5
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10718-10722, October 1996 Biochemistry Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: Implications for nucleotide excision repair and Cockayne syndrome YVETrE HABRAKEN, PATRICK SUNG, SATYA PRAKASH, AND LOUISE PRAKASH* Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061 Communicated by Stephen J. Lippard, Massachusetts Institute of Technology, Cambridge, MA, July 26, 1996 (received for review May 29, 1996) ABSTRACT Nucleotide excision repair (NER) of ultravi- olet light-damaged DNA in eukaryotes requires a large num- ber of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essen- tial NER factors. Neither of the two endonucleases, Radl- RadlO and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Radl-RadlO forms a ternary complex with the DNA damage recognition protein Radl4, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human R4D2 counterpart, XPG, result in Cockayne syn- drome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant <3.3 x 10-9 M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome. In humans, a defect in nucleotide excision repair (NER) of ultraviolet (UV)-damaged DNA results in the cancer prone syndrome xeroderma pigmentosum (XP). Because of the evolutionary conservation of NER factors, genetic and bio- chemical studies in the yeast Saccharomyces cerevisiae have been highly informative in helping delineate the mechanism of NER in eukaryotes. The ATP-dependent dual incision of UV-damaged DNA has been reconstituted in S. cerevisiae using the combination of the DNA damage recognition protein Radl4, the Rad4-Rad23 complex, the heterotrimeric replica- tion protein A (RPA), the six subunit transcription factor TFIIH, the Radl-RadlO endonuclease, and the Rad2 endo- nuclease (1). The combination of the human equivalents of these yeast proteins is also sufficient for dual incision of UV-damaged DNA (2). To understand the molecular details of how such a large body of NER proteins cooperate to accomplish incision of UV-damaged DNA, it is of fundamental importance to first determine whether the NER proteins essential for damage- specific incision are organized into distinct subassemblies, or whether they exist as one large physical complex or "repairo- some," as suggested by Svejstrup et al. in an earlier study (3). Our recent studies have provided strong support for the idea that NER occurs by the incorporation of distinct protein subassemblies at the damage site (4) rather than by a "pre- formed repairosome" consisting of TFIIH and all the other essential NER factors (3). One such protein subassembly that we have identified is the ternary complex of the damage recognition protein Rad14 and the Radl-RadlO endonucle- ase, and we have designated this subassembly as nucleotide excision repair factor 1 (NEF1) (4). Because the Radl-RadlO binary complex has no affinity for UV-damaged DNA (5), association with Radl4 provides an effective means for tar- geting the Radl-RadlO nuclease activity to the damage site. The Rad4 and Rad23 proteins form another subassembly, called NEF2 (1, 4). Because Rad2 shows no affinity for UV-damaged DNA (6), the manner by which this nuclease is recruited into the NER machinery has remained unclear. Here, we show that Rad2 in fact exists in a stable complex with TFIIH, which we designate as NEF3. The identification of Rad2 as a component of NEF3 completes the picture of organization of essential NER pro- teins into functional protein subassemblies and this enables us to suggest a plausible scheme by which the NER machinery assembles at the DNA damage site. In addition, our finding that Rad2 is specifically associated with TFIIH provides an explanation for the association of Cockayne syndrome (CS) with mutations inXPG, the human counterpart of yeast RAD2. MATERIALS AND METHODS Purification of NEF3. The buffers used were as described (7). Extract was prepared from 1 kg of yeast strain YPH/ TFB1.6HIS harboring the multicopy plasmid pRR162, which contains the RAD2 gene under the control of the alcohol dehydrogenase I (ADCI) promoter. Clarified cell lysate was subjected to fractionation in columns of Bio-Rex 70, DEAE Sephacel, and hydroxyapatite as described (7). Hydroxyapatite fractions 20-26, which contained the TFIIH peak and "40% of the total Rad2 protein, were pooled and dialyzed against buffer E. The dialysate was mixed gently with 1.1 ml of nickel NTA-agarose at 4°C for 3 h, centrifuged to collect the affinity matrix, which was transferred into a glass column with an internal diameter of 0.6 cm, and then washed with 4 ml each of 10, 20,30,40, and 100 mM imidazole in buffer E. The 40 mM and 100 mM imidazole eluates were combined, concentrated to 2 ml, diluted with two volumes of buffer F, and fractionated in a Mono S column (HR5/5), using a 20-ml gradient of 100-600 mM KOAc in buffer F. The Rad2-TFIIH peak (fraction V; eluting at -330 mM KOAc; total 3 ml) was applied Abbreviations: NER, nucleotide excision repair; XP, xeroderma pig- mentosum; NEF, nucleotide excision repair factor; CS, Cockayne syndrome; RPA, heterotrimeric replication protein A. *To whom reprint requests should be addressed at: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Med- ical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. e-mail: [email protected]. 10718 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on March 19, 2021

Transcription TFIIH DNA Rad2 3: Implications Cockayne · Rad2 protein was mixed with 50,ug bovine serum albumin (BSA)in200,ul ofbufferE,andthenfilteredthroughacolumn ofSephacrylS-300HR(1

  • Upload
    others

  • View
    2

  • Download
    0

Embed Size (px)

