Involvement of TFIIH in NER Factors Mediated Chromatin Remodeling Amita Singh

Involvement of TFIIH in NER factors mediated chromatin remodeling Amita Singh

To cite this version:

Amita Singh. Involvement of TFIIH in NER factors mediated chromatin remodeling. Genomics [q-bio.GN]. Université de Strasbourg, 2014. English. NNT : 2014STRAJ108. tel-01250872

HAL Id: tel-01250872 https://tel.archives-ouvertes.fr/tel-01250872 Submitted on 5 Jan 2016

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG

ÉCOLE DOCTORALE des Sciences de la Vie et de la Sante IGBMC - CNRS UMR 7104 - Inserm U 964

THÈSE présentée par :

Amita SINGH soutenue le : 29 Septembre 2014

pour obtenir le grade de : !"#$%&'($')R%+,-$&., té de Strasbourg

Discipline/ Spécialité : Sciences du Vivant/Aspects Moléculaires et Cellulaires de la Biologie

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THÈSE dirigée par :

EGLY Jean-Marc DRE INSERM, IGBMC, Strasbourg (Fr)

RAPPORTEURS :

SCHAEFFER Laurent DR, LBMC, Lyon (Fr) GELI Vincent DR, CRCM, Marseille (Fr) LAUGEL Vincent PUPH, CHU de Strasbourg (Fr)

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TABLE OF CONTENT 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

LIST OF FIGRES 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

LIST OF TABLES 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

LIST OF ABBREIATIONS 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

RESME OF THE THESIS IN ENGLISH 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888

RESME DE LA THESE DE DOCTORANT EN FRANÇAIS 888888888888888888888888888888888888888888888888888888888888

INTRODCTION 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

I8 THE TRANSCRIPTION MECHANISM 888888888888888888888888888888888888888888888888888888888888888888888888888888888

AT THE PROMOTER ELEMENTS AND TRANSCRIPTION COMPONENTS TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TW A8 T.V HQ`V ]`QIQ V` 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V TATA GQ688888888888888888888888888888888888888888888888 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V IJ1 1: Q` VCVIVJ ÎJ`_ 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V TFIIB `VHQ$J1 1QJ VCVIVJ s ^BRE% :JR BRER_ 888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V DQ1Js `V:I ]`QIQ V` VCVIVJ ^DPE_ 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V MQ 1` VJ VCVIVJ ^MTE_ 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V RQ1Js `V:I HQ`V VCVIVJ ^DCE_ 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V TCT IQ 1` ^]QC7]7`1I1R1JV 1J1 1: Q` IQ 1`_ 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 T.V HQ`V ]`QIQ V` VCVIVJ ^CPE _ 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 TATAR CVss ]`QIQ V`s 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 A8 RNA ]QC7IV`:sV II :JR T`:JsH`1] 1QJ M:H.1JV`7 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 RNA ]QC7IV`:sV II :JR CTD IQR1`1H: 1QJs 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 TFIIA 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 TFIIB 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 TFIID 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 TFIIE 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 TFIIF 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 TFIIH 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 MVR1: Q` 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 BT THE TRANSCRIPTION CYCLE TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT VT B8 P`V1J1 1: 1QJ HQI]CV6 :ssVIGC75 Q]VJ HQI]CV6 `QÌ: 1QJ :JR 1J1 1: 1QJ8 8888888888888888888888888888888888888888888888888 B8 P`QIQ V` HCV:`:JHV 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 B8 P`Q61I:C ]:%s1J$ :JR VCQJ$: 1QJ 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 B8 TVÌ1J: 1QJ :JR `V1J1 1: 1QJ 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 CT THE ACTIVATED TRANSCRIPTION TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT VY C8 O0V`01V1 Q` .V J%HCV:` `VHV] Q` `:I1C1Vs 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 C8 RV 1JQ1H :H1R `VHV] Q` 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 C8 T`:JsH`1] 1QJ Q` `V 1JQ1H :H1R `VHV] Q` :`$V $VJVs 8888888888888888888888888888888888888888888888888888888888888888888888888888888888

5 DT CHROMATIN AND GENE REGULATION TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT WY D8 E]1$VJV 1H IQR1`1H: 1QJ Q` H.`QI: 1J 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 PQs R `:JsC: 1QJ:C IQR1`1H: 1QJ Q` .1s QJVs 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 88:8 RV$%C: 1QJ G7 .1s QJV C7s1JV :HV 7C: 1QJ :JR RV:HV 7C: 1QJ8 88888888888888888888888888888888888888888888888888888888888 88G8 RV$%C: 1QJ G7 .1s QJV C7s1JVL:`$1J1JV IV .7C: 1QJ :JR RVIV .7C: 1QJ 888888888888888888888888888888888888888888 88H8 RV$%C: 1QJ G7 Q .V` .1s QJV IQR1`1H: 1QJs 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 DNA IV .7C: 1QJ 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 D8 C.`QI: 1J `V:``:J$VIVJ 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 II8 THE TFIIH AND NER FACTORS IN TRANSCRIPTION 88888888888888888888888888888888888888888888888888888888888

AT TFIIHS A MULTIFUNCTIONAL COMPLEX TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT YS A8 CQI]Qs1 1QJ Q` TFIIH 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 PB 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 PD 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 ] 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 ] 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 ] 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 ] 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 CR@ 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 C7C1J H 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 M: 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 ] LTTDRA 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 BT NER FACTORS TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT ZX CT TFIIHS BRIDGING TRANSCRIPTION AND REPAIR TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT Z[ III8 HMAN DISORDERS ASSOCIATED ITH MTATION IN TFIIH AND NER FACTORS 88888888888

AT XERODERMA PIGMENTOSUM TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT [V BT COCKYANE SYNDROME TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT [W CT XERODERMA PIGMENTOSUM C OCKYANE SYNDROME TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT [X DT TRICHOTHIODYSTROPHY TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT [Y RESLTS 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

A. INVOLVEMENT OF TFIIH IN NER FACTORS DEPENDENT CHROMATIN REMODELING TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT SRT BT ENZYMATIC ACTIVITY OF TFIIH TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT SV T B8 S%G%J1 HQI]Qs1 1QJ Q` TFIIH 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 B8 ATP:sV :JR .VC1H:sV :H 101 7 Q` TFIIH 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 B8 E6]V`1IVJ :C ]`QHVR%`Vs7 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 DISCSSION 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

AT TFIIHQ NER FACTORS AND CHROMATIN REMODELING TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT SWS A8 B1QH.VI1s `7 Q` TFIIH :JR .V Q0V`C:]]1J$ $VJV 1Hs Q` R1sV:sVs 88888888888888888888888888888888888888888888888888888888888888 A8 TFIIH RV]CQ7s .V `VH`%1 IVJ Q` NER `:H Q`s :JR H.`QI: 1J `VIQRVC1J$ V0VJ s 88888888888888888888888888888888888 BT FUTURE DIRECTION FOR TFIIH AND NER FACTORS TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT SWY CONCLSION 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

BIBLIOGRAPH 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

6 L.89=4+=F.,:7*8=

F1$%`V 8 CQ`V ]`QIQ V` IQ 1`s `Q` `:JsH`1] 1QJ G7 RNA ]QC7IV`:sV II8 8888888888888888888888888888888888888888888888888 F1$%`V 8 GVJV`:C `:JsH`1] 1QJ `:H Q`s Q` RNA ]QC7IV`:sV II 8888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 RNA ]QC7IV`:sV II `:JsH`1] 1QJ H7HCV8 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 S `%H %`V Q` `V 1JQ1H :H1R `VHV] Q` 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 CQ`V$%C: Q` V6H.:J$V : RRLRAR .V V`QR1IV`s8 888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 C`Qss :C@ GV 1VVJ .V RAR:H 10: VR ] MAPK ]: .1:7 :JR .V V6]`Vss1QJ Q` RAR :`$V $VJVs8 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 N%HCVQsQIV Q`$:J1<: 1QJ8 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 SH.VI: 1H H:` QQJ Q` HQ`V .1s QJV PTMs :JR .V1` ]Q VJ 1:C `%JH 1QJ 88888888888888888888888888888888888888 F1$%`V 8 DNA RVIV .7C: 1QJ P: .1:78 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 S `%H %`V Q` TFIIH 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 L1JV:` sH.VI: 1Hs Q` PB :JR PD .VC1H:sVs8 888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 PD :JR 1 s 1J V`:H 1QJs 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 RVC: 1QJs.1] Q` $VJV 1H R1sQ`RV`s Q IQCVH%C:` RV`VH s8 8888888888888888888888888888888888888888888888888888888888 F1$%`V 8 TFIIH s%G%J1 s 1J PB1 :JR PB I% :J HVCC C1JVs8 888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 TFIIH s%G%J1 s 1J PD1 5 PD I% :J 5 ] LTTDRA1 :JR ] LTTDRA I% :J HVCC C1JV8 8888888888888 F1$%`V 8 ATP:sV :H 101 7 Q` PB 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 ATP:sV :H 101 7 Q` PD 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 F1$%`V 8 HVC1H:sV :H 101 7 Q` PD 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

L.89=4+=T&'1*8=

T:GCV 8 CQJsVJs%s sV_%VJHV :JR ]Qs1 1QJ Q` .V R1``V`VJ HQ`V ]`QIQ V` VCVIVJ s8 8888888888888888888888888888 T:GCV 8 D1``V`VJ s%G%J1 s Q` RNA ]QC7IV`:sV II =1J V%@:`7Q Vs5 :`H.:V:5 G:H V`1: :JR .V1` `V: %`Vs8 88 T:GCV 8 CTD @1J:sVs :JR P.Qs].: :sV 1J I:II:Cs :JR 1J 7V:s _ 888888888888888888888888888888888888888888888888888888888888 T:GCV 8 GVJV`:C `:JsH`1] 1QJ `:H Q`s Q` RNA ]QC7IV`:sV II8 8888888888888888888888888888888888888888888888888888888888888888888 T:GCV 8 TFIIH HQI]QJVJ s :JR .V1` `%JH 1QJs 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 T:GCV 8 NER `:H Q`s :JR .V1` 1J V`:H 1QJ ]:` JV`s 8888888888888888888888888888888888888888888888888888888888888888888888888888888888 T:GCV 8 M% : 1QJ 1J PB5 PD :JR TTDRA $VJV s %R1VR R%`1J$ P.D 888888888888888888888888888888888888888888888888888888888 T:GCV 8 CQI]:`1sQJ Q` `V: %`Vs Q` P5 PRND5 TTD5 CS :JR PLCS 88888888888888888888888888888888888888888888888888888888888 T:GCV 8 F%JH 1QJs Q` TFIIH 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

L.89=4+=&''7*;.&9.438=

ATM======A9&=.&=9*1&3,.*(9&8.&=2:9&9*)= ATR= = = = = ATM=&3)=R&)-=7*1&9*)= BER======B&8*=*=(.8.43=7*5&.7= '5======B&8*=5&.7= BRCA======B7*&89=(&3(*7=5749*.3= BRE======TFIIB=7*(4,3.9.43=*1*2*39= BTF,======B&8&1=97&38(7.59.43=+&(947=,= CAF8+======C-742&9.3=&88*2'1>=+&(947=+= CAK======C)08A(9.;&9*)=K.3&8*= CDC======C*11=).;.8.43=(>(1*= CDK======C>(1.3=)*5*3)*39=0.3&8*= C-IP= = = = = C-742&9.3=.22:34857*(.5.9&9.43= CHO======C-.3*8*=-&289*7=4;&7>= COFS======C*7*'7484(:148+&(.4880*1*9&1=8>3)742*= CPD======C>(14':9&3*=5>7.2.).3*=).2*7= CTD= = = = = C89*72.3&1=D42&.3= CS======;40&>3*8=8>3)742* = CSA2B======C4(0&>3*=8>3)742*=5749*.3=,74:5=A2B= DNA======D*4=>7.'43:(1*.(=&(.)= DBD======DNA8'.3).3,=)42&.3= DCE== = = D4<3897*&2=(47*=*1*2*39= DDB======DNA=)&2&,*='.3).3,=5749*.3= DNA8PK= = = = DNA=5749*.3=0.3&8*= DPE======D4<3897*&2=574249*7=*1*2*39= DSB= D4:'1*=897&3)='7*&0= ERCC+= E=(.8.43=7*5&.7=(74888(4251*2*39.3,=74)*39= = 7*5&.7= FANC= F&3(43.=&3*2.&=(4251*2*39&9.43=,74:5= GFP= G7**3=+1:47*8(*39=5749*.3= GGR= G14'&1=,*342*=7*5&.7= GTF= G*3*7&1=T7&38(7.59.43=+&(947= GTF,H= G*3*7&1=T7&38(7.59.43=+&(947=IIH== HAT= H.8943*=&(*9>1897&38+*7&8*= HD= H*1.(&8*=)42&.3= HDAC= H.8943*=)*&(*9>1&8*== HP+= H*9*74(-742&9.3=5749*.3=+= = =

HR,-B== H42414,:*=R&),-B= I37= I3.9.&947= 0B= K.148'&8*= 0D&= K.148D&1943= LBD= L.,&3)='.3).3,=)42&.3= L.,= L.,&8*= MAT+= MS3&,*=A=974.8== MLL= M.=*)=1.3*&,*=1*:0*2.&= MSH= M:9=S=H42414,:*8= NER= N:(1*49.)*=*=(.8.43=7*5&.7= NR= N:(1*&7=7*(*59478= PARP= P41>=ADP87.'48*=541>2*7&8*= PARG== P41>8ADP87.'48*8,1>(4->)741&8*== PCNA= P741.+*7&9.3,=(*11=3:(1*&7=&39.,*3= PHD= P1*(0897.3=-42414,>=)42&.3= PIC== P7*.3.9.&9.43=(4251*== PPAR= P*74=.842*=P741.+*7&947=8A(9.;&9*)= = R*(*5947= P41= P41>2*7&8*= RAD= H42414,4:8=7*(42'.3&9.43=+&(947= RAR= R*9.34.(=A(.)=R*(*5947= 98RA= T7&388R*9.34.(=A(.)= RE= R*85438*=*1*2*39= RFC= R*51.(&9.43=+&(947=C= RING= R*&11>=.39*7*89.3,=3*<=,*3*=&=9>5*=4+=?.3(= = +.3,*7=249.+= RPA= R*51.(&9.43=5749*.3=A= RXR= R*9.34.)=X=R*(*5947= SAM= S8A)*348>1=2*9-.43.3*= 88DNA= S.3,1*=897&3)*)=DNA== SUMO= S:24>1&9.43=N82*9->15:7.3*=DNA= = ,1>(48>1&8*= SWI2SNF= S<.9(-2S:(748*=343=+*72*39&'1*= TBP= TATA='4=='.3).3,=5749*.3= TCR= T7&38(7.59.43=(4:51*)=7*5&.7=== TFIIA8H= T7&38(7.59.43=+&(947=II=A8H= T+'= T7&38(7.59.43=+&(947='= TTD= T7.(-49-.4)>89745->= TRF= T*142*7.(=7*5*&9='.3).3,=+&(947=

9 TSS= T7&38(7.59.43=89&9=8.9*= UV= U197&8;.41*9= VDR= V.9&2.3=D=R*(*5947= XP= X*74)*72&=P.,2*3948:2= XAB= XPA8='.3).3,=5749*.3= XPA8V= XP=(4251*2*39&9.43=,74:5=A8V= XRCC= X87&>=C7488=(4251*2*39&9.43=

