q) to stain DNA, soa ked in a large volume of I I water to reduce background staining, a nd th en photogr aphed th rough a ~ 1 ()n () Q xtono S red filter. (ii ) For the excision assay, reacti on mixtures (50 JLI, final volume) were assemb led in buffer R an d contained 500 ng of TFIIH, 250 B c D ng of RPA, 40 ng of Rad1 -Rad10 complex , 50 ng of Rad2, 100 ng of Rad 4-Rad 23 complex, a nd 50 ng of Rad 14. Th e combination of NER 97 ­ ~ ~ pr otei ns was incubated at 25 °C for 5 min before 1 JLg of undam aged or 68 - 116 - UV-da maged DNA was added in 10 JLl of TE. Th e complete reaction 200 - -Rad25 mixtures were incub ated at 30 °C for 60 min a nd th en depro tein ized by 97 - 45 - 116 ­ - Rad3 ext ract ion wit h a n equa l volum e of buffered ph en ol. Th e DNA was - -TFBI 97 - 68 - , precipitated by etha nol a nd red issolve d in 10 JLl of TE , a nd 5 JLI of 31- 68- which was treated wit h 7.5 units of calf thymus terminal tra nsfera se <Boehringer Ma nnheim l wit h 5 JLCi of [,,-""Pldideoxy ATP (Ame rsha rn 2 1- _ Corp.; 5000 Ci/rnrnol) for 60 min a t 37 °C in a final volume of20 JLI of th e 45 - 45 - buffer su pplied by th e vendor . Th e labeling mixtures wer e deprotein­ 14 - _ - p38 M 1 2 3 ized by trea tment with 0.5% SDS a nd 300 JLg/ml pr otein ase K for 10 min M 1 2 3 at 37 °C, followed by the precip itation of t he DNA. Th e pellets were dissolved in 4 JLl of bu ffer and a nalyze d on 15% seque ncing gels. The FIG. 1. Protein factors for in vitro reconstitution of NER. A, gels wer e dried a nd exposed to x-ray film s (Koda k Bio Ma x MR ) to pu rifi cation schemes for Rad1 4, Rad 4-Rad 23 complex, a nd TFIIH. B-D, reveal th e excision DNA fra gments. SDS-PAGE of pu rified NER factors. B, RadlO (1 JLg in lane t ), Rad1 4 (1 JLg in lane 2), and RPA (2 JLg in lane 3) consisti ng of the 69-, 36-, a nd RESULTS 13-kD a subuni ts each marked by an asterisk wer e ru n in a 12% den a­ turing polyacrylamide ge l a nd stained with Cooma ssie Blue. M, molec­ Purification of NER Factors-RadIO protein was purified ular size standards . C, 1 JLg each of Rad l tlane l ), Rad 2 (lane 2 ), and th e - l ,OOO-fold to near homo gen eity (Fig. lB , lan e 1) from yeast complex of Rad4 (upper band marked by asterisk in lane 3) and Rad23 strain CMY135 harboring the overproducing plasmid pSU C8 (lower band mark ed by asterisk in lane 3) were run in a 7% denaturing polyacrylam ide gel a nd stai ned with Cooma ssi e Blue. M, molecular size as described (22). Th e RAD14 gene contains a n intron and standa rds. D, TFIIH (500 ng total protein ) was run in an 8% den aturing encodes a protein with a pr edicted size of 43 kDa, and it polyacrylamide gel a nd silver-stained with a kit pu rchased from Bio­ exhibits a size of 48 kDa in SDS-PAGE (23). Rad14 protein has Rad . Th e va rious subu nits of TFIIH a re indicated . Th e identi ty of been purified - l ,OOO-fold to near homogen eity (Fig. lB , lan e 2 ) subunits was verified by immunoblotting usin g affinity-purified anti­ from yeast strain YRPll harboring th e overproducing plasmid Rad25, a nti- Rad3, anti-TFB1, a nd a nti-SS Ll a ntibodies. pR14.l5 containing RAD14 under th e control of the yeast alco­ ity-purified anti-Rad23 antibodies (25), we have verified that hol dehydrogenase I (ADC1) promoter, with th e purification scheme outlined in Fig. lA . Radl protein was purified - 2,000­ this 57-kDa species was indeed Rad23 protein . Because in wild