Citation preview

Page 1: Transcription TFIIH DNA Rad2 3: Implications Cockayne · Rad2 protein was mixed with 50,ug bovine serum albumin (BSA)in200,ul ofbufferE,andthenfilteredthroughacolumn ofSephacrylS-300HR(1

Proc. Natl. Acad. Sci. USAVol. 93, pp. 10718-10722, October 1996Biochemistry

Transcription factor TFIIH and DNA endonuclease Rad2constitute yeast nucleotide excision repair factor 3: Implicationsfor nucleotide excision repair and Cockayne syndromeYVETrE HABRAKEN, PATRICK SUNG, SATYA PRAKASH, AND LOUISE PRAKASH*Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061

Communicated by Stephen J. Lippard, Massachusetts Institute of Technology, Cambridge, MA, July 26, 1996 (received for review May 29, 1996)

ABSTRACT Nucleotide excision repair (NER) of ultravi-olet light-damaged DNA in eukaryotes requires a large num-ber of highly conserved protein factors. Recent studies in yeasthave suggested that NER involves the action ofdistinct proteinsubassemblies at the damage site rather than the placementthere of a "preformed repairosome" containing all the essen-tial NER factors. Neither of the two endonucleases, Radl-RadlO and Rad2, required for dual incision, shows any affinityfor ultraviolet-damaged DNA. Radl-RadlO forms a ternarycomplex with the DNA damage recognition protein Radl4,providing a means for targeting this nuclease to the damagesite. It has remained unclear how the Rad2 nuclease istargeted to the DNA damage site and why mutations in thehuman R4D2 counterpart, XPG, result in Cockayne syn-drome. Here we examine whether Rad2 is part of a higherorder subassembly. Interestingly, we find copurification ofRad2 protein with TFIIH, such that TFIIH purified from astrain that overexpresses Rad2 contains a stoichiometricamount of Rad2. By several independent criteria, we establishthat Rad2 is tightly associated with TFIIH, exhibiting anapparent dissociation constant <3.3 x 10-9 M. These resultsidentify a novel subassembly consisting of TFIIH and Rad2,which we have designated as nucleotide excision repair factor3. Association with TFIIH provides a means of targeting Rad2to the damage site, where its endonuclease activity wouldmediate the 3' incision. Our findings are important forunderstanding the manner of assembly of the NER machineryand they have implications for Cockayne syndrome.

In humans, a defect in nucleotide excision repair (NER) ofultraviolet (UV)-damaged DNA results in the cancer pronesyndrome xeroderma pigmentosum (XP). Because of theevolutionary conservation of NER factors, genetic and bio-chemical studies in the yeast Saccharomyces cerevisiae havebeen highly informative in helping delineate the mechanism ofNER in eukaryotes. The ATP-dependent dual incision ofUV-damaged DNA has been reconstituted in S. cerevisiaeusing the combination of the DNA damage recognition proteinRadl4, the Rad4-Rad23 complex, the heterotrimeric replica-tion protein A (RPA), the six subunit transcription factorTFIIH, the Radl-RadlO endonuclease, and the Rad2 endo-nuclease (1). The combination of the human equivalents ofthese yeast proteins is also sufficient for dual incision ofUV-damaged DNA (2).To understand the molecular details of how such a large

body of NER proteins cooperate to accomplish incision ofUV-damaged DNA, it is of fundamental importance to firstdetermine whether the NER proteins essential for damage-specific incision are organized into distinct subassemblies, orwhether they exist as one large physical complex or "repairo-some," as suggested by Svejstrup et al. in an earlier study (3).

Our recent studies have provided strong support for the ideathat NER occurs by the incorporation of distinct proteinsubassemblies at the damage site (4) rather than by a "pre-formed repairosome" consisting of TFIIH and all the otheressential NER factors (3). One such protein subassembly thatwe have identified is the ternary complex of the damagerecognition protein Rad14 and the Radl-RadlO endonucle-ase, and we have designated this subassembly as nucleotideexcision repair factor 1 (NEF1) (4). Because the Radl-RadlObinary complex has no affinity for UV-damaged DNA (5),association with Radl4 provides an effective means for tar-geting the Radl-RadlO nuclease activity to the damage site.The Rad4 and Rad23 proteins form another subassembly,called NEF2 (1, 4).

Because Rad2 shows no affinity for UV-damaged DNA (6),the manner by which this nuclease is recruited into the NERmachinery has remained unclear. Here, we show that Rad2 infact exists in a stable complex with TFIIH, which we designateas NEF3. The identification of Rad2 as a component of NEF3completes the picture of organization of essential NER pro-teins into functional protein subassemblies and this enables usto suggest a plausible scheme by which the NER machineryassembles at the DNA damage site. In addition, our findingthat Rad2 is specifically associated with TFIIH provides anexplanation for the association of Cockayne syndrome (CS)with mutations inXPG, the human counterpart ofyeast RAD2.