R*8:2*=4+=9-*=9-*8.8=.3=E3,1.8-== =

I3;41;*2*39=4+=TFIIH=.3=NER=+&(947=2*).&9*)=(-742&9.3=7*24)*1.3,=

TFIIHa=&=)>3&2.(=(4251*==

TFIIH= .8= &= 2:19.88:':3.9= (4251*=`= +.789= (-&7&(9*7.?*)= &3)= 5:7.+.*)= &8= &= ,*3*7&1= 97&38(7.59.43=

+&(947= 4+= RNA= 541>2*7&8*= II= RNA= 541= II_= O7.,.3&11>= TFIIH= <&8= 9-4:,-9= 94= '*= *=(1:8.;*1>= &=

'&8&1=97&38(7.59.43&1=+&(947=':9=1&9*7=.9=<&8=+4:3)=94='*=.3;41;*)=.3=DNA=7*5&.7=&3)=5488.'1>=.3=

(*11=(>(1*=7*,:1&9.43=&8=43*=4+=9-*=2&/47=(42543*39=*88*39.&1=+47=9-*=1.+*=4+=9-*=(*11_=I9=(438.898=4+=

9<4= 8:'8(4251*=*8a= (47*= (4251*== &3)= (>(1.38&(9.;&9.3,= 0.3&8*= CAK_= T-*= (47*= (4251*==

(438.898= 4+= 8.== 8:':3.98a= XPB`= 50,`=5/,`= 5..`= 5-.= &3)= 522TTD8A`= H4*./2&0*78= ,**+_ = CAK= .8=

(42548*)=4+=9-*=9-7**=8:':3.98a=CDK1`=(>(1.3=H=&3)=MAT+_= ==T-*=(47*=&3)=9-*=CAK=&7*=-*1)=

94,*9-*7= '>= 9-*= XPD`= 8:':3.9_= TFIIH= 5488*88*8= 8*;*7&1= *3?>2&9.(= &(9.;.9.*8= <-.(-= &7*=

.3).85*38&'1*=+47=3:(1*49.)*=*=(.8.43=7*5&.7=NER=&3)=97&38(7.59.43_=

TFIIH=):7.3,=NER=

L.;.3,=47,&3.828=&7*=(439.3:4:81>=*=548*)=94=)&2&,.3,=&,*398=8:(-=&8=UV87&>8`=X87&>8=<-.(-=

(7*&9*8=DNA=)&2&,*`=CPD8=5-494574):(98=.3=9-*=,*342*_=T4=2&.39&.3=,*342.(=89&'.1.9>=9-*8*=

DNA=1*8.438=&7*=7*24;*)='>=8*;*7&1=DNA=7*5&.7=5&9-<&>8_==NER=5&9-<&>=.8=43*=4+=9-*2=&3)=

8:').;.)*)= .394= ,14'&1= ,*342*= 7*5&.7= GGR= 5&9-<&>= &3)= 9-*= 97&38(7.59.438(4:51*)= 7*5&.7=

TCR= 5&9-<&>_= TFIIH= +:3(9.438= &+9*7= &= DNA= 1*8.43= -&8= '**3= 7*(4,3.?*)= '>= *.9-*7= 9-*= GGR=

5&9-<&>= 47= 9-*= TCR= 5&9-<&>= 4+=NER_= I3= GGR= 5&9-<&>8= 9-*= DNA= )&2&,*= .8= 7*(4,3.?*)= '>=

XPC2HR,-B=(4251*==<-.1*=.3=TCR=9-*=89&11*)=RNA=541=II=89&11*)=.3=+7439=4+=9-*=DNA=1*8.438=43=

9-*=97&38(7.'*)=DNA=897&3)=.3.9.&9*8=9-*=7*(7:.92*39=4+=9-*=CSA=&3)=CSB_=TFIIH=9-*3=:3<.3)8=

9-*= >?@= &74:3)= 9-*= 1*8.438= ;.&= .98= - 8/= -*1.(&8*2@6D&8*= &(9.;.9>= 4+= EDF= &3)= / 8= -= -*1.(&8*=

&(9.;.9>='>=XPD_=O5*3.3,=4+=9-*=':''1*=&74:3)=9-*=)&2&,*=.8=2&.39&.3*)='>=9-*=&77.;&1=4+=XPA=

&3)=RPA_=F.3&11>=9-*=)&2&,*)=DNA=.8=7*24;*)='>=XPG=&3)=XPF=*3)43:(1*&8*8=<-.(-=(7*&9*8=

(:9=&9=-=&3)=/=7*85*(9.;*1>_= =

TFIIH=.3=97&38(7.59.43=

E:0&7>49.(= ,*3*= *=57*88.43= .8= )7.;*3= '>= &= (4251*== 8*7.*8= 4+= *;*398= 9-&9= 89&798= <.9-= 9-*=

7*(7:.92*39= 4+= '&8&1= 97&38(7.59.43= +&(9478= TFIIF`= TFIIB`= TFIID= &3)= TFIIE= 94= 9-*= 574249*7=

7*,.43_=F4114<.3,=&)).9.43=4+=TFIIH`=574249*7=2*19.3,=&3)=9-*=45*3=(4251*==+472&9.43=9&0*=

51&(*`=1*&).3,=94=574249*7=*8(&5*`=&=97&38.9.43=&<&>=+742=574249*7=.394=*143,&9.43=(4251*=_==I3=

5&79.(:1&7`=9-*=XPB=*3?>2&9.(=&(9.;.9>=51&>8=&3=.25479&39=741*=.3=9-*=574249*7=*8(&5*=):7.3,=

97&38(7.59.43= .3.9.&9.43= '>= RNA=541= II= B7&)8-*7`= C4.3= *9= &1_= ,***_= T-*= XPB= &(9.;.9>= .38.)*= 4+=

TFIIH= .8= -.,-1>= 7*,:1&9*)= '>= 49-*78= TFIIH= 8:':3.98`= 8:(-= &8= ).7*(91>= '>= 5/,L**`= P&70= *9= &1_=

,**.`= 47= .3).7*(91>= '>= 522TTD8A= 9-74:,-= 5/,= K.2`= P&9*1= *9= &1_= +33/_= A349-*7= 8:':3.9= ()01=

2*).&9*8=9-*=5-485-47>1&9.43=4+=9-*=(&7'4=>1=9*72.3&1=)42&.3=CTD=4+=9-*=1&7,*89=8:':3.9=4+=

9-*= RNA= 541= II_= I9= &184= 2*).&9*8= 9-*= 5-485-47>1&9.43= 4+= 842*= 49-*7= 97&38(7.59.43= +&(9478= &3)=

3:(1*&7= 7*(*59478= &3)= 7*,:1&9*8= '&8&1= &8= <*11= &8= &(9.;&9*)= 97&38(7.59.43_= I9= -&8= '**3= 7*(*391>=

7*;*&1*)=9-&9=NER=+&(9478=XPC`=CSB`=XPA`= XPG=&3)=XPF=&143,=<.9-=TFIIH=8-&7*8=9-*=):&1=

&(9.;.9>= .3= DNA= 7*5&.7= &3)= 97&38(7.59.43_= I3)**)`= 9-*>= &7*= 7*(7:.9*)= &9= &(9.;*= 574249*78= &3)=

5&79.(.5&9*= .3= 9-*= 7*,:1&9.43= 4+= ,*3*= *=57*88.43= .3= 9-*= &'8*3(*= 4+= ,*3494=.(= &99&(0L*= M&>`=

M49&8F*73&3)*8= *9= &1_= ,*+*_= T-*8*= NER= +&(9478= (4397.':9*= 94= &(-.*;*= (-742&9.3= 7*24)*1.3,=

.3(1:).3,=-.8943*8=5489897&381&9.43&1=24).+.(&9.438=PTM8`=DNA='7*&08`=DNA=)*2*9->1&9.43=

&3)=,*3*=1445.3,=<-.(-=&114<8=&((:7&9*=&3)=459.2&1=97&38(7.59.43=L*=M&>`=F7&).3=*9=&1_=,*+,_=

M:9&9.43=.3=XPB`=XPD=&3)=522TTD8A=8:':3.9=4+=TFIIH=&3)=NER=+&(9478=XPA8G`=ERCC+`=TTD8

A`=CSA`=&3)=CSB`=-&;*='**3=&884(.&9*)=<.9-=9-*=-:2&3=,*3*9.(=).847)*78=8:(-=&8= X*74)*72&=

5.,2*3948:2 = XP`= 97.(-49-.4)>89745->= TTD`= (4(0&>3*= 8>3)742*= CS`= &3)= (*7*'7484(:148

+&(.4880*1*9&1= 8>3)742*= COFS_= T-*8*= 5&9.*398= 8-4<= &= 5*751*=.3,= (1.3.(&1= -*9*74,*3*.9>=

7&3,.3,=+742=+`***8+41)=.3(7*&8*)=+7*6:*3(>=4+=80.3=(&3(*78=K7&*2*7`=L**=*9=&1_=+321b=K7&*2*7`=

L*;>= *9= &1_= +33.`= 8-479= 89&9:7*= 94= 3*:7414,.(&1= 574'1*28= &3)= 57*2&9:7*= &,.3,_= T-*= (1.3.(&1=

+*&9:7*8= &7*= 34<= '*.3,= &997.':9*)= 94= 9-*= (42'.3*)= *++*(9= 4+= DNA= 7*5&.7= )*+.(.*3(>= &3)=

97&38(7.59.43&1= )>87*,:1&9.43_= S9:).*8= 4+= TFIIH= -&;*= )*243897&9*)= &= (433*(9.43= '*9<**3=

97&38(7.59.43=&3)=DNA=7*5&.7=&3)= -&;*=45*3*)=&=3*<=+.*1)=4+=97&38(7.59.43=).8*&8*8_= I9=-&;*=

'**3= 8-4<3= 9-&9= 2:9&9.438= .3= 9-*= C89*72.3&1= )42&.3= 4+= 9-*= XPD= 8:':3.9= ).89:7'8= 9-*=

&7(-.9*(9:7*=4+=TFIIH=&3)=.98=241*(:1&7=(422:3.(&9.43=<.9-=9-*=7*9.34.(=&(.)=7*(*5947= =RAR =

K*7.*1`=S9&7>=*9=&1_=,**,`=PPAR8=C425*`=D7&3*=*9=&1_=,**/`=9-*=*8974,*3=7*(*5947=C-*3`=R.*)1=

*9= &1_= ,***`= 9-*= 9->74.)= -47243*= 7*(*59478C425*`= M&1*7'&= *9= &1_= ,**1= &3)= 9-*= &3)74,*3=

7*(*5947= C->204<.9(-`= L*= M&>= *9= &1_= ,*++= 1*&).3,= 94= 9-*= )>87*,:1&9.43= 4+= ()0187*1&9*)=

5-485-47>1&9.43=&3)=(438*6:*391>=9-*=97&38&(9.;&9.43=4+=3:(1*&7=7*(*59478=9&7,*9*)=,*3*8_==

D:7.3,=2>=9-*8.8=I==.3;*89.,&9*)=)**5*7=241*(:1&7=.397.(&(.*8=4+=TFIIH=5&79.(:1&71>`=9-*=741*8=4+=

XPB`=XPD`=&3)=522TTD8A=8:':3.9=4+=TFIIH=):7.3,=97&38(7.59.43=9-&9=(4:1)=+.3&11>=1*&)=94='*99*7=

(4257*-*38.43= 4+= 9-*= *9.414,>= 4+= 9-*= (1.3.(&1= +*&9:7*8= &3)= 9-*= :3)*71>.3,= 241*(:1&7=

2*(-&3.828=&884(.&9*)=<.9-=).89.3(9=2:9&9.438=.3=TFIIH=+4:3)=.3=XP`=XP2CS`=47=TTD=5&9.*398_=

U8.3,= +.'74'1&898= (*11= 1.3*8= )*7.;*)= +742= XP`= XP2CS= &3)= TTD= 5&9.*398= -&7'47.3,= 9-*=

(477*8543).3,=2:9&9.438=.3=7*85*(9.;*=8:':3.98=4+=TFIIH`=I=89:).*)=).++*7*39=2:9&9.438=.3=XPB=

F33S= &3)= T++3P`= XPD= R++,H`= G0*,D`= R02-W`= R1,,W= &3)= 522TTD8A= L,+P`= R/08945=

8:':3.9= 4+= TFIIH_= A8= &= 24)*1`= 43*= 4+= 9-*= 3:(1*&7= 7*(*5947= 7*85438.;*= ,*3*8= RAR ,= <-.(-= .8=

9&7,*9*)=&3)=&(9.;&9*)='>=9-*=7*9.34.(=&(.)=7*(*5947=RAR=:543=97*&92*39=<.9-=&11897&38=7*9.34.(=

&(.)=RA=<&8=:8*)_=T7&38&(9.;&9.43= 4+=RAR ,=<&8=9-:8= &3&1>?*)= ):7.3,=9.2*=(4:78*`=<-.(-=

8-4<*)= 8.,3.+.(&39= ).89:7'&3(*= .3= 2RNA= *=57*88.43= 574+.1*= 4+= RAR ,= .3= XPB`= XPD= &3)=

522TTD8A= 2:9&39= (*11= 1.3*8= (425&7*)= 94= 9-*.7= 7*85*(9.;*= <.1)= 9>5*= (*11= 1.3*_= F:79-*7`= <*=

.3;*89.,&9*)=9-*=7*(7:.92*39=4+=9-*='&8&1=97&38(7.59.43&1=2&(-.3*7>=&3)=NER=+&(9478=&143,=9-*=

574249*7= &3)= 9-*= 9*72.3&947= 7*,.43= 4+= RAR ,= '>= C-742&9.3= I22:3457*(.5.9&9.43= C-IP=

&88&>_= I3= <.1)= 9>5*= WT= (*11= 1.3*8= <*= 4'8*7;*)= 9-*= 7*(7:.92*39= 4+= 9-*= '&8&1= 97&38(7.59.43&1=

2&(-.3*7>=RNA=541=II`=TFIIB=&3)=TFIIH=94,*9-*7=<.9-=9-*=NER=+&(9478=XPA`=CSB`=XPG`=&3)=

XPF= &9= &= 85*(.+.(= 9.2*= 54.39= <-.(-= (4.3(.)*8= <.9-= 9-*= RAR ,= 2RNA= *=57*88.43= 5*&0_=

A19-4:,-= *&(-= XPB= &3)= XPD= 2:9&9.438= 1*)= 94= ).++*7*39= &3)= 85*(.+.(= )>87*,:1&9.43= .3= 9-*=

+472&9.43=4+=9-*=97&38&(9.;&9.43=(4251*=`=':9=9&0*3=94,*9-*7=.9=8:,,*898=&=).89:7'*)=7*(7:.92*39=

4+= 9-*= 97&38(7.59.43&1= 2&(-.3*7>`= TFIIH= (4251*== &3)= 9-*=NER= +&(9478= &9= 9-*=&(9.;*= 574249*7_=

S:(-= 4'8*7;&9.438= 8:,,*89*)= &= 1.30= '*9<**3= 9-*= +472&9.43= 4+= TFIIH= (4251*== :543= .3.9.&9.43=

&3)= 9-*= (43(42.9&39= 7*(7:.92*39= 4+= 9-*= NER= +&(9478_= T-*= 7*(7:.92*39= 4+= 9-*= NER= +&(9478= &7*=

8-4<3= 94= '*= 57*7*6:.8.9*= +47= 9-*= (-742&9.3= 7*24)*1.3,= *;*398= <-.(-= .3(1:)*8= -.8943*= PTM8`=

DNA= '7*&08= &3)= &(9.;*= DNA= )*2*9->1&9.43= &9= 9-*= 574249*7_= W*= -*7*= 8-4<*)= 9-&9= -.8943*=

13 PTM8=.3=XPB<9`=XPD<9`=522TTD8A<9=).851&>*)=&=(1*&7=*37.(-2*39=.3=9-*=).82*9->1=4+=-.8943*=

H-K.=&3)=&(*9>1&9.43=4+=H-K3=<.9-=&=8.2:19&3*4:8=)*(7*&8*=.3=).82*9->1=4+=H-K3=2&708=<-.(-=

(4.3(.)*8=<.9-=2RNA=*=57*88.43=4+=RAR ,`=97&38(7.59.43=2&(-.3*7>`=NER=+&(9478_=T-*=-.8943*=

1&3)2&708= 1.30*)= 94=9-*= &(9.;*=97&38(7.59.43=<*7*=8.,3.+.(&391>=).89:7'*)=.3=&11=9-*=XPB`=XPD=

&3)=522TTD8A=2:9&39=(*11=1.3*8_==

C438.)*7.3,= 9-*= 741*= 4+= XPG= &3)= XPF= *3)43:(1*&8*8= .3= DNA= '7*&0= +472&9.43= &3)= DNA=

)*2*9->1&9.43= <-.(-= *;*39:&11>= 89&'.1.?*8= 9-*= (-742&9.3= 1445.3,= '*9<**3= 9-*= 574249*7= &3)=