fold to near homogen eity (Fig. l C, lan e 1) from yeast stra in type yeast Rad23 is more abunda nt th an Rad4 (25), only over­ CMY135 harboring th e overproducing plasmid pRR168 as de­ production of Rad4 was necessary to purify the Rad4-Rad2 3 complex (Fig. l C, lan e 3). TFIIH was purified > lO,OOO-fold to scribed previously (9). Rad2 protein wa s purified - 3,000-fold to near homogen eity (Fig. l C, lan e 2) from yeast strain LY2 har­ near homogen eity (Fig. lD) from yeas t strain YPH!I'FB1.6HIS boring th e overpro ducing plasmid pR2.26 as described (11). To that contains a 6-histidine tag in th e TFBI subunit of TFIIH overprodu ce th e RAD4-en coded protein in yeast for purifica­ (26) as outlined in Fig. lA . Du ring purification, the elution of TFIIH from various chromatographic matrices was monitored tion, th e pr otein coding frame of RAD4 was fused to the yeas t AD C1 promoter to yield plasmid pR4.l, whi ch wa s introduced by immunoblotting usin g affinity-purified anti bodies specific for th e Rad 3, Rad2 5, TFBl, and SSLI subunits (23). The TFIIH into yea st stra in YRPl 1. Th e scheme pr esented in Fig. lA was used to purify Rad4 protein - 4,000-fold to near homogen eity pr eparation used in this study conta ined six subunits, Rad25, Rad 3, TFBl, SSLl , p55, and p38 (Fig. iz». RPA, consisting of (Fig. l C, lan e 3) from YRPll(pR4.l). Th e elution of Rad4 pro ­ tein from various chroma togra phic matrices was monitored by three subunits of 69, 36, and 13 kDa, was purified to near hom ogeneity (Fig. lB , lane 3) from yeast strain LP27 49-9B immunoblotting usin g affinity- purified polyclonal antibodies raised against an inso luble form of Rad4 protein expressed in using th e procedure of Brill and Stillman (27). ATP-dependent In cision of UV-damaged DNA-To examine insect cells with th e use of baculovirus (data not shown). Rad4 protein has a pred icted molecular ma ss of 87 kDa (24), but it wheth er th e purified yeast factors Rad14, Rad4-Rad2 3 com­ plex, Radl-RadlO complex, Rad2, RPA , and TFIIH are suffi­ migrates in SDS-polyac ryla mide gels with a relative molecular mass of 116 kDa. Through out th e purification of Rad4, we cient for reconstituting NER in vitro, th ey wer e incubated at 30 °C in buffer containing ATP with plasmid DNA previously observed a precise co-elution of a protein with a relative mo- . lecular mass of 57 kDa, which is th e same as that described for irradiated with UV light to introduce photoproducts into the DNA. Reaction mixtures were deproteini zed and ana lyzed by the RAD23 gene produc t (25) that plays an important role in the proficien cy of NER <1 , 25). By immunoblotting using a ffin- agarose gel electrophoresis, followed by sta ining with ethidium Reconstitution of Yeast Nucleotide Excision Repa ir 12975 -A,AF .AAF A DNA • UV + UV A DNA .--I. i r----11 I R1 R1 Factor omitted Bl None 81 None 110 R2 R14 A4 DB RPA Factor omined Bl None 81 None /10 R2 R14 R4J23 UH RPA 123 ~ -oc -oc _ _ __ _ _ _ _ 0- 4 _ -sc -sc - - - ---- - - 3 4 8 9 10 BONA -w +IN · AAF + AAF B ONA r--11 i r---1 1 NER Factors -+-++++++++ _+e+++++++ + NER Factors Nucleotide ATP ATP ATP ATP ADP dATP 'Y$ e l P GfP UTP Nucleotide ATP ATP ATP ATP • ADP dATP "1'5 crs GYP UTP -oc -oc - - - - -sc - - - -- ---- -sc --- -- - - - - 2 3 4 5 6 7 8 9 10 11 2 3 4 5 6 7 8 9 10 11 F IG. 2. ATP·dependent in cis ion of UV-damaged DNA. A, recon­ FIG. 3. ATP·dependent incision of DNA containin