MATERIALS AND METHODSPurification of NEF3. The buffers used were as described

(7). Extract was prepared from 1 kg of yeast strain YPH/TFB1.6HIS harboring the multicopy plasmid pRR162, whichcontains the RAD2 gene under the control of the alcoholdehydrogenase I (ADCI) promoter. Clarified cell lysate wassubjected to fractionation in columns of Bio-Rex 70, DEAESephacel, and hydroxyapatite as described (7). Hydroxyapatitefractions 20-26, which contained the TFIIH peak and "40%of the total Rad2 protein, were pooled and dialyzed againstbuffer E. The dialysate was mixed gently with 1.1 ml of nickelNTA-agarose at 4°C for 3 h, centrifuged to collect the affinitymatrix, which was transferred into a glass column with aninternal diameter of 0.6 cm, and then washed with 4 ml eachof 10, 20,30,40, and 100mM imidazole in buffer E. The 40mMand 100 mM imidazole eluates were combined, concentratedto 2 ml, diluted with two volumes of buffer F, and fractionatedin a Mono S column (HR5/5), using a 20-ml gradient of100-600 mM KOAc in buffer F. The Rad2-TFIIH peak(fraction V; eluting at -330mM KOAc; total 3 ml) was applied

Abbreviations: NER, nucleotide excision repair; XP, xeroderma pig-mentosum; NEF, nucleotide excision repair factor; CS, Cockaynesyndrome; RPA, heterotrimeric replication protein A.*To whom reprint requests should be addressed at: Sealy Center forMolecular Science, University of Texas Medical Branch, 6.104 Med-ical Research Building, 11th and Mechanic Streets, Galveston, TX77555-1061. e-mail: [email protected].

10718

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

9, 2

021

Page 2: Transcription TFIIH DNA Rad2 3: Implications Cockayne · Rad2 protein was mixed with 50,ug bovine serum albumin (BSA)in200,ul ofbufferE,andthenfilteredthroughacolumn ofSephacrylS-300HR(1

Proc. Natl. Acad. Sci. USA 93 (1996) 10719

onto MonoQ (HR5/5), which was developed with a 12-ml,300-1500 mM KOAc gradient in buffer F, collecting 0.3-mlfractions. The peak of Rad2-TFIIH (fraction VI; 1.2 ml total),eluting at '1000 mM KOAc, was concentrated to 100,ul.

Molecular Sizing. For the sizing analysis, 5 jig of purifiedRad2 protein was mixed with 50,ug bovine serum albumin(BSA) in 200,ul of buffer E, and then filtered through a columnof SephacrylS-300 HR (1 x 42 cm; total 33 ml matrix) at 0.1ml/min in bufferE, collecting 0.5 ml fractions. For sizing theRad2-TFIIH complex, 150,ul of the combined and concen-trated 40 and 100 mM imidazole eluates from nickel NTA-agarose (see above) containing -5,ug of the Rad2-TFIIHcomplex was mixed with 50 Ag BSA and filtered through theS-300 column using the conditions described for Rad2 protein.

Immunoprecipitation. Affinity purified antibodies werecross-linked to protein A agarose beads at 2 mg antibodies perml matrix for use in immunoprecipitation, as described (8). InFig. 2C,S-300 fractions 29-34 from the Rad2-TFIIH complexsizing analysis were combined and 300,ul of the pool was mixedwith 6,ul of anti-Rad2, anti-Rad3, or anti-Rad5l immunobeadsfor 3 h at 4°C. Likewise,S-300 fractions 36-41 from the Rad2sizing analysis were combined and mixed with the variousimmunobeads. The beads were washed twice with 200,ul ofbuffer E and then once with buffer F, before bound proteinswere eluted using 10 Al of 2% SDS at 37°C for 10 min. In Fig.2E, 1 ml of the S-300 Rad2-TFIIH pool was mixed with 15,ulof anti-Rad2 immunobeads for 3 h at 4°C to bind the proteincomplex. The beads were then washed sequentially at 25°C for45 s each with 20,ul of 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, and 1%SDS. The various SDS eluates were analyzed by immunoblot-ting for their content of Rad2 and TFIIH.NER in Vitro. The NER reaction was carried out essentially

as described (1, 4, 7). Briefly, 80 ng RPA, 20 ng of theRadl-RadlO complex, 30 ng of the Rad4-Rad23 complex, 15ng of the Radl4 protein, 120 ng of the Rad2-TFIIH complex,and with or without 20 ng of the Rad2 protein, were prein-cubated at 25°C for 10 min in 9 Al of buffer R, followed by theincorporation of 100 ng ofM13mpl8DNA (>90% supercoiledform) that had or had not been irradiated with 400 J/m2 UVlight in 1,lA of H20. For detecting excision fragments, the NERreaction was scaled up as described (1, 4, 7). After incubationat 30°C, reaction mixtures were processed for agarose gelelectrophoresis and for 32P-labeling as described (1, 4, 7).