9*72.3&947= 7*,.43= 4+= RAR ,`= <*= &3&1>?*)= &884(.&9.43= 4+= 9-*8*= (-742&9.3= 7*24)*1.3,= *;*398=

<.9-= TFIIH_= W*= 9-:8= 8-4<*)= 9-*= 57*8*3(*= 4+= DNA= '7*&08= '49-= &9= 9-*= 574249*7= &3)= 9-*=

9*72.3&947=4+=XPB<9`=XPD<9=&3)=522TTD8A<9=(*118=:543=ATRA=97*&92*39=<.9-=(43(42.9&39=

7*(7:.92*39= 4+= XPG= &3)= XPF= &9= 7*85*(9.;*= 7*,.438_= I3= &11= 9-*= 2:9&39= (*118`= <*= 4'8*7;*)= &=

).89:7'*)= (477*1&9.43= '*9<**3= 9-*= XPG2= XPF= *3)43:(1*&8*8= <.9-= 9-*= DNA= '7*&08= &9= '49-=

574249*7=&3)=9*72.3&947=7*,.438_==

S:(-= DNA= '7*&0= +472&9.438= '>= XPG= &9= 574249*7= &3)= '>= XPF= &9= 9*72.3&947= -&;*= *;*39:&11>=

'**3=(477*1&9*)=<.9-=9-*=&(-.*;*2*39=4+=&(9.;*=DNA=)*2*9->1&9.43=&9=574249*7_=W*=9-:8=3*=9=

&3&1>?*)= 9-*= .251.(&9.43= 4+= TFIIH= .3= DNA= )*2*9->1&9.43= 574(*88_= U8.3,= U382*9->1= IP=

&5574&(-`= <*= 4'8*7;*)= 9-&9= &(9.;*= DNA= )*2*9->1&9.43= .8= 8.,3.+.(&391>= ).89:7'*)= &9= 9-*=

574249*7= 4+= RAR ,= .3= &11= 9-*= XPB`= XPD= &3)= 522TTD8A= 2:9&39= (*11= 1.3*8_= P7*8*3(*= 4+= &11= 9-*=

(42543*398`= 9-*= '&8&1= 97&38(7.59.43&1= 2&(-.3*7>`= NER= +&(9478`= CTCF`= DNA= '7*&0= &3)= DNA=

)*2*9->1&9.43= 94,*9-*7= -&;*= '**3= (477*1&9*)= +47= 9-*= 57*8*3(*= 4+= (-742&9.3= 1445.3,= '*9<**3=

574249*7=&3)=9*72.3&947_=F:79-*7247*=:8.3,=-C=C-7424842*=(43+.72&9.43=(&59:7*=9*(-3.6:*=

<*= )*243897&9*)= 9-&9= TFIIH= 2:9&9.438= ).89:7'= 9-*= CTCF= )*5*3)*39= ,*3*= 1445.3,= '*9<**3=

574249*7=&3)=9*72.3&947=7*,.43=4+=RAR ,=.3=9-*=XPB`=XPD=&3)=522TTD8A=(*118_==

14 C43(1:8.43=

A8= &= ,*3*7&1= (43(1:8.43`= 2>= 7*8:198= 89743,1>= 8:55479*)= &3= .3;41;*2*39= 4+= TFIIH= .3= 9-*=

7*(7:.92*39= 4+= 9-*= NER= +&(9478= &9= &(9.;*= 574249*7= &3)= (438*6:*391>= .3= 9-*.7= 741*8= .3= 9-*=

(-742&9.3= 7*24)*1.3,= .3(1:).3,= PTM8= 4+= -.8943*8`= &(9.;*= DNA= )*2*9->1&9.43`= DNA= '7*&08=

.3):(9.43=&3)=CTCF8)*5*3)*39=,*3*=1445.3,=4+= RAR ,_=S.,3.+.(&391>=2>=<470=-&8=(4397.':9*)=

94= :3;*.1= 0*>= 741*8= 4+= TFIIH= .3= 97&38(7.59.43`= 9-*7*'>= 574;.).3,= &= 89*5= +47<&7)= 94<&7)8= 9-*=

:3)*789&3).3,=4+=97&38(7.59.43&1=).8*&8*8a=XP`=XP2CS=&3)=TTD_==

RS8:2*=)*=1&=9-T8*=)*=)4(947&39=*3=+7&3O&.8= =

C4397.':9.43= )*= TFIIH= )&38= 1*= 7*24)*1&,*= )*= 1&= (-742&9.3*= )S5*3)&39= )*8=

+&(9*:78=NER=1478=)*=1&=97&38(7.59.43=

TFIIHa=:3=(4251*=*=)>3&2.6:*=

TFIIH= *89= :3= (4251*=*= A= 51:8.*:78= 84:88:3.9S8`= (&7&(9S7.8S= *9= 5:7.+.S= *3= 9&39= 6:*= +&(9*:7= )*=

97&38(7.59.43=,S3S7&1*=)*=1234=541>2S7&8*=77= C43&<&>=&3)=C43&<&>=+323b=G*7&7)`=F.8(-*7=*9=

&1_= +33+_= 819S7.*:7*2*39= .1= &= S9S= )S24397S= 6:.1= 8&,.88&.9= ):3= +&(9*:7= 2:19.+43 (9.433*1=

.251.6:S=)&38=51:8.*:78=574(*88:8=(*11:1&.7*8=9*18=6:*=1&=7S5&7&9.43=)*=12<4=5&7=*=(.8.43=)*=

3:(1S49.)*8=NER=*9=1&=7S,:1&9.43=):=(>(1*=(*11:1&.7*_=I1=(4257*3)=)*:==84:88(4251*=*8`=1*=(47*=

(4389.9:S= )*= 8.== 84:88:3.9S8=a= XPB`= 50,`= 5/,`= 5..`= 5-.= *9= 522TTDAK**3*>`= C-&3,= *9= &1_= +33-b=

S(7.2&`= K43.(04;&= *9= &1_= ,**2= *9= 1*= CAK= C>(1.38&(9.;&9.3,= 0.3&8* = (42548S= )*= 974.8= 84:88

:3.9S8a=CDK1`=(>(1.3*=H=&3)=MAT+_=L&=84:8=:3.9S=XPD=5*72*9=)*=7*1.*7=1*=(47*=*9=1*=CAK_=TFIIH=

5488T)*= 51:8.*:78= &(9.;.9S8= *3?>2&9.6:*8= .3).85*38&'1*8= 54:7= 1&= 7S5&7&9.43= 5&7= *=(.8.43= )*=

3:(1S49.)*8=NER=*9=54:7=1&=97&38(7.59.43=F.,:7*=&_==

F.,:7*=&_=S4:88:3.9S8=)*=TFIIH=

TFIIH=)&38=1&=7S5&7&9.43=NER=

>*8= 47,&3.82*8= ;.;&398= 8439= (4389&22*39= *=548S8= A= )*8= &,*398= *3)422&,*&39= 12<4`=

574;*3&39=)*=1*3;.7433*2*39=9*18=6:*=1*8=7&>438=8B=4:=7S8:19&39=):=2S9&'41.82*=(*11:1&.7*=9*1=

6:*=1*=8 97*88=4=>)&9.+_=2+.3=)*=2&.39*3.7=1.39S,7.9S=):=,S342*=.1=*=.89*=51:8.*:78=8>89T2*8=)*=

7S5&7&9.43= 6:.= 5*72*99*39= )S1.2.3*7= (*8= )422&,*8= )*= 12<4_= = F&72.= (*8= 8>89T2*8= )*=

7S5&7&9.43`=1&=;4.*=NER=(.'1*=1*8=1S8.438=.3):.8&39=:3*=).89478.43=)*=1&=)4:'1*=-S1.( *=)*=12<4b=

*11*= *89= 8:').;.8S*= *3= )*:== ;4.*8= 1&= GGR= ,14'&1= ,S342*= 7*5&.7= *9= 1&= TCR= 97&38(7.59.43=

(4:51*)=7*5&.7_=D&38=1&=;4.*=GGR`=1&=1S8.43=*89=7*(433:*=5&7=1*=(4251*=*=XPC2-HR,-B=&1478=

6:*=)&38=1&=;4.*=HI3=(*89=1234=541>2S7&8*=77='146:S*=*3=(4:78= )*=97&38(7.59.43=5&7=1&=1S8.43=

6:.= 5*72*9= ).3.9.*7= 1&= 7S5&7&9.43= *3= 7*(7:9&39= 1*8= +&(9*:78= IJ2= *9= IJK_= I*99*= S9&5*= )*=

7*(433&.88&3(*= *89= 8:.;.*= 5&7= 1*= 7*(7:9*2*39= )*= HL77M=)439= = 1*= 71*= *89= 14:;*79:7*= )*= 12<4=

&:94:7=)*=1&=1S8.43=,7B(*=A=1&(9.;.9S=2HF&8*=)*=1&=84:8=:3.9S=PFK=*9=1&(9.;.9S=-S1.(&8*=)*=PF<_=

E38:.9*=.1=>=&=7*(7:9*2*39=)*8=&:97*8=+&(9*:78=XPA`=RPA`=1*8=*3)43:(1S&8*8=XPF2ERCC+=*9=XPG=

6:.= .3):.8*39= :3*= )4:'1*= .3(.8.43= ):= '7.3= (439*3&39= 1&= 1S8.43`= *9= *3+.3= 1&= 2&(-.3*7.*= )*=

7*8>39-T8*=*9=1.,&9.43_=

TFIIH=)&38=1&=97&38(7.59.43=

>&= 97&38(7.59.43= )*8= ,T3*8= )*= (1&88*= 77= .251.6:*= :3*= 8S7.*= )S;T3*2*398= *9= *89= .3.9.S*= 5&7= 1*=

7*(7:9*2*39=)*8=+&(9*:78=,S3S7&:==)*=97&38(7.59.43=TFIID`=TFIIB`=TFIIE===TFIIF=*9=TFIIH=8:7=1*=

574249*:7b= 1&77.;S*= )*= HL77M= 5*72*9= 14:;*79:7*= )*= 12<4`= 1S(-&55S*= ):= 574249*:7= *9=

1.3.9.&9.43= )*= 1S143,&9.43_= >&(9.;.9S= *3?>2&9.6:*= )*= PFK= /4:*= :3= 71*= .25479&39= )&38=

1S(-&55S*=):=574249*:7=):7&39=1.3.9.&9.43=)*=1&=97&38(7.59.43=5&7=1234=541=77= B7&)8-*7`=C4.3=

*9= &1_= ,*** _= I*99*= &(9.;.9S= *89= 7S,:1S*= 5&7= )&:97*8= 84 :8= :3.9S8= )*= TFIIH`= ).7*(9*2*39= 5&7= 5/,=

T&0*)&(-.`=S&./4=*9=&1_=,*+*`=4:=.3).7*(9*2*39=5&7=522TTDA=A=97&;*78=843=.39*7&(9.43=&;*(=5/,=

S:,&8&<&`=O0:)&=*9=&1_=,**/_=C)01=:3*=&:97*=84:8=:3.9S=)*=TFIIH=&88:7*=1&=5-485-47>1&9.43=

):= )42&.3*= C8 9*72.3&1= )*= 1&= 51:8= ,7&3)*= 84:8= :3.9S= )*= 1234= 541= 77 =b= *11*= 5*72*9= &:88.= 1&=

5-485-47>1&9.43=)&:97*8=+&(9*:78=)*=97&38(7.59.43=.3(1:&39=349&22*39=1&=+&2.11*=)*8=7S(*59*:78=

3:(1S&.7*8_=

RS(*22*39`=.1=&=S9S=)S24397S=6:*=1*8=+&(9*:78=NER=XPC`=CSB`=XPA`=XPG=&3)=XPF=94:9=(422*=

TFIIH= 5&79&,*39= :3*= )4:'1*= &(9.;.9S= )&38= 1&= 7S5&7&9.4 3= )*= 12<4= *9= 1&= 97&38(7.59.43 L*= M&>`=

M49&8F*73&3)*8= *9= &1_= ,*+*_= E3= *++*9`= .18= 8439= 7*(7:9S8= &:= 3.;*&:= )*8= 574249*:78= )*= ,T3*8=

.3):.98= (.'1S8= *9= &(9.;S8= 5&7= )*8= 7S(*59*:78= 3:(1S&.7*8= *9= 5&79.(.5*39= A= 1&= 7S,:1&9.43= )*= 1&=

97&38(7.59.43=*3=&'8*3(*=)*=)422&,*8=)*=12<4_=I*8=+&(9*:78=4T3=(4397.':*39=&:=7*24)*1&,*=

)*=1&=(-742&9.3*== (4389.9:S=5&7=)*8=24).+.(&9.438=5489=97&):(9.433*11*8=FHV8=)-.8 943*8`=)*8=

(&88:7*8=)*=12<4=&:=3.;*&:=):=574249*:7`=1&=)*2*9->1&9.43=&(9.;*=)*=12<4=*9=1&=+472&9.43=