g AAF ad­ sti tution of the incision reaction using purified protein facto rs. Th e ducts. A, the NER factors incise AAF-cont ai ning DNA. The und amaged DNA used was either not treated (- UV , lan es 1 and 2) or trea ted with DNA (lan e 2) OJ' damaged DNA tla ne 4) was incubated with the Radl ­ UV (+UV , lan es 3- 10). The full complement of NE R factors <Rad l ­ Rad lO complex, Rad2, Rad4·Rad23 complex, Radl4, TF IIH , and RPA at RadI O complex, Rad2, Rad4-Rad 23 complex, Radl4, TFIIH, and RPA) 30 °C for 10 min in buffer containing ATP. The reaction mixtur es in was incubated with und am aged DNA (lane 2) and UV-damaged DNA lanes 5- 10 conta ined AAF·damaged DNA and ATP , but one of the NER (lane 4) in buffer containing ATP at 30 °C for 10 min . One of the factors was omitted; Rad l -RadlO complex was absent in lane 5, Rad2 afore ment ioned NE R factors was absent in th e reaction mixtu res in was absent in lan e 6, Rad14 was absent in lane 7, Rad4-Rad 23 complex lan es 5- 10 th at contai ned UV-damaged DNA and ATP; Radl-Radl0 was absent in lan e 8, TF IIH was absent in la ne 9, and RPA was absent complex was omitted in lan e 5, Rad2 was omitted in lan e 6, Rad1 4 was in lane 10. Bl , DNA incubated in buffer with out any of the NER factors itted in lan e 7, Rad4-Rad 23 complex was omitted in la ne 8, TF IIH om ilanes I an d 3). B, incision of AAF-cont aining DNA requi res ATP. The was omitted in lan e 9, an d RPA was omitted in lan e 10, as indica ted at full complement of NER factors (lane 2 and lanes 4 - 11) was incu bated th e top of the figu re. Bl , DNA incubated in buffer without any of the with either undam aged DNA (- AAF, lane 2 ) or with AAF-containing NER factors (lanes 1 an d 3). B, incision of UV-da mage d DNA requires DNA (+AAF, la nes 4 -11) in the presence of ATP (lan es 2 and 4 ), ADP ATP. Th e full complement of NER factors (lane 2 an d lanes 4-11) was (lane 6), dATP tlane 7), ATP yS ilane 8), CTP (lan e 9), GTP ilane 10), incubated wit h eit her und am aged DNA (- UV, lane 2) or with UV­ UTP (lan e 11), or without any nucleotid e tlane 5) at 30 °C for' 10 min. In dam aged DNA (+UV , lan es 4· 11) in the pre sence of ATP (lanes 2 and 4), lanes 6 and 8, creatine kina se was omitted to inacti vate the ATP­ ADP (lan e 6), dATP (lan e 7), ATPyS (lane 8), CTP (lane 9), GTP (lane rege nerating sys tem. No NER factors wer e added in lanes I and 3. S C, 10), UTP (lane 11), or without any nucleotide (lane 5) at 30 °C for 10 supercoiled form; ac, open circular form. min. In lanes 6 and 8, creatine kinase was omitted to inactivate the ATP-regenerating system. No NER factors were added in lan es 1 and 3 . 2B) . Interestingly, th e non-hydro lyzable ATP ana log ATPyS is SC, supercoiled form; ac, open circula r form . also inactive (Fig. 2B , lan e 8), suggesting that ATP hydrolysis is in fact required for the incision reaction. Furth ermore, Mg + br omide to exa mine the fate of the DNA substrate. Fig. 2A is also required for incision (data not shown). shows th at whe n a ll the purified NER factors were present, In cision ofDNA Damaged by AAF-The NER machinery has greate r than 80% of the UV-da maged superco iled plasmid DNA specificity for a variety of DNA lesions. We examined whether was converte d to the open circular form (lane 4), indicating th at our pu rified NER protein s would also incise DNA dam aged by incision of th e dam aged DNA had occurred. By contrast, plas­ N-acetoxy-2-aminoacetylflu oren e. As shown in Fig. 3A, plas­ mid DNA that had not been subjecte d to UV treatme nt was not mid DNA containing the AAF adduct was incised when all th e acte d on by th e same protein factors (Fig . 