RESULTS

Copurification of Rad2 with TFIIH. In normal yeast cells,Rad2 protein is of much lower abundance than other NERproteins (data not shown). However, the levels of the RAD2transcript and protein are elevated upon exposure of cells toDNA damaging agents (9, 10). We observed that some prep-arations of TFIIH from normal yeast cells contained a sub-stoichiometric amount of Rad2 protein, but no Radl, Rad4,RadlO, or Radl4 (data not shown). This suggested to us thatperhaps Rad2 was physically associated with TFIIH, and thatthe sub-stoichiometric level of Rad2 might have been due tothe lower cellular abundance of Rad2.To investigate this issue further, we used yeast strain YPH/

TFB1.6HIS (11) containing a 6-histidine tag in the aminoterminus of the TFB1 protein and also harboring the plasmidpRR162 (ADCI-RAD2), which expresses the Rad2 proteinfrom the alcohol dehydrogenase I (ADCI) promoter. As aresult of Rad2 overproduction, the level of Rad2 protein is nolonger limiting relative to TFIIH. Extract from YPH/TFB1.6HIS (pRR162) was subjected to fractionation in col-umns of Bio-Rex 70, DEAE Sephacel, and hydroxyapatite asdescribed (7). Immunoblot analysis of the 50 hydroxyapatitefractions revealed that TFIIH, as identified by the content ofthe Rad3, Rad25, TFB1, and SSL1 proteins, was present infractions 20-28, and that the peak of Rad2 protein was in

fractions 20-34. Fractions 20-26 containing the bulk of TFIIHand -40% of total Rad2 protein were pooled, dialyzed, andmixed with nickel agarose to immobilize TFIIH via the 6-his-tidine tag on TFB1. After affinity binding, the nickel matrixwas washed sequentially with 10, 20, 30, 40, and 100 mMimidazole, and the various eluates were subjected toimmu-noblotting to determine their content of TFIIH and Rad2protein. As shown in Fig.1A, the bulk of the onput TFIIH wasfound in the 40 mM and 100 mM imidazole eluates, repre-senting 15% and 75% of the total, respectively. Interestingly,a substantial proportion ('60%) of the Rad2 protein in theonput coeluted with TFIIH in the 40 mM and 100 mMimidazole washes, representing -10% and '50% of the total,respectively. Since purified Rad2 protein binds nickel agarosevery weakly and is eluted quantitatively from the affinitymatrix by 10-20 mM imidazole under the same conditions(Fig. 1B), it seemed likely that Rad2 protein present in the 40mM and 100 mM imidazole eluates during TFIIH purificationwas physically associatedwith TFIIH.Rad2 Is Stably Associated with TFIIH. Purified Rad2

protein, Mr 118,000 (9), behaves as a monomer in molecularsizing (6), emerging from the SephacrylS-300 HR column (seeMaterials and Methods) in fractions 36-42, corresponding to54.5%-63.6% of the column volume (Fig. 2A). Because anassociation of Rad2 with TFIIH would add '400 kDa (the sumof the molecular weights of all the known TFIIH subunits) tothe former, we could use molecular sizing to address whetherthere is a complex of Rad2 with TFIIH. To do this we subjectedthe combined 40 mM and 100 mM imidazole eluates from thenickel agarose step, which contained TFIIH and Rad2 protein(Fig. 1A), to sizing in the S-300 column. As shown in Fig. 2B,immunoblotting of the S-300 fractions revealed that (i) theRad2 protein peak emerged much earlier than free Rad2, infractions 30-34 corresponding to 45.4%-51.5% of the columnvolume and (ii) the content of TFIIH, as judged by the levelsof Rad3 and TFB1 proteins, paralleled the amount of Rad2 inthe S-300 fractions; observations that were consistent withRad2 being complexed with TFIIH.To demonstrate that Rad2 was in fact associated with

TFIIH, the S-300 pool containing TFIIH and Rad2 was

A Imidazole (mM)ST SP 10 20 30 40100

.'..'.. .: , .........

- Rad2- Rad25- Rad3-TFBI- SSLI

B Imidazole (mM)ST SP 10 20 30 40 100O Sam--Rad2

FIG. 1. Affinity chromatography on nickel agarose reveals copu-rification of Rad2 with TFIIH. (A) A hydroxyapatite pool containingTFIIH and Rad2 protein was mixed with nickel agarose and cen'tri-fuged to collect the affinity matrix, which was washed in a column with10, 20, 30, 40, and 100 mM imidazole, as described. The startinghydroxyapatite pool (ST), the supernatant from the centrifugation step(SP), and the various imidazole eluates were analyzed by immuno-blotting for their content of Rad2, Rad3, Rad25, TFB1, and SSLiproteins. (B) Purified Rad2 protein (20 ,ug in buffer E) was mixed withnickel agarose and processed as described in A. The starting material(ST), the supernatant from the centrifugation step (SP), and thevarious imidazole washes were analyzed by immunoblotting for theircontent of Rad2.

Biochemistry: Habraken et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

9, 2

021

Page 3: Transcription TFIIH DNA Rad2 3: Implications Cockayne · Rad2 protein was mixed with 50,ug bovine serum albumin (BSA)in200,ul ofbufferE,andthenfilteredthroughacolumn ofSephacrylS-300HR(1

10720 Biochemistry: Habraken et al.

AS-300 Fractions

OP 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

. :... ..um.::.;' -Rad2

BS-300 Fractions

OP 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50- Rad2

-Rad3TFBI

C

Antibody aR2 aR3 aR51 aR2aR3aR51

.up..NW

1 23 456D kDa

3- Rad2

116-1-_ -Rad2597

-Rad3- TFB1

68 -

_- p55-* SSLI

45- ,J,]-CCLI

- p38

- KIN28

E SDm

....................... ...... ............