)*= 7S&77&3,*2*39= (-742&9.3.*3`= 5*72*99*39= :3*= 97&38(7.59.43= 57S(.8*= *9= 459.2&1*= L*= M&>`=

F7&).3=*9=&1_=,*+,_==

L*8=2:9&9.438=)&38=1*8=84:88:3.9S8=XPB`=XPD=*9=522TTDA=)*=TFIIH`=*9=)*8=&:97*8=+&(9*:78=NER`=

439=S9S=&884(.S*8=&;*(=)*8=2&1&).*8=,S3S9.6:*8=7&7*8=9*11*8=6:*=1*= X*74)*72&=5.,2*3948:2 =XP`=1&=

97.(-49-.4)>89745-.*=TTD`=1*=8>3)742*=)*=(4(0&>3*=CS`=*9=1*=8>3)742*=(S7S'7484(:148+&(.48

86:*1*9.6:*= IXLJ_= I*8= 5&9.*398= 24397*39= :3*= -S9S74,S3S.9S= (1.3.6:*= &11&39= ):3*= 97T8= +479*=

57S).8548.9.43=A=)S;*1455*7=)*8=(&3(*78=)*=1&=5*&:K7&*2*7`=L**=*9=&1_=+321b=K7&*2*7`=L*;>=*9=

&1_= +33.= A= )*8= 574'1T2*8= 3*:7414,.6:*8= 4:= :3= ;.*.11.88*2*39= 57S2&9:7S_= C*8= 8>2592*8=

(1.3.6:*8= 8439= &997.':S8= A= 1*++*9= (42'.3S= ):3= )S+&:9= )*= 1&= 7S5&7&9.43= 4T3= *9= ):3*=

)S7S,:1&9.43=)*=1&=97&38(7.59.43_==

>S9:)*= ):= (4251*=*= HL77M= &= )S24397S= 1&= 57S8*3(*= ):3= 1.*3= *397*= 1&= 97&38(7.59.43= *9= 1&=

7S5&7&9.43=)*=12<4=*9=&=4:;*79=:3=34:;*&:=(43(*59=)*=2&1&).*8=)*=1&=97&38(7.59.43_=71=&=S9S=

)S24397S= 6:*= 1*8= 2:9&9.438= &:= )42&.3*= C89*72.3&1= )*= 1&= 84:8= :3.9S= XPD= 5*79:7'&.*39=

1&7(-.9*(9:7*= ):= HL77M= *9= 843= .39*7&(9.43= 241S(:1&.7*= &;*(= 51:8.*:78= 7S(*59*:7= 3:(1S&.7*8 `=

(422*= 1*= 7S(*59*:7= )*= 1&(.)*= 7S9.346:*= RAR= K*7.*1`= S9&7>= *9= &1_= ,**,`= = 1*8= 7S(*59*:78=

5*74=.842*=5741.+*7&9478&(9.;&9*)=PPAR8=C425*`=D7&3*=*9=&1_=,**/ `=1*=7S(*59*:7=)"8974,T3*=

ER=C-*3`=R.*)1=*9=&1_=,***`=1*= 7S(*59*:78=)*=1-47243*=9->74).*33*=H38= C425*`=M&1*7'&=

*9=&1_=,**1`=1*8=7S(*5 9*:78=)*=1-47243*=&3)74,T3*= C->204<.9(-`=L*=M&>=*9=&1_=,*++`=7S8:19&39=

84:;*39=):3*=)S7S,:1&9.43=)&38=1*=2S(&3.82*=)*=5-485-47>1&9.43=5&7=()01 =b=.1=*3=7S8:19*=:3=

)S+&:9=)*=97&38&(9.;&9.43=)*8=,T3*8=(.'1S8=5&7=(*8=7S(*59*:78=3:(1S&.7*8_==

18 D:7&39= 2&= 9-T8*`= /*= 2*= 8:.8= .39S7*88S*= &:== .39*7&(9.438= 241S(:1&.7*8= )*= TFIIH`= *9=

5&79.(:1.T7*2*39=&:==71*8=)*8=84:8=:3.9S8=XPB`=XPD=*9=522TTDA=):7&39=1&=97&38(7.59.43`=&+.3=

)*= 2.*:== (4257*3)7*= 1*8= 2S(&3.82*8= 241S(:1&.7*8= 84:88/&(*398= &884(.S8= &:== 2:9&9.438=

(43(*73&39= (*8= 5749S.3*8`= (*= 6:.= 5*72*997&.9= )*= 2.*:== *=51.6:*7= 1*8= 8>2592*8= (1.3.6:*8=

4'8*7;S8=(-*?=1*8=5&9.*398=XP`=XP2CS`=*9=TTD_=

F.,:7*='_=M:9&9.438=S9:).S*8=)&38=XPB`=XPD=*9=52=2=TTD8A=84:88:3.9S8=)*=TFIIH=

N4:8=&;438=:9.1.8S=)*8=+.'74'1&89*8=574;*3&39=)*8=5&9.*398=(.9*8=(.8 )*88:8`=54:7=S9:).*7=1*++*9=)*=

).++S7*39*8=2:9&9.438=)*8=84:8=:3.9S8=(43(*73S*8=)*=TFIIHa=XPB=F33S=&3)=T++3P`=XPD=R++,H`=

G0*,D`=R02-W`=R1,,W=*9=522TTD8A=L,+P`=R/08945_=N497*=24)T1*= (438.89&.9=*3=1S;&1:&9.43=

)*=1&=97&38&(9.;&9.43=):3=,T3*=(.'1*=):=7S(*59*:7=3:(1S&.7*=323 ,=&57T8=97&.9*2*39=)*8=(*11:1*8=

*3=(:19:7*=5&7=1&(.)*=7S9.346:*_=44:8=&;438=4'8*7;S=:3*=5*79:7'&9.43=8.,3.+.(&9.;*=):=574+.1=

)*=57*88.43=)*=12342=)*=323 ,=)&38=1*8=(*11:1*8=2:9&39*8=54:7=XPB`=XPD=*9=522TTDA=*3=

(425&7&.843=&;*(=1&=1.,3S*=8&:;&,*_=E38:.9*=34:8=&;438=S9:).S=5&7=C-IP=I22:3457S(.5.&9.43=

)*=1&=(-742&9.3*=1*=7*(7:9*2*39=)*=1&=2&(-.3*7.*=)*=97&38(7.59.43=*9=)*8=+&(9*:78=NER=8:7=1*=

574249*:7=*9=1*=9*72.3&9*:7=):=,T3*=RAR ,_=D&38=1&=1.,3S*=8&:;&,*`=34:8=&;438=4'8*7;S=6:*=1*=

9*258=)*=7*(7:9*2*39=)*=1234=541=77`=)*8=+&(9*:78=)*=97&38(7.59.43=HL77K=*9=HL77M=&.38.=6:*=1*8=

+&(9*:78=4T3=PF2`=IJK`=PF]=*9=PFL=(43(.)&.9=&;*(=1*=5.(=)*=57*88.43=)*=12342=)*=3 AR ,_=

A:=(4397&.7*=)&38=1*8=1.,3S*8=2:9&39*8=54:7=XPB=*9=XPD`=.1=>=&;&.9=:3=)S+&:9=)*=7*(7:9*2*39=)*=

19 (*8=).++S7*398=+&(9*:78=8:7=1*=574249*:7=&(9.+=6:.=;&7.&.9=8*143=1*=9>5*=)*=,U3*=*9=1&=548.9.43=)*=1&=

2:9&9.43_= C*8= 4'8*7;&9.438= 8:,,T7*39= :3= 1.*3= *397*= TFIIH= *9= 1*= 7*(7:9*2*39= (43(42.9&39= )*8=

+&(9*:78=4T3=1478=)*=1.3.9.&9.43=)*=1&=97&38(7.59.43_=<&:9&39=51:8=6:.1=&=S9S=)S24397S=6:*=(*=

7*(7:9*2*39=)*8=+&(9*:78=4T3=1478=)*=1&=97&38(7.59.43=*3=&'8*3(*=)*=)422&,*8=)*=12<4=S9&.9=

:3*=(43).9.43=57S&1&'1*=&:==S;S3*2*398=)*=7*24)*1&,*=(-742&9.3.*3=.3(1:&39=1*8=24).+.(&9.438=

)-.8943*8`=1*8=(&88:7*8=)2<4=*9=1&=)S2S9->1&9.43=&(9.;*=&:=3.;*&:=):=574249*:7_=44:8=&;438=

.(.= 24397S= 6:*= )&38= 1*8= (*11:1*8= 8&:;&,*8= = .1= >= &;&.9= :3*= &:,2*39&9.43= )*8= 24).+.(&9.438=

)-.8943*8 = H-K.2*,= *9= H-K3&(= &;*(= :3*= 7S):(9.43= )*= H-K32*= 8.2:19&3S2*39= &:= 5.(=

)*=57*88.43=)*=12342=)*= RAR ,`=):=7*(7:9*2*39=)*8=+&(9*:78=)*=97&38(7.59.43=*9=)*8=+&(9*:78=

4T3_=I*8=).++S7*39*8=24).+.(&9.438=)-.8943*8=(&7&(9S7.89.6:*8=)*=1&=97&38(7.59.43=&(9.;*=439 =S9S=

8.,3.+.(&9.;*2*39=5*79:7'S*8=)&38=1*8=1.,3S*8=)*=(*11:1*8=2:9&39*8=54:7==XPB`=XPD=*9=522TTDA_=

P7*3&39=*3=(438.)S7&9.43=1*=71*=)*8=*3)43:(1S&8*8=XPG=*9=XPF=)&38=1&=+472&9.43=)*8=(&88:7*8=

)2<4=*9=1&=)S2S9->1&9.43=6:.=5*72*997&.9=)*=89&'.1.8*7=1&=+472& 9.43=)*=897:(9:7*8=*3='4:(1*8=

,T3*= 1445.3,= *397*= 1*= 574249*:7= *9= 1*= 9*72.3&9*:7= ):= ,T3*= RAR ,`= 34:8= &;438= &3&1>8S=

1&884(.&9.43=*397*=(*8=S;S3*2*398=)*=7*24)*1&,*=(-742&9.3.*3=*9=1S9&9=)*=HL77M_=44:8=&;438=

&.38.=4'8*7;S=*3=51:8=):=7*(7:9*2*39=)*=XPG=*9= PFL`=1&=57S8*3(*=)*=(&88:7*8=)2<4=&:9&39=9&39=

&:=3.;*&:=):=574249*:7=6:&:=3.;*&:=):=9*72.3&9*:7=8:.9*=&:=97&.9*2*39=5&7=1&(.)*=7S9.346:*=

)&38= 1*8= (*11:1*8= WT`= &1478= 6:*= )&38= 1*8= (*11:1*8= 2:9&39*8`= (*99*= (477S1&9.43= *397*= 1*8= )*:==

*3)43:(1S&8*8=*9=1*8=( &88:7*8=)2<4=S9&.9=5*79:7'S*_= =

71=&=S9S=24397S=6:*=1*8=(&88:7*8=)*=12<4=.3):.9*8=5&7=PF]=&:=3.;*&:=):=574249*:7`=*9=5&7=PFL=

&:= 3.;*&:= ):= 9*72.3&9*:7`= S9&.*39= (477S1S*8= &:= 574(*88:8= )*= )S2S9->1&9.43= &(9.;*`= (*8=

S;T3*2*398= 8439= -&:9*2*39= (447)433S8= &+.3= )*= 5*72*997*= :3*= 97&38(7.59.43= 459.2&1*_= N4:8=

&;438= &3&1>8S= 1.251.(&9.43= )*= HL77M= )&38= (*= 574(*88:8= *3= :9.1.8&39= 1&5574(-*= 832*9->1 8IP_=

N4:8=&;438=&.38.=4'8*7;S=6:*=)&38=1*8=(*11:1*8=2:9&39*8`=1&=)S2S9->1&9.43=&(9.;*=&:=3.;*&:=):=

574249*:7= )*= RAR ,= S9&.9= 8.,3.+.(&9.;*2*39= 5*79:7'S*_= N4:8= &;438= &:88.= )S24397S= 5&7= 1&=

9*(-3.6:*= -C= C-7424842*= (43+472&9.43= (&59:7*= 6:*= (*8= 2:9&9.438= )*= TFIIH= &19S7&.*39= 1&=

+472&9.43=)*8='4:(1*8=)*=(-742&9.3*=*397*=1*=574249*:7=*9=1*=9*72.3&9*:7=):=RAR ,=&.)S*=5&7=

147,&3.8&9*:7= )*=1&=(-742&9.3*=CTCF_=

20 C43(1:8.43=

M*8=7S8:19&98=8:,,T7*39=+479*2*39=:3*=.251.(&9.43=)*=TFIIH=)&38=1*=7*(7:9*2*39=)*8=+&(9*:78=

NER= 8:7= 1*= 574249*:7= &(9.+= &.38.= 6:*= 54:7= 1*:78= 71*8= 1478= ):= 7*24)*1&,*= )*= 1&= (-742&9.3*=

.3(1:&39= 1*8= 24).+.(&9.438= )*8= -.8943*8 `= 1&= )S2S9->1&9.43= &(9.;*= )*= 12<4`= 1.3):(9.43= )*8=

(&88:7*8=)2<4=*9=1&=+472&9.43=)*8='4:(1*8=)S5*3)&39*=)*=IHIL_= =

C-&6:*= 2:9&9.43= )&38= TFIIH= .(.= S9:).S*= 57S8*39*= :3*= )S7S,:1&9.43= 85S(.+.6:*= .11:897&39= 1&=

(4251*=.9S=)*8=)S+&:98=97&38(7.59.433*18=4'8*7;S8= (-*?=1*8=5&9.*398=XP`=TTD=*9=CS=.88:8=)*=1&=

(42'.3&.843= = )*8= +43(9.438= *3?>2&9.6:*8= )*= TFIIH= *9= 1*8= S9&5*8= )*= 7*24)*1&,*= )*= 1&=

(-742&9.3*= )&38= 1&= (&8(&)*= )*8= S;T3*2*398= 6:.= (4389.9:*39= 1&= 7S,:1&9.43= )*= 1*=57*88.43= )*8=

,T3*8_=_==

F.,:7*=(_=C-&6:*=2:9&9.43=)&38=1*8=84:88:3.9S8=)*=TFIIH=7*57S8*39*=85S(.+.6:*=)S7S,:1&9.43=)*=

97&38(7.59.43=

21 R*+*7*3(*8a=

+= C43&<&>`= J_= W_= ]= C43&<&>`= R_= C_= A= 2:19.8:':3.9= 97&38(7.59.43= +&(947= *88*39.&1= +47=

&((:7&9*=.3.9.&9.43='>=RNA=541>2*7&8*=II_= J_=B.41_=C-*2_ =,0.`=,-/18,-0,=+323_=

,= G*7&7)`=M_ =*9=&1_ =P:7.+.(&9.43=&3)=.39*7&(9.43=5745*79.*8=4+=9-*=-:2&3=RNA=541>2*7&8*=

BII=,*3*7&1=97&38(7.59.43=+&(947=BTF,_= T-*=J4:73&1=4+='.414,.(&1=(-*2.897> =,00`=,*3.*8,*3./_=+33+_=

-= G.,1.&8M&7.`=G_ =*9=&1_ =A=3*<`=9*39-=8:':3.9=4+=TFIIH=.8=7*85438.'1*=+47=9-*=DNA=7*5&.7=

8>3)742*=97.(-49-.4)>89745->=,74:5=A_= N&9:7*=,*3*9.(8 =-0`=1+.81+3=,**._=

.= C4.3`= F_ = *9= &1_ = 522TTD8A= &8= &= R*5&.78S5*(.+.(= TFIIH= S:':3.9_= M41*(:1&7= (*11 = ,+`= ,+/8,,0=

,**0_=

/= B7&)8-*7`=J_`=C4.3`=F_=]=E,1>`=J_=M_=D.89.3(9=741*8=+47=9-*=-*1.(&8*8=4+=TFIIH=.3=97&38(7.59=

.3.9.&9.43=&3)=574249*7=*8(&5*_= J=B.41=C-*2 =,1/`=,/-,8,/-2=,***_=

0= C4.3`= F_`= O08*3>(-`= V_= ]= E,1>`= J_= M_= D.89.3(9= R41*8= +47= 9-*= XPB25/,= &3)= XPD25..=

S:'(4251*=*8= 4+= TFIIH= .3= D&2&,*)= DNA= O5*3.3,= ):7.3,= N:(1*49.)*= E=(.8.43= R*5&.7_=

M41*(:1&7=(*11 =,0`=,./8,/0=,**1_=

1= L*=M&>`=N_ =*9=&1_ =NER=+&(9478=&7*=7*(7:.9*)=94=&(9.;*=574249*78=&3)=+&(.1.9&9*=(-742&9.3=

24).+.(&9.43=+47=97&38(7.59.43=.3=9-*=&'8*3(*=4+=*=4,*34:8=,*3494=.(=&99&(0_= M41*(:1&7=(*11 =-2`=/.8

00`=)4.aS+*318,10/+***,++8X= 5.. =+*_+*+02/_241(*1_,*+*_*-_**.=,*+*_=

2= L*=M&>`=N_`=F7&).3`=D_`=I19.8`=I_`=B4:,3*7*8`=P_=]=E,1>`=J_=M_=XPG=&3)=XPF=E3)43:(1*&8*8=

T7.,,*7= C-742&9.3= L445.3,= &3)= DNA= D*2*9->1&9.43= +47= A((:7&9*= E=57*88.43= 4+= A(9.;&9*)=

G*3*8_= M41=C*11 =.1`=0,,80-,`=)4.aS+*318,10/+,**/**8X= 5.. =+*_+*+02/_241(*1_,*+,_*/_*/*=,*+,_=

3= K7&*2*7`= K_= H_`= L**`= M_= M_= ]= S(4994`= J_= X*74)*72&= 5.,2*3948:2_= C:9&3*4:8`= 4(:1&7`=

&3)=3*:7414,.(=&'3472&1.9.*8=.3=2-*=5:'1.8-*)=(&8*8_= A7(-=D*72&941 =+,-`=,.+8,/*=+321_=

+*= K7&*2*7`= K_= H_ = *9= &1_ = X*74)*72&= 5.,2*3948:2= &3)= 7*1&9*)= ).847)*78a= *=&2.3.3,= 9-*=