2A, lane 2). Thus, the purified protein factors were pr esent (lane 4) bu t not whe n any combination of NER protein s carries out an incision reaction of th e fact ors was omitte d from th e reaction mixture (lanes that is highl y specific for UV dam age. Importantly, omission of 5- 10). Th e incision of AAF-modified DNA also has a specific any of the comp onents Radl-RadlO complex, Rad 2, Radl4, requirement for ATP or dATP (Fig. 38). ATP yS did not promote Rad4-Rad 23 complex , TFIIH, or RPA from the reaction mixture incision (Fig. 38), again suggesting a requirement for ATP abolishe d the formation of the open circular form (Fig. 2A, hydrolysis in the repair of AAF-dam aged DNA. lan es 5-10), indicating that incision of UV-dam aged DNA re­ S ize of Excision DNA Fragments- NE R in human s has been qui res all of th ese pu rifi ed pr otein factors . It is of particular shown to occur by way of du al incision of th e DNA strand that interest that nicking of the UV-damaged DNA did not occur contains th e lesion, resulting in th e release of a DNA fragment whe n eithe r the Radl-RadlO endonuclease or th e Rad2 endo­ 27- 29 nucl eotides in length (28). To detect the excision DNA nucl ease was omitte d from th e reaction mixture (Fig. 2A, lanes fragment, we incubated plasmid DNA dam aged by UV light in 5 and 6). Th is obse rvation strongly suggests th at both th e the reconstituted re pair syste m. Following phenol extraction Radl-Radl0 complex and the Rad 2 protein , in addition to bein g to remove repai r pr otein s, the DNA was purified and treated th e endonucleolyt ic comp onents, have a pivotal role in th e with calf th ymus terminal transferase in the presence of 32P proper assembly of the incision enzyme compl ex at the damage [a- ldid eoxy ATP to label any excision DNA fragments that site, thus ensuring that th e two incision nicks are mad e in a might have been generate d. As shown in Fig. 4, a series of DNA coordinated fashion. fragments rangin g in size from 25 to 28 nucleotid es was de­ Because Rad 3 and Rad2 5 protein s both possess an ATP tected in the reaction mixture th at conta ined UV-da maged (dATP)-dependent helicase activity th at is required for NER (7, DNA as substrate (lane 5) but not in th e cont rol that contained 13, 14), it was of considerabl e importance to determine whether undam aged DNA (lane 2). Importantly, th e DNA fragments the incision of dam aged DNA in our reconstituted sys te m re­ were not produced if ATP was absent (Fig. 4, lane 6) or whe n qui res ATP . We found th at in the abse nce of ATP , the inci sion TFIIH was omitted from the repair reaction (Fig. 4, lan e 4 ). of UV-da maged DNA does not occur (Fig. 2B , compare lanes 4 Because the :J2P-labeling protocol added one nucl eotide to the and 5). Whereas dATP is as effective as ATP in pr omoting th e excision DNA fra gment s, the actual size ra nge of these frag­ incision reaction, ADP , CTP, GTP, and UTP are inactive (Fig. ments is 24-27 nucleotid es. 12976 Reconstitution of Yeast Nucleotide Excision Repair - Uv + UV DNA th at th e en donucleolytic sciss ions at th e dam age site occur in a ,------, AlP + + nated manner, as well as to minimize gratuitous