- -:

1 2 34 56 7

- Rad2

- Rad25

- Rad3-TFBI

- SSLI

- Rad2

- Rad25- Rad3% TFBI

FIG. 2. Rad2 is stably associated with TFIIH. (A) Purified Rad2protein was filtered through a Sephacryl S-300 column, and columnonput (OP) and fractions 22-50 from this analysis were subjected toimmunoblotting to determine their content of Rad2. (B) The pool of40 mM and 100 mM imidazole eluates from the nickel agarose step thatcontained Rad2 and TFIIH was subjected to sizing in the same S-300column, and column onput (OP) and fractions 22-50 from this analysiswere subjected to immunoblotting to determine their content of Rad2,Rad3, and TFB1 proteins. (C) Purified Rad2 protein alone (lanes 1-3)and Rad2-TFIIH complex (lanes 4-6) were subjected to immunopre-cipitation with protein A agarose beads bearing anti-Rad2 (aR2)(lanes 1 and 4), anti-Rad3 (aR3) (lanes 2 and 5), and anti-Rad5l(aR51) (lanes 3 and 6) antibodies. Immunoprecipitates were treatedwith 2% SDS to elute bound proteins and the SDS eluates weresubjected to immunoblot analysis. (D) Rad2-TFIIH complex (500 ngof protein) from the Mono Q step was subjected to electrophoresis ina SDS/9% polyacrylamide gel and then silver stained. Rad2, the

subjected to immunoprecipitation with protein A agarosebeads bearing affinity purified anti-Rad2 and anti-Rad3 anti-bodies (6, 12), and as control, with beads bearing anti-Rad5lantibodies (13), which show no cross-reactivity with Rad2 orwith TFIIH (Fig. 2C). In addition to precipitating TFIIH,anti-Rad3 beads also quantitatively precipitated Rad2 protein(Fig. 2C, lane 5). Precipitation of Rad2 by anti-Rad3 beads wasdue to physical association of Rad2 with TFIIH because thesame anti-Rad3 beads did not bring down free Rad2 protein(Fig. 2C, lane 2). Conversely, anti-Rad2 beads precipitated; inaddition to Rad2 protein, TFIIH as well (Fig. 2C, lane 4). Incontrol experiments, we confirmed that the anti-Rad2 beadsdid not cross-react with free TFIIH in the absence of Rad2(data not shown). Thus, Rad2 protein exists as a higher ordercomplex with TFIIH, which is designated as NEF3.

In NEF3, Rad2 protein is strongly associated with TFIIH, asindicated from the following observations. When we subjectedthe nickel agarose NEF3 pool (Fig. 1A) to sequential chro-matographic fractionation in columns of Mono S and Mono Qdesigned originally to purify TFIIH (1, 7), we again foundcopurification of Rad2 protein with TFIIH in both instances(data not shown). As judged by silver staining, the Mono Qpool of highly purified NEF3 contained a stoichiometricamount of Rad2 protein (Fig. 2D). Since the Rad2 protein inthe Mono Q pool remains associated with TFIIH in thepresence of 1 M potassium acetate, it is clear that NEF3 isstable to high salt. As shown in Fig. 2E, when NEF3 isimmobilized on anti-Rad2 beads and then subjected to washingwith increasing concentrations of SDS, we found that -2, 18,40, 25, and 15% of the total TFIIH was eluted by 0.05, 0.1, 0.2,0.3, and 0.5% of the protein denaturant, respectively, indicat-ing that the protein complex is in fact partially stable to thedenaturant. In this experiment, because Rad2 was bounddirectly by its cognate antibodies in the immunobeads,0.5%-1% SDS was necessary for its elution from the immu-nocomplex (Fig. 2E).

Because no dissociation of Rad2 from TFIIH occurredduring molecular sizing on Sephacryl S-300 (Fig. 2B) andduring the final stages of purification of the complex (Mono Sand Mono Q), it can be deduced that the apparent dissociationconstant of the complex must be significantly lower than 3.3 x10-9 M, corresponding to the concentration of NEF3 in theS-300 pool.NEF3 Is the Functional Entity in NER. In the reconstituted

NER system, incision of UV-damaged DNA is monitored byexamining the conversion of supercoiled UV-irradiated plas-mid to the open circular form. To detect the excision DNAfragments, reaction mixtures are treated with calf thymusterminal transferase in the presence of [a-32P] dideoxy ATP tolabel the fragments, followed by electrophoresis on a sequenc-ing gel and autoradiography to visualize the radiolabeledfragments. As assayed by the agarose gel method (Fig. 3A) and32P-labeling of excision fragments (Fig. 3B), NEF3 togetherwith NEF1 (Radl-RadlO-Radl4), NEF2 (Rad4-Rad23), andRPA promote NER (lane 6), and as expected, the addition ofRad2 protein does not further enhance the extent of thedamage specific incision reaction (lane 5).