1.30&,*='*9<**3=)*+*(9.;*=DNA=7*5&.7=&3)=(&3(*7_= J_=I3;*89_=D*72&941_ =+*-`=30S8+*+S=+33._=

22 ++= K*7.*1`=A_`=S9&7>`=A_`=S&7&8.3`=A_`=R4(-*99*8E,1>`=C_=]=E,1>`=J_=M_=XPD=2:9&9.438=57*;*39=

TFIIH8)*5*3)*39=97&38&(9.;&9.43='>=3:(1*&7=7*(*59478=&3)=5-485-47>1&9.43=4+=RAR&15-&_= C*11 =

+*3`=+,/8+-/=,**,_=

+,= C425*`=E_ =*9=&1_ =D>87*,:1&9.43=4+=9-*=P*74=.842*=P741.+*7&9478A(9.;&9*)=R*(*5947=T&7,*9=

G*3*8='>=XPD=M:9&9.438_= M41=C*11=B.41 =,/`=0*0/80*10=,**/_=

+-= C-*3`=D_ =*9=&1_ =A(9.;&9.43=4+=*8974,*3=7*(*5947=&15-&='>=S++2=5-485-47>1&9.43=.3;41;*8=&=

1.,&3)8)*5*3)*39=.39*7&(9.43=<.9-=TFIIH=&3)=5&79.(.5&9.43=4+=CDK1_= M41=C*11 =0`=+,18+-1=,***_=

+.= C425*`= E_ = *9= &1_ = N*:7414,.(&1= )*+*(98= .3= 97.(-49-.4)>89745->= 7*;*&1= &= (4&(9.;&947=

+:3(9.43=4+=TFIIH_= N&9:7*=3*:748(.*3(* =+*`=+.+.8+.,,=,**1_=

C->204<.9(-`=P_`=N_=L*=M&>`=*9=&1_=,*++_=T-*=5-485-47>1&9.43=4+=9-*=&3)74,*3=7*(*5947=

'>=TFIIH=).7*(98=9-*=:'.6:.9.325749*&842*=574(*88_=EMBO=J=-*-a=.028.13_=

23 I3974):(9.43=

24 INTRODUCTION=

T7&38(7.59.43=.8=&=+:3)&2*39&1=574(*88=9-&9=51&>8=&3=.25479&39=741*=.3=)*;*1452*39=&3)=

(*11:1&7= 7*85438*8_= I9= .8= )*+.3*)= &8= 9-*= 8>39-*8.8= 4+= RNA= +742= &= DNA= 9*251&9*_= I3=

'&(9*7.&= &3)= &7(-&*&`= 97&38(7.59.43= 4+= &11= ,*3*8= .8= (&9&1>?*)= '>= &= 8.3,1*= RNA=

541>2*7&8*`=':9=.3=*:0&7>49.(=,*342*=9-*=).++*7*39=RNA=541>2*7&8*8=541=I`=541=II`=&3)=

541= III= 97&38(7.'*= ).++*7*39= (1&88*8= 4+= ,*3*8= <-.(-= <*7*= ).8(4;*7*)= ):7.3,= +30/8+31*=

R4*)*7=&3)=R:99*7=+31*_=RNA=541=I=8>39-*8.?*=9-*=,/S=7RNA=57*(:78478=&3)=541=III=

8>39-*8.?*= 9-*= /S= 7RNA= &3)= 97&38+*7= RNA8= 9RNA8_= B>= (4397&89= RNA= 541= II= .8=

7*85438.'1*=+47=9-*=97&38(7.59.43=4+=2RNA8=&3)=8*;*7&1=82&11=3:(1*&7=RNA8=&3)=)4*8=

84= :8.3,= 5749*.3= 2&(-.3*7>= (4257.8.3,= &5574=.2&9*1>= 0*= 541>5*59.)*8_= I3= 9-.8= 9-*8.8=

)*9&.1*)=)*8(7.59.43=4+=RNA=541=II=2&(-.3*7>=.8=5&79.(:1&71>=).8(:88*)_=

I_ T-*=97&38(7.59.43=2*(-&3.82=

T-*=7*,:1&9.43=4+=97&38(7.59.43=.8=.397.(&9*`=4+9*3=.3;41;.3,=.39*751&>='*9<**3=574249*78=

&3)=;&7.4:8=7*,:1&947>=*1*2*398_=A=57*7*6:.8.9*=+47=:3)*789&3).3,=9-*=2*(-&3.828=&3)=

57.3(.51*8= 4+= 9-.8= 574(*88= 7*6:.7*8= .)*39.+.(&9.43= 4+= 9-*8*= *1*2*398= &3)= 9-*.7=

7*1&9.438-.58=<.9-=8*;*7&1=,*3*7&1=97&38(7.59.43=+&(9478=GTF8_=T-*8*=*1*2*398=.3(1:)*=

9-*= (47*= 574249*7`= &)).9.43&1= (.88&(9.3,= DNA= 8*6:*3(*8= 8:(-= &8= 574=.2&1= 574249*7=

*1*2*398`=*3-&3(*78`=8.1*3(*78`=&3)=.38:1&9478_=

AT T&# .0-+-2#0 #*#+#,21 ," 20 ,1!0'.2'-, !-+.-,#,21

A_+=T-*=(47*=574249*7=

T-*=RNA=541=II=(47*=574249*7=1.*8=&9=9-*=(*39*7=4+=9-*=97&38(7.59.43=574(*88_=T-*=(47*=

574249*7=8*7;*8=&8=9-*='&8*=+47=9-*=&88*2'1>=4+=RNA=541=II=&3)=&11=9-*=89.2:1&947>=&3)=

7*57*88.;*=5749*.3=+&(9478=9-&9=&7*=.3;41;*)=.3=9-*=7*,:1&9.43=4+=97&38(7.59.43&1=&(9.;.9>_=

I9=.8=)*+.3*)=&8=9-*=DNA=8*6:*3(*=<-.(-=).7*(98=9-*=.3.9.&9.43=4+=9-*=97&38(7.59.43='>=

25 F'%30# WT C-0# .0-+-2#0 +-2'$1 $-0 20 ,1!0'.2'-, 7 RNA .-*7+#0 1# IIT

T# +-2'$1 0# 27.'! **7 $-3," ', $-!31#" !-0# .0-+-2#01T T&'1 "' %0 + '1 0-3%&*7 2- 1! *# ," " .2#" $0-+ T-)313+'Q M #2 *T XVV]R J34#,VG#01&-, ," K "-, % XVWV

ECVIVJ s PQs1 1QJ CQJsVJs%s sV_%VJHV B1JR1J$ `:H Q`s BRE% R Q R SSGRCGCC TFIIB TATA RL Q R TATAAR TBP BRER R Q R RTDKKKK TFIIB PCE R Q U DSGGGRASM J@JQ1J ^.%I:J_ IJ` R Q U TCAKT ^ D`QsQ].1C: _ TAFLTAF AN ^.%I:J_ TCT R Q U CTTT J@JQ1J MTE U Q U CSARCSSAACGS TFIID DPE U Q U RGCGTG TAF LTAF DCE SI U Q U CTTC SII U Q U CTGT TAF SIII U Q U AGC

T *# ST C-,1#,131 1#/3#,!# ," .-1'2'-, -$ 2&# "'$$#0#,2 !-0# .0-+-2#0 #*#+#,21T =

S-C GR W - A TR Y-C TR R-A GR M-C AR K-T GR D-T G AR N-A C G TT A" .2#" $0-+ J34#,VG#01&-, ," K "-, % TRSRT =

26 RNA= 541= II_= I9= (4257.8*8= 4+= 9-*= 97&38(7.59.43= 89&79= 8.9*= TSS= &3)= (&3= *=9*3)= 5= .*= '5=

:5897*&2= &3)= 247= )4<3897*&2= 4+= 9-*= (+= 548.9.43_= P7*;.4:81>= .9= <&8= 9-4:,-9= 9-&9= 9-*=

(47*=574249*7=.8=&=8.251*=&3)=&=8.3,1*=249.+=':9=34<=.9=.8=(1*&7=9-&9=9-*7*=.8=(438.)*7&'1*=

).;*78.9>=.3=9-*=(47*=574249*7=&3)=897:(9:7*_=T-*7*=&7*=9<4=0.3)=4+=574249*78b= +4(:8*) =

&3)= ).85*78*) = S2&1*= &3)= K&)43&,&= ,**-_= F4(:8*) = 574249*78= (439&.38= *.9-*7= 8.3,1*=

TSS= 47= ).89.3(9= (1:89*78= 4+= 8-479= 7*,.43= 4;*7= 8*;*7&1= 3:(1*49.)*8`= <-*7*&8= ).85*78*) =

574249*78= -41)8= 3:2'*7= 4+= TSS= ).897.':9*)= 4;*7= &= '74&)= 7*,.43= 4+= /*= 94= +**= '5= &3)=

9>5.(&11>=7*8.)*8=.3=9-*=C5G=.81&3)8=.3=;*79*'7&9*8_=T-*=+4(:8*)=(47*=574249*78=&7*=247*=

&3(.*39=&3)=<.)*857*&)=9-74:,-4:9=9-*=3&9:7*=9-&3=9-*=).85*78*)=574249*7_=H4<*;*7=

.3=;*79*'7&9*8`=).85*78*)=574249*78=&7*=247*=(42243=9-&3=9-*=+4(:8*)=574249*78_=T-*=

(47*=574249*7=*1*2*398=8:(-=&8=9-*=TATA='4=`=BRE:=9-*= :5897*&2=TFII B=7 *(4,3.9.43=

*1*2*39`=IN7= .3 .9.&94 7`=DPE=)4<3897*&2= 574249*7= *1*2*39`=MTE= 249.+= 9*3= *1*2*39`=

DCE=)4<3897*&2= (47*= *1*2*39`=TCT=249.+=541>5>7.2.).3*=.3.9.&947=249.+=&3)=XCPE=

+= X= (47*= 574249*7= *1*2*39= += &7*= &11= +4:3)= 94= '*= 9-*= 5&79= 4+= +4(:8*)= 574249*7_= I3=

(4397&89`= ).85*78*)= (47*= 574249*7= ,*3*7&11>= 1&(08= 9-*= &'4;*= 2*39.43*)= *1*2*398=

C&73.3(.`=S&3)*1.3=*9=&1_=,**0_=T-*7*=&7*=-4<*;*7=34=:3.;*78&1=(47*=574249*7=*1*2*398_=

T-*= *1*2*398= )*8(7.'*)= '*14<= &7*= .3= (439*=9= 4+= +4(:8*)= (47*= 574249*7= F.,:7*= += &3)=

T&'1*=+`=&)&59*)=+742=J:;*38G*78-43=&3)=K&)43&,&=,*+* =

WTW T&# TATA -6 =

T-.8=8*6:*3(*=*1*2*39=<&8=9-*=+.789=(47*=574249*7=249.+=9-&9=<&8=).8(4;*7*)=&3)=(&11*)=

&8=9-*=H4,3*88='4==.3=41)=1.9*7&9:7*8_=T-*=249.+=-&8=&=(438*38:8=8*6:*3(*=TATATAA=

&3)=.8=14(&9*)=,/8=-*='5=:5897*&2=4+=9-*=TSS_==T-*=TATA='4==.8=7*(4,3.?*)=&3)='4:3)=

'>= 9-*= TATA8'.3).3,= 5749*.3= TBP`= 8:':3.9= 4+= 9-*= TFIID= (4251*=_= A19-4:,-= TATA=

'4== <&8= 9-*= +.789= 034<3= (47*= 574249*7= *1*2*39`= .9= .8= 57*8*39= 431>= .3= +*8+/= = 4+= 9-*=

2&22&1.&3=(47*=574249*78=C&73.3(.`=S&3)*1.3=*9=&1_=,**0_=M489=4+=9-*=-4:8*0**5.3,=

,*3*8`=43(4,*3*8`=,74<9-=+&(9478`=&3)=TF8=&7*=4+9*3=TATA81*88_=

27 WTX T&# I,'2' 2-0 #*#+#,2 I,0

T-*=.3.9.&947=.8=9-*=2489=(422431>=4((:77.3,=249.+=.3=9-*=+4(:8*)=(47*=574249*7=&3)=

(4;*78= 9-*= TSS= (+_= T-*= +:3(9.43= 4+= I37= &8= ).++*7*39= (47*= 574249*7= *1*2*39= <&8= +.789=

4'8*7;*)='>=S2&1*= *9=&1 _=S2&1*=&3)=B&19.247*=+323_=TFIID=-&8='**3=+4:3)=94=.39*7&(9=

'*89= <.9-= 9-*= I37= ;.&= .98= TBP8= &884(.&9*)= +&(9478= TAF8_= T-*= +:3(9.43&1= &3&1>8*8= 4+=

2&22&1.&3=574249*78=-&;*=8-4<3=&='74&)*7=2&22&1.&3=(438*38:8=YR=<-*7*=R=.8=(+=

C&73.3(.`= S&3)*1.3= *9= &1_= ,**0= <-.(-= .8= ).++*7*39= +742= 9-*= (1&88.(&1= I37= (438*38:8=

8*6:*3(*= YYANWYY_= = I3= D74845-.1& = 9-*= I37= (438*38:8= .8= TCAKTY`= <-.1*= .3= 7.(*= &3)=

A7&'.)458.8` =&=YR=I37=249.+=<-*7*=R=.8=(+=.8=4'8*7;*)=Y&2&2494`=I(-.)&=*9=&1_=,**1_=

WTY T&# TFIIB 0#!-%,'2'-, #*#+#,21 BRE3 ," BRE"