nicking coordi FaclorOmitted BI none 81 lUi none none of DNA. In summary, the followin g major conclusion s emerge from this work. First, th e damage recognition factor Radl4, the Rad4-Rad2 3 complex, the Radl-Radl0 endonuclease, th e Rad2 endonuclease, RPA, and TFIIH are all essential for the incision step ofNER. Second , becau se of th e high degr ee of purity of th e protein fact ors used, we infer that th e combination of th ese protein s is sufficient for th e incision reaction. Thi rd , th e inci­ sion reaction occurs only in the pr esence of ATP , and our - 30 results strongly sugges t a requirement of ATP hydrolysis in this reaction. Finally, our obse rvation that th e size of the excision fragme nt produced by th e yeas t incision enzyme com­ -25 plex resembles that in human s indicates that th e yeast and human NER machineri es act in a highly similar manner. Ackn owledgm ent s-We are very grateful to A. Sancar and colleagues -20 for communicat ion of th eir results prior to public ati on and to W. J . Feaver and R. D. Kornberg for yea st stra in YPH!I'FB1.6HIS, for th e E. coli plasmid th at expresses 6-histidine tagged SSLl, and for a sa mple of ank S. Wilson for help­ purified 6-histidine tagged SSLI protein . We th ful discussions and A. McCullough for anti-SSLI antibodies. 2 3 4 5 6 REFERENCES FIG. 4. Size of the excision DNA fragments. UV-irra diated (lanes 1. Prak ash , S., Sung, P., a nd Pr ak ash , L. (1993) Annu. Rev. Genet. 27, 33- 70 3-6) and uni rr adi at ed (lanes 1 and 2) plasmid DNAs were incubated 2. Guzder, S. N., Sung, P., Pr akash . L., and Pra kash , S. (1993) Proc. Na tl. Acad . with the full complement of NER factors (lanes 2, 5, and 6) in th e Sci. U. S. A. 90, 5433- 5437 3. Su ng, P., Pra kash , L., Weber, S., and Prak ash , S. (1987) Proc. Na tl. A cad . Sci. presence of ATP ttanes 2 and 5) or in its absence (lane 6). Th e reacti on U. S. A. 84, 6045-6049 lane 4 contained ATP but lacked TFIIH. The numbers to th e mixtu re in 4. Sung, P., Pr ak ash , L., Matson, S. W., a nd Pr akas h, S. (1987) Proc. Na tl. Acad . right of the autora diogra m indicate th e positi ons in nuc1eotides of th e Sci. U. S. A. 84, 8951- 8955 DNA marker s used. Bl , DNA incubat ed in buffer without any of th e 5. Bailly, V., Sung, P., Prakash, L., and Prak ash , S. (1991) Proc. Na tl. Acad . Sci. NER factors (lanes 1 and 3). U. S. A. 88, 9712-9716 6. Sung, P., Watkins, J . F., Pra kash, L., and Pr akash, S. (1994) J. Bioi. Chem. 269, 8303-8308 DISCUSSION 7. Guzder, S., Sung, P., Bailly, V., Prakash , L., a nd Prakash, S. (1994) Na ture 369, 578 - 581 In this work we have achi eved th e reconstitution of a syste m 8. Bailly, V., Sommer s, C. H., Sung, P., Pr ak ash , L., a nd Prakash , S. (1992 ) Proc. th at mediates du al inci sion of DNA damaged eithe r by UV or N atl. A cad . Sci. U. S. A. 89, 8273- 8277 9. Sung, P., Reynolds, P., Prakash, L., and Prak ash , S. (1993)J. Bivl. Chern. 268, AAF, by combining the following essentially homogeneous 2639 1-26399 S. cerevisiae factors: Radl-Radl0 complex, Rad2, Rad4-Rad23 10. Tomki nson, A. E., Bard well, A. J ., Bardwell, L., Tap pe, N. J ., and Fri edberg, E. complex, Radl4, RPA, and TFIIH consisting of Rad3, Rad25, C. (1993 ) Nature 362, 860-862 11. Habraken , Y., Su ng, P., Prak ash, L., a nd Pr a kash, S. (1993 ) Na ture 366, TFBl, SSLl, p55, and p38 subunits . Sinc e all these purified 365-368 protein factors are indispen sable for damage-specific inci sion , 12. Guzder , S. N., Qiu, H., Sommer s, C. H., Sung, P., Prakash , L., and Pr ak ash , S. (1994) Na ture 367, 91-94 th ey represent th e minimum set of factors for accomplishing 13. Qiu, H., Park, E., Pra kash, L., a nd Pra kash, S. (1993) Gelles & Dev. 7, th is reaction. 