- DISCUSSIONTo achieve the goal of defining the manner by which the NERmachinery assembles at the DNA damage site, we show here

various TFIIH subunits, and the two CCL1 isoforms and KIN28, whichare constituents of TFIIK, are also indicated. Because Rad25 does notstain well with silver, its actual amount is higher than the stainingintensity would suggest. (E) Rad2-TFIIH complex (NEF3) was im-mobilized on anti-Rad2 protein A agarose beads and then eluted with0.05%, 0.1%, 0.2%, 0.3%, 0.5%, and 1% SDS. Free NEF3 (lane 1) andthe SDS eluates (lanes 2-7) were subjected to immunoblot analysis.

Proc. Natl. Acad. Sci. USA 93 (1996)

...... .......

Dow

nloa

ded

by g

uest

on

Mar

ch 1

9, 2

021

Page 4: Transcription TFIIH DNA Rad2 3: Implications Cockayne · Rad2 protein was mixed with 50,ug bovine serum albumin (BSA)in200,ul ofbufferE,andthenfilteredthroughacolumn ofSephacrylS-300HR(1

Biochemistry: Habraken et al.

A

ATPRad2NEF3

-UV +UVI I I

- oc

SC

1 2 3 4 5 67

B-uv

ATP + +

Rad2 - +

NEF3 - +

uv

- +UVf + ---I

-30

-25

-20

1 2 3 4 5 6 7

FIG. 3. The NER activity of purified NEF3. (A) Unirradiated M13DNA (-UV) and UV-irradiated M13 DNA (+UV) were incubatedat 30°C for 10 min with RPA, NEF1 (Radl-RadlO-Radl4 complex),NEF2 (Rad4-Rad23 complex), and NEF3 (Rad2-TFIIH complex),with and without Rad2 protein or ATP, as indicated (lanes 2, 3, and5-7). The DNAs were also incubated in buffer without any NER factor(lanes 1 and 4). Reaction mixtures were processed for agarose gelelectrophoresis and the DNA species stained with ethidiut bromide.Incision of the supercoiled DNA (SC) generated open circular form(OC). (B) Unirradiated M13 DNA (-UV) and UV-irradiated DNA(+UV) were incubated at 30°C for 30 min without any NER factors(lanes 1 and 4) and with (lanes 2, 3, and 5-7) the same combinationof NER factors as described in A. Excision DNA fragments werepurified from the reaction mixtures, labeled with [a-32P] dideoxy ATPand terminal transferase, and then processed for electrophoresis in a

DNA sequencing gel.

that Rad2 protein is physically associated with TFIIH in anovel subassembly, which we have designated -as NEF3. Inmolecular sizing in Sephacryl S-300, while Rad2 alone behavesas a monomer, Rad2 in the 40 mM and 100 mM imidazoleeluates from the nickel agarose step comigrated with TFIIH ata much earlier position than free Rad2, and Rad2 was coim-munoprecipitated with TFIIH from S-300 fractions containingthese proteins (Fig. 2). In NEF3, Rad2 is very tightly bound toTFIIH, as they copurify through six chromatographic steps,and the Rad2-TFIIH association is stable to at least 1 Mpotassium acetate and even partially stable to the proteindenaturant SDS (Fig. 2). In previous studies, TFIIH wassuggested to interact with various NER proteins includingRad2 (14), Rad4 (14), Rad23 (15), and also XPA (16), whichis the human counterpart of yeast Radl4 (17). However,because these studies do not exclude weak and transientprotein-protein interactions, the significance of these interac-tions in the formation of stable NER protein subassemblies hasremained unclear. In fact, the finding that none of the Rad4,Rad23, Radl4 proteins or Radl and RadlO is stably associatedwith TFIIH (4) strongly suggests that a majority of the abovenoted interactions are weak and transient, and are not involvedin the formation of stable protein subassemblies.

Proc. Natl. Acad. Sci. USA 93 (1996) 10721

Table 1. Functional subassemblies in NER

Repair factor Components Function or activityNEF1 Radl (XPF), RadlO DNA endonuclease,

(ERCC1), Radl4 (XPA) DNA damagerecognition

NEF2 Rad4 (XPC), Rad23 Tethering of NEF1(HHR23) with NEF3

NEF3 Rad2 (XPG), Rad3 (XPD), DNA endonuclease,Rad25 (XPB), TFB1, DNA helicase,SSL1, p55, p38 DNA damage

recognitionRPA p69, p36, p13 DNA damage

recognitionThe human equivalents of Rad proteins are indicated in parenthe-

ses.

NEF3 has multiple biochemical activities: the Rad3. andRad25 subunits in TFIIH both possess a DNA helicase func-tion that is fueled by ATP hydrolysis (18, 19); Rad3 proteinalso has an affinity for UV-damaged DNA that is dependenton ATP binding (20), and Rad2 protein has a structure-specificDNA endonuclease activity that cleaves 5' overhanging single-stranded DNA adjacent to duplex DNA (21). Our recentTFIIH reconstitution studies have provided direct evidencethat Rad3 and Rad25, as well as TFIIHi, which contains theremaining four subunits, are all indispensable for the incisionof UV-damaged DNA (7). Furthermore, the helicase defectiverad3 Arg-48 and rad25 Arg-392 mutant proteins are inactive inthe incision step of NER (7), indicating that both Rad3 andRad25 helicases function in DNA unwinding during incision ofUV-damaged DNA.