T-*= TFIIB= 7*(4,3.9.43= *1*2*39= <&8= .)*39.+.*)= &8= TFIIB= '.3).3,= 8*6:*3(*= '>= E1'7.,-9=

&3)=(411*&,:*8=L&,7&3,*`=K&5&3.).8=*9=&1_=+332_=T-.8=7*,.43=<&8=.3.9.&11>=.)*39.+.*)=&8=

:5897*&2=4+=9-*=TATA='4==&3)=-*3(*=3&2*)=BRE:_=T-*=49-*7=8*6:*3(*=.)*39.+.*)=&8=

)4<3897*&2= TFIIB= 7*(4,3.9.43= *1*2*39= .8= 3&2*)= &8= BRE)_= B49-= BRE:= &3)= BRE)=

7*,:1&9*=9-*=1*;*1=4+='&8&1=97&38(7.59.43=.3=(43/:3(9.43=<.9-=9-*=TATA8'4=_=H4<*;*7`=

BRE:= &3)= BRE)= (&3= -&;*= 548.9.;*= &3)= 3*,&9.;*= *++*(98= 43= 9-*= 97&38(7.59.43= .3= &=

574249*7=(439*=9=)*5*3)*39=2&33*7=D*3,`=M&1*(4;@=*9=&1_=,**3_=

WTZ T&# D-5,120# + .0-+-2#0 #*#+#,2 DPE

T-*=DPE=.8=&=(47*=574249*7=*1*2*39=14(&9*)=)4<3897*&2=(,2=94=(--=94=A(+=.3=9-*=I37=

K&)43&,&=,**,_=I9=.8=7*(4,3.?*)='>=TAF0=&3)=TAF3=8:':3.9=4+=TFIID=(4251*==&3)=.8=

(438*7;*)= +742= D74845-.1& = 94= -:2&38_= T-*= DPE= (438*38:8= .3= D74845-.1& = .8= RGWYVT`=

<-.1*=.3=-:2&38=.9=-&8=>*9=94='*=)*9*72.3*)_=DPE=+:3(9.438=.3=(445*7&9.43=<.9-=9-*=I37=

&3)=9-*=85&(.3,='*9<**3=9-*=DPE=&3)=I37=.8=.25479&39=+47=9-*=459.2&1=97&38(7.59.43_=

28 WT[ T&# M-2'$ 2#, #*#+#,2 MTE

T-*=MTE=.8=&349-*7=+:3(9.43&11>=&(9.;*=(47*=574249*7=*1*2*39_=I9=.8=14(&9*)=:5897*&2=4+=

9-*=DPE=+742=(+2=94=(,1=7*1&9.;*=94=9-*=TSS=&3)=.8=(438*7;*)=+742= D74845-.1& =94=-:2&38_=

L.0*=DPE`=MTE=.8=&184=&=7*(4,3.9.43=8.9*=+47=TFIID_=MTE=&3)=DPE=(&3=<470=.3=8>3*7,>=

':9=MTE=(&3=&184=+:3(9.43=.3=(445*7&9.43=<.9-=I37=.3)*5*3)*39=4+=DPE=&3)=TATA='4=_=

WT\ T&# "-5,120# + !-0# #*#+#,2 DCE

T-*=DCE=<&8=+.789=+4:3)=.3=-:2&3= 8,14'.3=574249*7=L*<.8`=K.2=*9=&1_=,***_=I9=4((:78=

2:9:&11>= *=(1:8.;*= 4+= DPE_= T-*= DCE= (438.898= 4+= 9-7**= 8-479= 8:'= *1*2*398= 3&2*1>= SI=

CTTC=+742=(0=94=(++`=SII=CTGT=+742=(+0=94=(,+=&3)=SIII=AGC=+742=(-*=94=(-._=

WT] T&# TCT +-2'$ .-*7.70'+'"',# ','2' 2-0 +-2'$

T-*=TCT=249.+=*3(425&88*8=9-*=97&38(7.59.43=89&79=8.9*=4+=3*&71>=&11=7.'4842&1=5749*.3=

,*3*=574249*78=.3= D74845-.1& =&3)=2&22&18_=I9=.8=8.2.1&7=94=9-*=.3.9.&947=I37`=':9=.8=349=

7*(4,3.?*)= '>= 9-*= (&343.(&1= TFIID= (4251*== <-.(-= '.3)8= 94= 9-*= I37= &3)= (&3349=

+:3(9.43=.3=1.*:=4+=&3=I37_=H4<*;*7=&=8.3,1*=T8948A=3:(1*49.)*=8:'89.9:9.43=(43;*798=9-*=

TCT=*1*2*39=.394=&=+:3(9.43&11>=&(9.;*=I37=K&)43&,&=,*+,_=TCT=249.+=85&38=+742=8,=94=

(0=7*1&9.;*=94=TSS`=5>7.2.).3*=3:(1*49.)*=*3(425&88=9-*=C(+=89&79=8.9*=<-.(-=.8=).++*7*39=

+742=9-*=(&343.(&1=A2G=(=+=89&79=8.9*8_==

WT^ T&# X !-0# .0-+-2#0 #*#+#,2 W XCPE W

T-*= XCPE= += .8= &= 7&7*= *1*2*39= 4+= 574249*7= 14(&9*)= +742= 82= 94= (,= 7*1&9.;*= 94= TSS_= T-*=

8*6:*3(*=4+=XPCE+=.8=DSGYGGRASM=T40:8:2.`=M&=*9=&1_=,**1=&3)=.8=57*8*39=.3=431>=

+=4+=9-*=-:2&3=(47*=574249*78`=2489=4+=<-.(-=&7*=)*;4.)=4+=TATA8'4=_=I9=)4*8=349=

+:3(9.43='>=.98*1+`=.389*&)=7*6:.7*8=9-*=8*6:*3(*=85*(.+.(=&(9.;&9478=8:(-=&8=NRF+`=NF8+=

&3)=S5+_=

29 A= TFIID TAF TAF TAF F TFIIB TBP MTE DPE BRE% TATA BRER IJ` DCE

MVR1: Q` TFII TFIIHTF IIHH TATA TBP TFIIE PQCPQ II TFIIAA TFIIB B TFII

F'%30# XT G#,#0 * 20 ,1!0'.2'-, $ !2-01 -$ RNA .-*7+#0 1# II

A B',"',% -$ GTF1 2 .0-+-2#0 B T&# GTF1 -$ RNA .-*7+#0 1# II

30 WT_ TATAV *#11 .0-+-2#01

A19-4:,-=*&71>=<470=8:,,*89*)=9-&9=TATA8'4==.8=&=:'.6:.94:8=+*&9:7*=4+=RNA=P41=II=

574249*78`= 7*(*39= ,*342*= <.)*= 89:).*8= 8-4<8= 9-&9= .3)**)= 247*= 9-&3= 2*= 4+= 9-*=

2&22&1.&3=574249*78=&7*=TATA81*88=S&3)*1.3`=C&73.3(.=*9=&1_=,**1_=T-*=2&/47.9>=4+=

9-*= *:0&7>49.(= -4:8*0**5.3,= ,*3*8= (439&.3= 9-*= 574249*78= (-&7&(9*7.?*)= '>= 9-*= C5G=

.81&3)8_= T-*8*= 574249*78= 9>5.(&11>= 1&(0= 9-*= (&343.(&1= TATA8'4=*8`= DPE8= &3)= 9-*= I37=

249.+8_=B.4.3+472&9.(8=&3&1>8.8=8:,,*898=9-&9=BRE=*1*2*398=&7*=+7*6:*391>=+4:3)=.3=9-*=

C5G(= DNA= 9-&3= .3= 9-*= C5G8= DNA= G*78-*3?43= &3)= I48-.0-*8= ,**/_= T-*8*= C5G=

.81&3)8= -&;*= 2:19.51*= '.3).3,= 8.9*8= +47= 9-*= 97&38(7.59.43= +&(947= S5+= &3)= .98= '.3).3,=

+:79-*7=7*(7:.98=9-*=TFIID`=TFIIB`=RNA=541=II=&3)=49-*7='&8&1=97&38(7.59.43=+&(9478_==

A_,=RNA=541>2*7&8*=II=&3)=T7&38(7.59.43=M&(-.3*7>=

T7&38(7.59.43='>=RNA=541=II=.8=-.,-1>=(4251*==&3)=9.,-91>=7*,:1&9*)=574(*88_=RNA=541=

II=7*6:.7*8=8*;*7&1=97&38(7.59.43=+&(9478=94=7*(4,3.?*`='.3)=&3)=(1*&7=9-*=(47*=574249*78=

4+= 9-*= ,*3*8= F.,:7*= ,_= T-*8*= 97&38(7.59.43= +&(9478= TFIIA`= TFIIB`= TFIID`= TFIIE`= TFIIF=

&3)=TFIIH=&7*=(&11*)=,*3*7&1=97&38(7.59.43=+&(9478=GTF8=&3)=<*7*=3&2*)=&((47).3,=94=

9-*= 8:'8*6:*39= 5749*.3= +7&(9.438= 4'9&.3*)= ):7.3,= 3:(1*&7= *=97&(9= S&<&)4,4= &3)=

S*39*3&(=+33*b=C43&<&>`=H&31*>=*9=&1_=+33+b=G*7&7)`=F.8(-*7=*9=&1_=+33+b=F147*8`=L:=*9=&1_=

+33,_= T-*= TF= 7*57*8*398= 9-*= T7&38(7.59.43= +&(9478`= 9-*= R42&3= 3:2*7&1= .3).(&9*8= 9-*=

RNA=541=II=)7.;*3=97&38(7.59.43=&3)=9-*=1*99*78=7*57*8*398=9-*=(-742&94,7&5-.(=+7&(9.43=

4+=9-*=85*(.+.(=GTF8_=

XTW RNA .-*7+#0 1# II ," CTD +-"'$'! 2'-,1

O:9=4+=9-*=9-7**=RNA=541>2*7&8*8`=RNA=541=II=.8=9-*=2489=*=9*38.;*1>=89:).*)_=RNA=

541=II=(438.898=4+=&=+*8541>5*59.)*=(&9&1>9.(=(47*=&3)=9-*=-*9*74).2*7.(=R5'.21=(4251*=`=

949&1=4+=/+-=KD&=1.89*)=.3=T&'1*=,_=T-*=8:':3.98=&7*=(&11*)=R5'+=94=R5'+,`=R5'=89&3)8=+47=

RNA=541>2*7&8*=B`=&8=&349-*7=342*3(1&9:7*=8>89*2=7*+*77*)=RNA=541>2*7&8*='>=A`=B=

31 S%G E%R A`H.:V:C B:H V`1:C FV: %`Vs S1

S%G:ssVIGC7 R]G R]Q R CQIIQJ 1J ]QI I5 ]QC II5 8 ]QC III R]G R]Q [ CQIIQJ 1J ]QI I5 ]QC II5 8 ]QC III R]G R]Q R CQIIQJ 1J ]QI I5 ]QC II5 8 ]QC III R]G R]G R]Q > NTP G1JR1J$ s1 V 8 S%G:ssVIGC7 R]G R R 8 R]G R]G R]Q < P`QIQ V` `VHQ$J1 1QJ 8 S%G:ssVIGC7 R]G R]Q < CQIIQJ 1J ]QI I5 ]QC II5 8 ]QC III R]G R]Q R 8 R]G R]Q R CQIIQJ 1J ]QI I5 ]QC II5 8 ]QC III S :C@ R]G R]Q R 8 R]G R]Q R J1_%V Q ]QC II 8

T *# TT D'$$#0#,2 13 3,'21 -$ RNA .-*7+#0 1# II =', #3) 07-2#1Q 0!& # Q !2#0' ," 2&#'0 $# 230#1T

A" .2#" $0-+ T&-+ 1 ," C&' ,% TRRXR W'*" ," C0 +#0 TRST

&3)=C=.389*&)=4+=I`=II=&3)=III_=F.;*=8:':3.98=4+=RNA=541=II=&7*=(42243=94=&11=9-*=RNA=

541>2*7&8*8_=F:79-*7247*=R5'+`=,`=-2++`=.=&3)=0=.3=>*&89=&7*=(438*7;*)=&8=8:':3.98= `=

`= =-424).2*7`= =&3)= =7*85*(9.;*1>=.3='&(9*7.&_=

T-*=&88*2'1>=4+=RNA=541=II=4((:78=.3=&=85*(.+.(=47)*7=<-.(-=-&8='**3=)*243897&9*)=.3=

>*&89= &3)= '&(9*7.&= C7&2*7`= A72&(-*= *9= &1_= ,**2b= D*(0*7= &3)= H.3943= ,*+-_= T-*8*=

89:).*8= 8-4<= 9-&9= 9-*= ,*3*7&1= &7(-.9*(9:7*= 4+= 9-*= RNA= 541= II= .8= '&8*)= 43= 9-*= (47*=

8:':3.98_= T-*= 9<4= 1&7,*= 8:':3.98= R5'+2= = &3)= *5',2= `= +472= 9-*= (*397&1= 2&88= 4+= 9-*=

*3?>2*=&3)=1.*=43=9-*=45548.9*=8.)*=4+=&=548.9.;*1>=(-&7,*)=(1*+9_=T-*8*=1&7,*=8:':3.98=

&7*=89&'.1.?*)='>=9-*=R5'-8=R5'++=-*9*74).2*7=.3=>*&89=&3)= =-424).2*7=.3='&(9*7.&_=

R5'02 `=R5'+*=&3)=R5'+,=+:79-*7=89&'.1.?*=9-*=1&7,*=8:':3.98=A72&(-*`=K*99*3'*7,*7=

*9= &1_= ,**-b= W.1)= &3)= C7&2*7= ,*+,_= T-*= &88*2'1>= 4+= RNA= 541= II= .8= )43*= +:11>= .3=

(>9451&82= '*+47*= .9= ,*98= .25479*)= 94= 9-*= 3:(1*:8= B4:143`= P7&)*98B&1&)*= *9= &1_= ,*+*_=

L&9*7=).884(.&9.43=&3)=7*(>(1.3,=4+=9-*=+&(9478=4((:78=&8=9-*.7=+:3(9.43=.8=(4251*9*)=.3=

3:(1*:8_==

T-*= C89*72.3&1= )42&.3= CTD= 4+= R5'+= .8= &= :3.6:*= +*&9:7*= 4+= RNA= 541= II= C47)*3`=

C&)*3&= *9= &1_= +32/= <-.(-= .8= 349= 57*8*39= .3= &3>= 49-*7= RNA= 541>2*7&8*_= T-*= CTD=

(438.898= 4+=9&3)*2=-*59&)=7*5*&98=<.9-=&=(438*38:8= 8*6:*3(*=4+=T>78S*78P748T-78S*78

P748S*7= Y +S,P-T.S/P0S1_= T-*= 3:2'*7= 4+= 7*5*&98= .8= 85*(.*88)*5*3)*39= &3)= ,*3*7&11>=

7*+1*(98= 9-*= (4251*=.9>= 4+= 9-*= 47,&3.82= 7&3,.3,= +742= ,0= .3= >*&89`= ./= .3= +1.*8= 94= /,= .3=

;*79*'7&9*8_=T-.8=)42&.3=.8=.25479&39=+47=9-*=574(*88.3,=4+=2RNA=P74:)+449`=F:7,*7=

*9= &1_= ,**,`= +47= 7*85438*= 4+= RNA= 541= II= <.9-= 9-*= *3-&3(*78= S(&+*`= C-&4= *9= &1_= +33*b=

G*7'*7`= H&,2&33= *9= &1_= +33/= &3)= +47= 9-*= 47,&3.?&9.43= 4+= 97&38(7.59.43= +4(.= .3= 9-*=

3:(1*:8=M.89*1.=,***_=A((47).3,1>`=CTD=24).+.(&9.438=(&3=:3)*7,4=)7&2&9.(=(-&3,*8=

):7.3,= 97&38(7.59.43= 94= 7*(7:.9= +&(9478= 3**)*)= .3= ).++*7*39= 5-&8*8= 4+= 9-*= 97&38(7.59.43=

(>(1*_=O:9=4+=8*;*3=&2.34=&(.)8`=+.;*=4+=9-*=->)74=>1&9*)=&2.34=&(.)8=&7*=9-*=549*39.&1=

9&7,*9=+47=9-*=5-485-47>1&9.43='>=8*;*7&1=CTD=0.3&8*8=&3)=CTD=5-485-&9&8*8= L.89*)=.3=

33 CTD IQR1`1H: 1QJs F%JH 1QJ M:II:Cs V:s

SV` K1J:sVs CR@ ^PRTEFG_5 CR@L `Q` s%GsV CR@ 5 Ls@5 B%`5 C @ Q` $VJVs P.Qs].: :sV FH]5 CRH FH] SV` K1J:sV CR@ 5 CR@ K1J 5 CR@ 5 MHs P.Qs].: :sV RPAP5SH]5Ss% R `5 Ss%