2161-2171 14. Sung, P., Higgins, D., Prakash, L., and Prakash, S. (1988) EMBO J. 7, In our reconstituted system, inci sion of dam aged DNA shows 3263-3269 a strict dependenc e on ATP or dATP, whose hydrolysis is re­ 15. Feaver , W. J ., Svejst rup , J . Q., Bard well, L., Bardwell, A. J ., Bura towski, S., quired for th e repair reaction because the non-hydrolyzable Gulyas, K D., Donah ue, T. F., Fri edberg, E. C., a nd Korn berg, R. D. (1993) Cell 75, 1379 - 1387 ana log ATP yS does not promote th e inci sion of damaged DNA. 16. Wan g, Z., Svejstrup, J . Q., Feaver , W. J ., Wu, X., Kornb erg, R D., a nd Th e requi rement for ATP and its hydrolysis in th e incision ste p Friedb erg, E. C. (1994) Na ture 368, 74- 76 of NER is consistent with the results from genetic studies 17. Drapkin, R , Reardon, J . T., Ansa ri, A., Huan g, J . C., Zawel, L., Ahn, K , Sa ncar, A., an d Reinberg, D. (1994) Na ture 368, 769-772 conducted with mutan t varia nts of Rad3 and Rad25 protein s 18. van Vuu ren , A. J ., Verm eulen, W., Ma, L., Weeda, G., Appeldoorn , E., J aspers, that are defective in ATP hydrolysis (7, 13, 14). We suggest that N. G. J ., va n del' Eb, A. J ., Bootsma , D., Hoeijm ak er s, J . H. J ., Humbert , S., Schaeffer, L., and Egly, J .-M. (1994) EMBO J . 13, 1645-1653 at the expense of ATP hydrolysis, th e combined helicase func­ 19. Coverley, D., Kenn y, M. K , Lan e, D. P., a nd Wood, R D. (1992) N ucleic Acid s tion of Rad 3 and Rad25 create s a single-stranded region at the Res. 20, 3873-3880 dam age site for du al incisi on by th e Radl-Radl0 and Rad2 20. Clugston, C. K , McLaughlin, K , Kenny, M. K , and Brown , R. (1992) Cancer Res. 52, 6375-6379 endonucleases . 21. Mu, D., Pa rk, C.-H., Matsunaga, T., Hsu , D. S., Reardon, J. T., a nd Sa ncar, A. It is intriguing that nickin g of th e damaged DNA does not (1995 ) J . BioI. Chem . 270, 2415- 2418 22. Sung, P., Prak ash , L., a nd Pr ak ash , S. (1992 ) Nature 355, 743- 745 occur if either th e Radl-Radl0 endonuclease or the Rad2 en­ 23. Guzder, S. N., Bailly, V., Sung , P., Prakash, L., and Pra kash, S. (1995)J. BioI. donu clease is present alone with th e remainder of th e incision Chern. 270 , 8385-8388 NER components. Thi s finding st rongly suggests th at in addi­ 24. Gietz, R D., a nd Prakash , S. (1988 ) Gene (A rnst.) 74, 535-54 1 25. Wat kin s, J . F., Sung, P., Prakash, L., and Prakash, S. (1993) Mol. Cell. BioI. tion to providing th e endonucleolytic activities for dual incision , 13, 7757- 7765 th e Radl-Radl0 protein comp lex and th e Rad2 protein are also 26. Svejst rup, J . Q., Feav er , W. J ., LaP oint e, J ., and Korn berg, R. D. (1994 )J . BioI. Chern. 269 , 28044 - 28048 required for th e proper assembly of th e NER ensemble at th e 27. Brill, S. J ., an d Stillman, B. (1989) N ature 342, 92- 95 dam age site . Th e involvement of the two endonucleolytic com­ 28. Hua ng, J. C., Svoboda , D. L., Reardon, J . T., a nd Sa nca r, A. (1992) Proc. Na tl. ponents in as sembling the NER complex may serve to ensure Acad. Sci. U. S. A. 89, 3664 - 3668

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