All of the NER proteins known to be essential for incisionof UV-damaged DNA in yeast are constituents of NEFi,NEF2, NEF3, and RPA (Table 1). The properties of theseprotein complexes suggest a sequence of assembly of the NERmachinery at the DNA damage site (Fig. 4). The Radl4protein in NEFi is a zinc metalloprotein with affinity forUV-damaged DNA (22), and RPA too has affinity for DNAdamaged by UV (23), by cis-diamminedichloroplatinum (II)(24), and by N-acetoxy-2-acetylaminofluorene (25). HumanRPA has been shown to interact with XPA (25-27), and thesetwo proteins together have higher affinity for damaged DNAthan either protein alone (25, 26). These results suggest that aninteraction between yeast RPA and NEF1 via Radl4 proteinmay enhance the ability of these entities to recognize the DNA

S. 3'

NEF2 RPNEF3

ATP

Rad 3-Rad25 -. ADP +pi

5'-53'

IV Rad 1 - Radlo1O Rad 2

FIG. 4. Sequence of assembly of NER factors at the damage site.Steps I-IV are discussed in the text.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

9, 2

021

Page 5: Transcription TFIIH DNA Rad2 3: Implications Cockayne · Rad2 protein was mixed with 50,ug bovine serum albumin (BSA)in200,ul ofbufferE,andthenfilteredthroughacolumn ofSephacrylS-300HR(1

10722 Biochemistry: Habraken et al.

damage, resulting in their stable binding to the damage site.Following the binding of damaged DNA by NEF1 and RPA(Step I), NEF2, consisting of the Rad4 and Rad23 proteins,could be brought to the damage site because of the ability ofRad23 to interact with Radl4 (15) (Step II). Subsequently, viainteractions of TFIIH with the Rad23 component of NEF2(15), and the ability of Rad3 protein to bind damaged DNA(20), NEF3 could become incorporated into this proteinassembly (Step II). As RPA interacts with XPG (25), a similarinteraction of Rad2 in NEF3 with yeast RPA may also aid inthe assembly process. Following ATP dependent unwinding ofdamaged DNA by the Rad3 and Rad25 helicases (7) in NEF3(Step III), the Radl-RadlO and Rad2 nucleases, respectively,would mediate the 5' and 3' incisions of the damaged DNAstrand (21, 28, 29) (Step IV). RPA stimulates the junctioncutting activity of XPF-ERCC1 and XPG nucleases (29),suggesting that interactions with RPA may enhance the activityof Radl-RadlO and Rad2 nucleases.

In humans, mutations in the TFIIH subunitsXPD and XPB,the respective counterparts of the yeast RAD3 and RAD25genes, cause CS, which is characterized by severe growthdefects, mental retardation, and cachexia. Mutations in XPG,the human counterpart of yeast RAD2, can also result in CS(30). By contrast to Rad3 and Rad25 and their human coun-terparts, which are components of TFIIH, the lack of availableevidence linking Rad2 or XPG to RNA polymerase II tran-scription has rendered it difficult to assign transcriptionaldeficiency as a possible cause of CS. The association of Rad2with TFIIH documented here is consistent with the observa-tion that XPG copurifies with TFIIH (31). Based upon thesefindings, we propose that CS causative mutations inXPB,XPD,and XPG all impair TFIIH function in a similar manner.UV-induced cyclobutane pyrimidine dimers are removed by

the NER machinery at a faster rate from the transcribed strandof an active gene than from the nontranscribed strand (32, 33).Mutations in the human CSA and CSB genes cause CS and, inaddition, they confer a defect in preferential repair of thetranscribed strand (34). However, the lack of this preferentialrepair is unlikely to be the cause of CS, because even thoughmutations in XPA confer a total defect in the repair of thetranscribed strand as well as the nontranscribed strand, XPApatients do not exhibit CS symptoms. We suggest that muta-tions in XPB, XPD, and XPG that cause CS engender a defectin interaction of their encoded proteins with CSA, CSB, orboth proteins, and this in turn reduces the efficiency with whichRNA polymerase II is displaced not only from the damage sitesbut also from some natural transcription pause sites. At thesetranscriptional pause sites, the human equivalent of NEF3,along with CSA and CSB proteins, may facilitate the resump-tion of transcript elongation, without aborting the associatednascent transcript, by pushing the stalled RNA polymeraseeither forward or backward. Such a hypothetical role of thecombination of NEF3, CSA, and CSB may be important formaintaining the transcriptional efficiency of certain genes, inparticular, those that have long transcripts. A deficiency in therate of elongation of certain transcripts, stemming from themutational inactivation of XPB, XPD, XPG, CSA, and CSB,could be the basis of CS.

We thank Phil Hanawalt for reinforcing the idea that defective RNApolymerase II displacement may be the cause of CS. This work wassupported by Grant CA35035 from the National Cancer Institute andGrant DE-FGO3-93ER61706 from the Department of Energy.