SV` K1J:sV CR@ 5 CR@ K1J P.Qs].: :sV Ss% Ss% 5 B%`

T7` K1J:sV HAGC J@JQ1J @1J:sV 1J 7V:s

T.` K1J:sV PQCQRC1@V @1J:sV5 CR@ ^JQ 0V`7 1VCC NQ RV`1JVR RV`1JVR_

T *# UT CTD )', 1#1 ," P&-1.& 2 1# ', + ++ *1 ," ', 7# 12 _=

A" .2#" $0-+ E%*-$$Q D'#,12 '#0 #2 *T TRSTR H1',Q X' ,% #2 *T TRSV

34 T&'1*= -= E,14++`= D.*389'.*7= *9= &1_= ,*+,b= H8.3`= X.&3,= *9= &1_= ,*+. _= B&8*)= 43= 9-*=

5-485-47>1&9.43=89&9*`=RNA=541=II=(&3=*=.89=.3=&=-.,-1>=5-485-47>1&9*)=CTD=II4=+472=

&3)=.3=&=34385-485-47>1&9*)=CTD=II&=+472_=T-.8=5-485-47>1&9.43=4((:78=57.3(.5&11>=

43= 8*7.3*= ,`= /= &3)= 1= 4+= 9-*= CTD= 4+= 9-*= RNA= 541= II= <-.(-= .8= *89&'1.8-*)= '>= .397.(&9*=

.39*751&>='*9<**3=CTD=0.3&8*8=&3)=CTD=5-485-&9&8*8=&8=RNA=541=II=24;*8=&143,=9-*=

,*3*8_=A3&1>8*8=+742=&=3:2'*7=4+=5749*.38(4).3,=,*3*8= -&;*=.3).(&9*)=9-&9= 9-*=CTD=

5-485-47>1&9.43= 5&99*73= (-&3,*8= &8= 9-*= 97&38(7.59.43= 574,7*88*8_= I3= +&(9`=

5-485-47>1&9*)=7*7/=43=*9:=541=;;=.8=+4:3)=3*&7=9-*=/)=*3)=4+=,*3*8=&3)=.9=&((:2:1&9*8=

5-485-47>1&9*)=S*7,21=94<&7)8=9-*=*3)=4+=9-*=97&38(7.59.43=:3.9=B:7&94<80.=,**3_=S*7/=

&3)= S*7,= 5-485-47>1&9.43= &55*&78= 94= '*= 7*6:.7*)= +47= &11= ,*3*= 9>5*8`= <-.1*= 9-*= S*71=

5-485-47>1&9.43= ).851&>8= ,*3*885*(.+.(= +:3(9.43_= O9-*7= 24).+.(&9.438= .3(1:)*=

,1>(48>1&9.43=4+=9-*=8*7.3*8=&3)=5-485-47>1&9.43=4+=T>7+`=-4<*;*7=*=&(9=741*=4+=9-*8*=

24).+.(&9.438=.8=349=>*9=034<3_=

XTX TFIIA

TFIIA= <&8= 47.,.3&11>= 7*+*77*)= 94= &8= AB= 47= STF= &3)= <&8= ).8(4;*7*)= &8= &3= .39*7&(9.43=

5&793*7=4+=TFIID=L.3)&-1=+31.b=S&2:*18`=F.7*=*9=&1_=+32,_=Y*&89=TFIIA=(438.898=4+=9<4=

8:':3.98`= 9-*= 1&7,*= 8:':3.9= TOA+= &3)= 9-*= 82&11= 8:':3.9= TOA,`= <-*7*&8= .3= -:2&38=

9-*7*= &7*= 9-7**= 8:':3.98`= TFIIA `= TFIIA = &3)= TFIIA _= P7*;.4:8= 89:).*8= 8-4<8= 9-&9=

TFIIA=<&8=*88*39.&1=+47= .38;.974 =97&38(7.59.43=R*.3'*7,=&3)=R4*)*7=+321`=<-.1*=1&9*7= .38

;.974 =89:).*8=8-4<8=9-&9=TFIIA=<&8=).85*38&'1*=+47='&8&1=1*;*1=97&38(7.59.43=V&3=D>0*`=

R4*)*7=*9=&1_=+322_=S42*=89:).*8=&184=8-4<=9-&9=TFIIA=89.2:1&9*8='&8&1=&3)=&(9.;&9*)=

97&38(7.59.43= .38;.974 `= ,*3*7&11>= '>= 8:'89.9:9.3,= TBP= <.9-= .39&(9= TFIID= (4251*==

C&1)*(499`=M(K*4<3=*9=&1_=+33.b=H&258*>=+332_=L&9*7=842*=89:).*8=8-4<*)=9-&9=TFIIA=

<&8= 349= 7*6:.7*)= +47= *.9-*7= '&8&1= 47= &(9.;&9*)= 97&38(7.59.43= P*9*72&33`= Z.*,1*7= *9= &1_=

,**-_=

35 F:H Q`s P`Q V1Js HQI]Qs1 1QJ F%JH 1QJs 1J RNA ]QC II `:JsH`1] 1QJ

TFIIA TFIIA 5 TFIIA 5 TFIIA AJ 1`V]`Vss1QJ5 s :G1C1<: 1QJ Q` TATARTBP HQI]CV65 HQ:H 10: 1QJ

TFIIB SVCVH 1QJ Q` TSS5 s :G1C1<: 1QJ Q` TATARTBP HQI]CV65 `VH`%1 IVJ Q` RNA ]QC IILTFIIF

TFIID TBP5 TAFRTAF CQ`V ]`QIQ V` G1JR1J$5 HQ:H 10: Q`5 ]`Q V1J @1J:sV5 %G1_%1 1J :H 10: 1J$LHQJ=%$: 1QJ :H 101 75 .1s QJV :HV 7CR `:Js`V`:sV :H 101 7

TFIIE TFIIE 5 TFIIE RVH`%1 IVJ Q` TFIIH Q .V ]`QIQ V`5 ]`QIQ V` VsH:]V :JR HCV:`:JHV

TFIIF RAP5 RAP SVCVH 1QJ Q` TSS5 RVH`%1 IVJ Q` RNA ]QC II5 TFIIE5 TFIIH Q .V ]`QIQ V`5 ]`QIQ V` VsH:]V5 VCQJ$: 1QJ

TFIIH PB5 PD5 ] 5 ] 5 ]5 ATP:sV :JR .VC1H:sV :H 101 7 `Q` ]`QIQ V` Q]VJ1J$ :JR ]5 ] 5 CR@ 5 C7HC1J H5 HCV:`:JHV8 K1J:sV :H 101 7 `Q` CTD Q` RNA ]QC II :JR J%HCV:` M: `VHV] Q`s

MVR1: Q` MVRRMVR5 CR@ 5 H7HC1J CQQ]V`: 10V G1JR1J$ 11 . RNA ]QC II5 @1J:sV :JR :HV 7CR C ^:`1:GCV HQI]Qs1 1QJ_ `:Js`V`:sV :H 101 75 HQJ `QCs G:s:C :JR :H 10: VR `:JsH`1] 1QJ

T *# VT G#,#0 * 20 ,1!0'.2'-, $ !2-01 -$ RNA .-*7+#0 1# IIT

A" .2#" $0-+T&-+ 1 ," C&' ,% TRRX

36 N*;*79-*1*88= TFIIA= .3)**)= '*(42*= *88*39.&1= +47= 9-*= 97&38(7.59.43= .3= &= 7*(4389.9:9*)=

8>89*2=<-.(-=(439&.38=5&79.&11>=5:7.+.*)=+7&(9.438_=T4,*9-*7`=9-*8*=89:).*8=8:,,*89=9-&9=

TFIIA= -&8= 89.2:1&947>= *++*(98= 574'&'1>= '>= 7*;*78.3,= 9-*= .3-.'.947>= *++*(98= 4+= 9-*=

3*,&9.;*= (48+&(9478= 1.0*= NC+`= D7+2NC,`= D7,2NC,`= &3)= HMG+_= TFIIA= 89.2:1&9*8=

97&38(7.59.43= '>= 89&'.1.?.3,= TBP= '.3).3,= 94= TATA= '4== &3)= .8= *88*39.&1= +47= 9-*= 57*8=

.3.9.&9.43=(4251*==PIC=&88*2'1>=B:7&94<80.`=H&-3=*9=&1_=+323_==I9='*(42*8=*88*39.&1=

.3=(&8*=4+=TATA81*88=574249*78=.38;.974_=D*51*9.43=4+=TFIIA=-&8='**3=8-4<3=94=)*(7*&8*=

9-*=RNA=541=II=97&38(7.59.43='49-=.3=TATA8(439&.3.3,=&3)=TATA81*88=574249*78=K&3,`=

A:'1*= *9= &1_= +33/_= TFIIA= &184= (422:3.(&9*8= <.9-= 8*;*7&1= +&(9478= .3(1:).3,= TFIID`=

SAGA=(42543*398=&3)=(4&(9.;&9478_=

XTY TFIIB

TFIIB= <&8= +.789= (-&7&(9*7.?*)= '>= R*.3'*7,= &3)= R4*)*7= .3= +321_= I3= -:2&38`= TFIIB= .8=

(42548*)=4+=-+0=&2.34=&(.)8=&3)=*=.898=&8=&=8.3,1*=--8KD&=541>5*59.)*=<-.(-=8-&7*8=

8*6:*3(*= -42414,>= <.9-= D74845-.1& = &3)= >*&89_= TFIIB= (438.898= 4+= 9<4= )42&.38`= C8

9*72.3&1= )42&.3`= 7*+*77*)= &184= &8= 9-*= (47*= )42&.3= TFIIB(= &3)= N89*72.3&1= )42&.3=

TFIIB3_=T-*=C89*72.3&1=)42&.3=.39*7&(98=<.9-='49-=9-*=TBP=&3)=DNA=*3(425&88.3,=

9-*=2&/47=,74;*=:5897*&2=&3)=9-*=2.347=,744;*=)4<3897*&2=94=9-*=TATA='4=_=T-*=N8

9*72.3&1=)42&.3=(439&.38=&=?.3(=7.''43=249.+=<-.(-=.39*7&(98=<.9-=9-*=R5'+=&3)=R5',=

8:':3.98= 4+= RNA= 541= II= &3)= <.9-= RAP-*= RNA= 541>2*7&8*= II= &884(.&9.3,= 5749*.3= -*=

8:':3.9=4+=TFIIF_=T-*=C=&3)=N8=9*72.3&1=)42&.3=&7*=(433*(9*)=<.9-=+1*=.'1*=1445=<-.(-=

.8= -.,-1>= (438*7;*)= 7*,.43= (&11*)= (-&7,*)= (1:89*7= )42&.3= CCD= 47= B8+.3,*7_= T-*= B8

+.3,*7=.8='*1.*;*)=94=7*,:1&9*=9-*=(43+472&9.43&1=(-&3,*=4+=TFIIB=<-*3=.9=.39*7&(98=<.9-=

DNA=47=&(9.;&9478=&3)=-*3(*=24):1&9*=.98=+:3(9.43_=B8+.3,*7=7*,:1&9*8=9-*=+:3(9.43=4+=

TFIIB= .3= 574249*7= 7*(4,3.9.43= '>= .39*7&(9.3,= <.9-= TBP`= 51&>8= &3= .25479&39= 741*= .3=

7*(7:.9.3,= RNA= 541= II2TFIIF= &9= 9-*= TSS= .3= 97&38(7.59.43= &(9.;&9.43= &3)= -*3(*= -&8= &=

37 (7:(.&1=741*=.3=9-*=&88*2'1>=4+=PIC=O75-&3.)*8`=L&,7&3,*=*9=&1_=+330b=H&258*>=+332b=

H&-3= ,**.b= T-42&8= &3)= C-.&3,= ,**0_= TFIIB= (&3= &184= &:94= &(*9>1&9*= .98*1+= &3)= 9-.8=

24).+.(&9.43=89&'.1.?*8= .39*7&(9.43= 4+=TFIIB= &3)=TFIIF= &3)=&(9.;&9*8= 97&38(7.59.43='49-=

.38;.974 =&3)= .3=;.;4 _=TFIIB=&184=24):1&9*8=97&38(7.59.43=&+9*7=9-*=PIC=&88*2'1>=9-74:,-=

.98=B8+.3,*7='>='14(0.3,=9-*=*=9*38.43=4+=3*<1>=8>39-*8.?*)=RNA=97&38(7.59=B:8-3*11`=

W*894;*7=*9=&1_=,**._=

XTZ TFIID

T7&38(7.59.43=+&(947=II=D=TFIID=(4251*==-&8='**3=89:).*)=4;*7=+47=247*=9-&3=,*=>*&78_=

I3= *&71>= 1.9*7&9:7*8= .9= <&8= &184= (&11*)= &8= DB`= BTF+= &3)= D= M&98:.`= S*,&11= *9= &1_= +32*b=

S&2:*18`=F.7*=*9=&1_=+32,_=I9=(438.898=4+=TATA8'.3).3,=5749*.3=TBP=H&-3`=B:7&94<80.=

*9= &1_= +323b= H47.048-.`= Y&2&2494= *9= &1_= +33*= &3)= &74:3)= +.= TBP8&884(.&9*)= +&(9478=

TAF8_= T-*=TFIID=(4251*== .8=<*11= (438*7;*)='*9<**3=).++*7*39=85*(.*8= 7&3,.3,=+742=

-:2&3=94=>*&89_=I9=(438.898=4+=9-7**=14'*8`=+472.3,=&=-478*88-4*=897:(9:7*`=<.9-=TFIIA=

&3)= TFIIB= 43= 9-*= 45548.9*= 8.)*8= 4+= 9-*= (*397&1= (&;.9>_= = T-*= TBP= &3)= 842*= TAF=

(42543*398=4+=TFIID='.3)=).++*7*39=(47*=574249*7=*1*2*398=<-.(-=(1&88.+.*8=TFIID=&8=&=

(47*= 574249*78'.3).3,= +&(947= 2*39.43*)= .3= T&'1*+_= T-*= +:3(9.43= 4+= TBP= 8:':3.9= 4+=

TFIID= .8= 94= (439&(9= 9-*= TATA= '4== &114<.3,= 9-*= TFIID= 94= 7*(4,3.?*= TATA8(439&.3.3,=

574249*78_= F:79-*7247*`= 9-*= .39*7&(9.43= '*9<**3= TAF8= &3)= ).++*7*39= (47*= 574249*7=

*1*2*398=TAF8I37`=TAF8DPE`=&3)=TAF8DCE=&184=(43+*7=TFIID=9-*=&'.1.9>=94=7*(4,3.?*=

TATA81*88=574249*78_=TAF8=51&>=&=;.9&1=741*=.3=9-*=8*1*(9.43=4+=9-*=574249*7=&3)=(48

&(9.;&9*= 9-*= '&8&1= 97&38(7.59.43= 574(*88= 9-74:,-= 7*(4,3.9.43= &3)= '.3).3,= 94= (47*=