1. Guzder, S. N., Habraken, Y., Sung, P., Prakash, L. & Prakash, S.(1995) J. Biol. Chem. 270, 12973-12976.

2. Mu, D., Hsu, D. S. & Sancar, A. (1996) J. Biol. Chem. 271,8285-8294.

3. Svejstrup, J. Q., Wang, Z., Feaver, W. J., Wu, X., Bushnell, D. A.,Donahue, T. F., Friedberg, E. C. & Kornberg, R. D. (1995) Cell80, 21-28.

4. Guzder, S. N., Sung, P., Prakash, L. & Prakash, S. (1996) J. Biol.Chem. 271, 8903-8910.

5. Sung, P., Reynolds, P., Prakash, L. & Prakash, S. (1993) J. Biol.Chem. 268, 26391-26399.

6. Habraken, Y., Sung, P., Prakash, L. & Prakash, S. (1993) Nature(London) 366, 365-368.

7. Sung, P., Guzder, S. N., Prakash, L. & Prakash, S. (1996) J. Biol.Chem. 271, 10821-10826.

8. Bailly, V., Sommers, C. H., Sung, P., Prakash, L. & Prakash, S.(1992) Proc. Natl. Acad. Sci. USA 89, 8273-8277.

9. Madura, K. & Prakash, S. (1986) J. Bacteriol. 166, 914-923.10. Robinson, G. W., Nicolet, C. M., Kalainov, D. & Friedberg, E. C.

(1986) Proc. Nati. Acad. Sci. USA 83, 1842-1846.11. Svejstrup, J. Q., Feaver, W. J., LaPointe, J. & Kornberg, R. D.

(1994) J. Biol. Chem. 269, 28044-28048.12. Sung, P., Prakash, L., Weber, S. & Prakash, S. (1987) Proc. Natl.

Acad. Sci. USA 84, 6045-6049.13. Sung, P. (1994) Science 265, 1241-1243.14. Bardwell, A. J., Bardwell, L., Iyer, M., Svejstrup, J. Q., Feaver,

W. J., Kornberg, R. D. & Friedberg, E. C. (1994) Mol. Cell. Biol.14, 3569-3576.

15. Guzder, S. N., Bailly, V., Sung, P., Prakash, L. & Prakash, S.(1995) J. Biol. Chem. 270, 8385-8388.

16. Park, C.-H., Mu, D., Reardon, J. T. & Sancar, A. (1995) J. Biol.Chem. 270, 4896-4902.

17. Bankmann, M., Prakash, L. & Prakash, S. (1992) Nature (Lon-don) 355, 555-558.

18. Sung, P., Prakash, L., Matson, S. W. & Prakash, S. (1987) Proc.Natl. Acad. Sci. USA 84, 8951-8955.

19. Guzder, S. N., Sung, P., Bailly, V., Prakash, L. & Prakash, S.(1994) Nature (London) 369, 578-581. -

20. Sung, P., Watkins, J. F., Prakash, L. & Prakash, S. (1994) J. Biol.Chem. 269, 8303-8308.

21. Habraken, Y., Sung, P., Prakash, L. & Prakash, S. (1995) J. Biol.Chem. 270, 30194-30198.

22. Guzder, S. N., Sung, P., Prakash, L. & Prakash, S. (1993) Proc.Natl. Acad. Sci. USA 90, 5433-5437.

23. Burns, J. L., Guzder, S. N., Sung, P., Prakash, S. & Prakash, L.(1996) J. Biol. Chem. 271, 11607-11610.

24. Clugston, C. K., McLaughlin, K., Kenny, M. K. & Brown, R.(1992) Cancer Res. 52, 6375-6379.

25. He, Z., Henricksen, L. A., Wold, M. S. & Ingles, C. J. (1995)Nature (London) 374, 566-569.

26. Li, L., Lu, X., Peterson, C. A. & Legerski, R. J. (1995) Mol. Cell.Biol. 15, 5396-5402.

27. Matsuda, T., Saijo, M., Kuraoka, I., Kobayashi, T., Nakatsu, Y.,Nagai, A., Enjoji, T., Masutani, C., Sugasawa, K., Hanaoka, F.,Yasui, A. & Tanaka, K. (1995) J. Biol. Chem. 270, 4152-4157.

28. Bardwell, A. J., Bardwell, L., Tomkinson, A. E. & Friedberg,E. C. (1994) Science 265, 2082-2085.

29. Matsunaga, T., Park, C.-H., Bessho, T., Mu, D. & Sancar, A.(1996) J. Biol. Chem. 271, 11047-11050.

30. Hanawalt, P. C. (1994) Science 266, 1957-1960.31. Mu, D., Park, C.-H., Matsunaga, T., Hsu, D. S., Reardon, J. T. &

Sancar, A. (1995) J. Biol. Chem. 270, 2415-2418.32. Mellon, I., Spivak, G. & Hanawalt, P. C. (1987) Cell 51, 241-249.33. Mellon, L& Hanawalt, P. C. (1989) Nature (London) 342, 95-98.34. van Hoffen, A., Natarajan, A. T., Mayne, L. V., van Zeeland,

A. A., Mullenders, L. H. F. & Venema, J. (1993) Nucleic AcidsRes. 21, 5890-5895.

Proc. Natl. Acad. Sci. USA 93 (1996)

Dow

nloa

ded

by g

uest

on

Mar

ch 1

9, 2

021