574249*7= 249.+8B:71*>= &3)= R4*)*7= +330_= TAF+= &3)= TAF,= 9-&9= (&3= '.3)= I37= ).7*(91>=

C-&101*>=&3)=V*77./?*7= +333= &3)=49-*7= 9<4=TAF8b= TAF0= &3)=TAF3=<*7*=7*5479*)=94=

.39*7&(9=<.9-=DPE_==S42*=(4251*=*8=49-*7=9-&3=TFIID=(439&.3.3,=TAF8=8:(-=&8=TFTC`=

SAGA`= STAGA= &3)= PCAF= -&;*= &184= '**3= 7*5479*)= B7&3)`= L*:7*39= *9= &1_= +333_=

M47*4;*7`=9-*=897:(9:7*=4+=TAF=0`=3=&3)=+,=-&8=2&3>=8.2.1&7.9.*8=<.9-=9-&9=4+=-.8943*=

38 H-`=H,B=&3)=H.= 8:,,*89.3,= 9-*=*=.89*3(*=4+=&3=4(9&2*7= 897:(9:7*= <-.(-=.8=8.2.1&7=94=

3:(1*4842*8= T&'1*=.`=T-42&8=&3)=C-.&3,=,**0 _=

XT[ TFIIE

TFIIE=897:(9:7&11>=(438.898=4+=9<4=8:':3.98`= =&3)= `=<-.(-=+472=&= ,,=-*9*749*97&2*7=

O-0:2&=*9=&1_`=+33+_=F742=9-*=+:3(9.43&1=54.39=4+=;.*<=TFIIE=51&>8=&3=*88*39.&1=741*=.3=

9-*= .3.9.&9.43= 4+= 97&38(7.59.43= '>= .39*7&(9.3,= ).7*(91>= <.9-= TFIIF`= TFIIB`= RNA= 541= II`=

574249*7=DNA=&3)=-*158=+:79-*7=94=7*(7:.9=9-*=TFIIH==M&=43`=G44)7.(-=*9=&1_=+33._=

TFIIE= (&3= 89.2:1&9*= 9-*= ATP&8*`= 9-*= 0.3&8*= &3)= 9-*= -*1.(&8*= &(9.;.9.*8= 4+= 9-*= TFIIH=

+&(.1.9&9.3,= 9-*= +472&9.43= 4+= .3.9.&9.438(425*9*39= RNA= 541= II= (4251*== O-0:2&= &3)=

R4*)*7=+33._=TFIIE='.3)8=+742=8+*=94=(+*=7*,.438=43=9-*=574249*7=DNA`=9-:8=.3.9.&9.3,=

9-*=574249*7=2*19.3,=&74:3)=9-*=TSS=94=(7*&9*=&=97&38(7.59.43=':''1*_==S5*(.+.(&11>`=9-*=

N89*72.3&1=-&1+=4+=TFIIE =.39*7&(98=<.9-=9-*=TFIIE =&3)=RNA=541=II=;.&=34384;*71&55.3,=

7*,.438=<-.(-=7*8:198=.3=CTD=5-485-47>1&9.43=9-:8=+&(.1.9&9.3,=97&38.9.43=+742=.3.9.&9.43=

94=*143,&9.43=.3='&8&1=97&38(7.59.43_=T-*=C89*72.3&1=7*,.43=4+=TFIIE =.39*7&(98=<.9-=50,=

8:':3.9=4+=TFIIH=O0:)&`=T&3&0&=*9=&1_=,**.=&3)=+&(.1.9&9*8=9-*=*397>=4+=TFIIH=.3=PIC=

&88*2'1>_= S.2.1&7= 94= TFIIE `= 9-*= N89*72.3&1= 4+= TFIIE = *3-&3(*8= 9-*= TFIIH8= 2*).&9*)=

CTD=5-485-47>1&9.43=<-.1*=9-*=C89*72.3&1=)42&.3=.8=.3;41;*)=.3=9-*=97&38.9.43=+742=

97&38(7.59.43=.3.9.&9.43=94=*143,&9.43='>=RNA=541=II_=

XT\ TFIIF

TFIIF=<&8=.)*39.+.*)='&8*)=43=.98=89743,`=5->8.(&1=.39*7&(9.43=<.9-=RNA=541=IIB:7943`=

K.11**3=*9=&1_=+322_=TFIIF=.8=-*9*7489*97&2*7=(438.89.3,=4+=9-*=7*5*9.9.43=4+=9<4=8:':3.98a=

RAP-*= ,0= 0D&= &3)= RAP1.= /20D&_= RAP-*= 8:':3.9= 4+= -:2&3= TFIIF= .8= (&5&'1*= 4+=

.39*7&(9.3,=<.9-=9-*=RNA=541=II`=DNA=&3)=8:':3.9=RAP1._=RAP1.=-&8=9-7**=+:3(9.43&1=

)42&.38=(&5&'1*=4+=.39*7&(9.3,=<.9-=RAP-*`=9-*=TAF+=8:':3.9=4+=TFIID`=TFIIB`=RNA=541=

II=&3)=FCP+=5-485-&9&8*=R:55*79=&3)=T/.&3=+33/_=I3=5&79.(:1&7`=RAP1.=.39*7&(9=<.9-=

9-*=R5'3=8:':3.9=4+=RNA=541=II`=<-.(-=+&(.1.9&9*8=9-*=7*(7:.92*39=4+=RNA=541=II=94=9-*=

39 574249*7=(4251*=2TFIID2TFIIB=F147*8`=L:=*9=&1_=+33+_=F:79-*7247*`=TFIIF=(4397.':9*8=

94= 45*3= (4251*== +472&9.43= '>= *3&'1.3,= *397>= 4+= TFIIE= &3)= TFIIH= 94= 9-*= 8(&++41)=

M&=43`=G44)7.(-=*9=&1_=+33.b=T.74)*`=B:884=*9=&1_=+333_=TFIIF=7*2&.38='4:3)=94=RNA=

541=II=):7.3,=RNA=*143,&9.43`=*3-&3(.3,=.98=574(*88.;.9>=&3)=9-*=541>2*7.?&9.43=7&9*=

L*.`=R*3=*9=&1_=+333b=Y&3`=M47*1&3)=*9=&1_=+333_=B*8.)*8`=TFIIF=(&3='*=5-485-47>1&9*)=

'>=5749*.3= 0.3&8*= CK,=&3)= 89.2:1&9*=*143,&9.43= /;@7.`=P&1=*9=&1_=,*++_= TFIIF=.8=&184=

.251.(&9*)= .3= 9-*= 574(*88= 4+= RNA= 541= II= 7*.3.9.&9.43= '>= 7*(7:.9.3,= &3)= *3-&3(.3,= 9-*=

&(9.;.9>=4+=5-485-&9&8*=FCP+`=<-.(-=.8=7*6:.7*)=+47=)*5-485-47>1&9.43=4+=9-*=CTD=4+=

RNA=541=II=&3)=.8=&=57*7*6:.8.9*=+47=9-*=3*=9=74:3)=4+=97&38(7.59.43=A7(-&2'&:19`=P&3=

*9=&1_=+332_=

XT] TFIIH

TFIIH=<.11='*=*=9*38.;*1>=57*8*39*)=.3=C-&59*7=II`=8.3(*=9-*=TFIIH=.8=&=2&/47=8:'/*(9=4+=

9-*=9-*8.8=&3)=7*6:.7*8=+:79-*7=).8(:88.43_==

XT^ M#"' 2-0

M*).&947=.8=&=1&7,*=(4251*==(42548*)=4+=,/8-*=5749*.3=&77&3,*)=.3=897:(9:7&1=24):1*8=

9-&9=.8=9-4:,-9=94=&(9=&8=&=241*(:1&7='7.),*='*9<**3=DNA='.3).3,=97&38(7.59.43=+&(947=

&3)= RNA= 541= II_= T-*= M*).&947= <&8= ).8(4;*7*)= &3)= +.789= 5:7.+.*)= +742= S&((-&742>(*8=

(*7*;.8.&* = &3)= .8= 47,&3.?*)= .3= +4:7= 8:'(4251*=*8`= 9-*= -*&)`= 2.))1*`= 9&.1`= &3)= C)02=

24):1*8= P488`= E'2*.*7= *9= &1_= ,*+-_= M*).&947= &184= 8-4<8= 8.,3.+.(&39= *;41:9.43&7>=

(438*7;&9.43=7&3,.3,= +742=>*&89=94=-:2&3=M&1.0=&3)=R4*)*7`=,***`=':9=9-*=8:':3.9=

(42548.9.43= 4+= 2*).&947= +742= ).++*7*39= 85*(.*8= (&3= ;&7>= )*5*3).3,= 43= 9-*= 47,&3.82=

&3)=9-*=89&,*=4+=(*11=(>(1*_=A884(.&9.43=4+=2*).&947=<.9-=RNA=541=II=.8=.3-.'.9*)=89743,1>=

.38;.974 = '>= 5-485-47>1&9.43= 4+= 9-*= CTD= '>= K.3,2= <-.(-= 8:,,*898= 9-&9= 2*).&947= .8= &=

(42543*39=4+=9-*=PIC=&3)=9-&9=.98=5-485-47>1&9.43=.8=1.30*)=94=574249*7=*8(&5*=G:.).`=

B/4738)499.7=*9=&1_=,**._=T-*=2*).&947=349=431>='.3)8=94=RNA=541=II=':9=&184='.3)8=94=9-*=

97&38&(9.;&9.43= )42&.3= 4+= 9-*= 1&7,*= 3:2'*7= 4+= 9-*= 97&38(7.59.43= +&(9478= &3)= 94= 2&3>=

40 GTF8_= T-74:,-= 9-*8*= .39*7&(9.438`= 2*).&947= .8= (&5&'1*= 4+= 574249.3,= 97&38(7.59.43=

.3.9.&9.43='>=RNA=541=II=&3)=+&(.1.9&9*8=9-*=&88*2'1>=4+=+:3(9.43&1=PIC_=T-*=C)02=0.3&8*=

24):1*= 4+= 2*).&947= -&8= &184= '**3= 8-4<3= 94= 7*,:1&9*= 97&38(7.59.43= '>= 9&7,*9.3,= 9-*=

CDK12(>(1.3= H= 8:':3.98= 4+= TFIIH`= 9-:8= 24):1&9.3,= 9-*= 97&38.9.43= +742= .3.9.&9.43= 94=

*143,&9.43='>=89.2:1&9.3,=9-*=CTD=0.3&8*=&(9.;.9>=4+=TFIIH=K.2=*9=&1_`=+33._==

W-41*=,*342*=C-IP=&3&1>8.8=-&8=8-4<3=9-&9=2*).&947=(&3='.3)=94=9-*=*3-&3(*7=7*,.43=

&3)=&9=9-*=574249*7=7*,.43`=<-*7*=.9=(414(&1.?*8=<.9-=9-*=RNA=541=II=A3)7&:`=;&3=)*=

P&8(-=*9=&1_=,**0_=M*).&947=<&8=&184=8-4<3=94='*=.251.(&9*)=.3=97&38&(9.;&9.43=4+=RNA=

541= II= G= <-.(-= .8= &= ).89.3(9= +472= 4+= RNA= 541= II= 9-&9= (439&.38= 9-*= 9.,-91>= &884(.&9*)=

G)4<3+= 541>5*59.)*= *3(4)*)= '>= POLR,M= J.8-&,*`= M&1.0= *9= &1_= ,*+,_= M*).&947= .8=

&184=.3;41;*)=.3=DNA=1445.3,=7*8:19.3,=+742=9-*=2*).&9478=(4-*8.3=.39*7&(9.43=<-.(-=

574;.)*8= 9-*= (-7424842&1= &7(-.9*(9:7*= 7*6:.7*)= +47= 9-*= ,*3*= 97&38(7.59.43= K&,*>`=

N*<2&3=*9=&1_=,*+*b=P488`=E'2*.*7=*9=&1_=,*+-_==

M:9&9.43= .3= 2*).&947= 1*&)= 94= ).847)*78= <-.(-= 8-4<8= (1.3.(&1= +*&9:7*8= ;*7>= 8.2.1&7= 94=

9-48*=+4:3)=.3=TFIIH87*1&9*)=).8*&8*=1.0*=XP`=TTD=&3)=CS=M*39.43=.3=C-&59*7=III=.3=

)*9&.1=

41 BT T&# 20 ,1!0'.2'-, !7!*#

E:0&7>49.(=97&38(7.59.43=.8=&=57*(.8*1>=9.2*)=*;*39`=<-.(-=(&3='*=).;.)*)=.394=3:2'*7=

4+=).89.3(9=89*58a=P74249*7='.3).3,=&3)=57*.3.9.&9.43=(4251*==&88*2'1>`=45*3=(4251*==

+472&9.43`= .3.9.&9.43`= 574249*7= (1*&7&3(*`= *143,&9.43= &3)= 9*72.3&9.43= H&-3= ,**.b=

L&:,*1`=D&114?=*9=&1_=,*+*b=S-&3).1>&=&3)=R4'*798=,*+,`=F.,:7*=-_

B_+=P7*.3.9.&9.43=(4251*==&88*2'1>`=45*3=(4251*==+472&9.43=&3)=.3.9.&9.43_=

T-*= +.789= 89*5= .3= 9-*= RNA= 541= II= 2*).&9*)= 97&38(7.59.43= .8= 9-*= '.3).3,= 4+= 9-*= ,*3*=

85*(.+.(=7*,:1&947>=+&(9478=&74:3)=9-*=97&38(7.59.43=.3.9.&9.43=8.9*_==T-*8*=+&(9478=*.9-*7=

(&3= &(9= ).7*(91>= '>= .39*7&(9.3,= <.9-= (42543*398= 4+= 9-*= 97&38(7.59.43= 2&(-.3*7>= 47=

.3).7*(91>='>=7*(7:.9.3,=(-742&9.3=24).+>.3,=5749*.38=43=9-*=97&38(7.59.43=2&(-.3*7>_=

T-.8=&88*2'1>=4+=9-*=+&(9478=4((:78=&9=9-*=(47*=574249*7=;.&=9<4=).++*7*39=2*(-&3.828b=

'>=8*6:*39.&1=&88*2'1>=5&9-<&>29-*=89*5<.8*=24)*1=47='>=9-*=-414*3?>2*=5&9-<&>_=I3=

89*5<.8*=24)*1`=57*8=.3.9.&9.43=(4251*==PIC=&88*2'1>=+472&9.43=4((:78=.3=8*6:*39.&1=

2&33*7_= I9= 89&798= <.9-= 9-*= 7*(4,3.9.43= 4+= 9-*= TATA8+1&30.3,= 7*,.43= '>= TBP= &3)= .98=

&884(.&9*)= +&(9478b= TFIID= &3)= 9-*3= 9-*= +&(9478= 8:(-= &8= TFIIA`= TFIIB= (4397.':9*= 94= 9-*=

9*73&7>=(4251*==+472&9.43=&9=9-*=574249*7_=TFIIF`=RNA=541=II`=+:79-*7=/4.38=9-.8=9*73&7>=

(4251*==<-.(-=&114<8=9-*=7*(7:.92*39=4+=TFIIE=&3)=TFIIH=43=9-*=574249*7=94=+472=&=

(4251*== <-.(-= .8= (425*9*39= +47= 97&38(7.59.43_= W-*7*&8`= .3= -414*3?>2*= 5&9-<&>`= 9-*=

&88*2'1>= 4+= &= 57*.3.9.&9.43= (4251*== 2.,-9= -&55*3= .3= &= 8.3,1*= (43(*79*)= 7*(7:.92*39=

89*5= <-.(-= '7.3,8= &= 1&7,*= 57*&88*2'1*)= RNA= 541= II= -414*3?>2*= (439&.3.3,= 9-*=

2*).&947=(4251*==&3)=2489`=.+=349=&11`=4+=9-*=GTF8=94=9-*=574249*7_=

I3=9-*=(4251*9*=PIC`=RNA=541=II=.8=.39*79<.3*)='>=2&3>=.39*7&(9.438_=TFIIB=.39*7&(9=;.&=

.98= B8+.3,*727*&)*7= 8*,2*39= <.9-= 9-*= RNA= 541= II= 9-&9= <.11= :19.2&9*1>= 574;.)*= 9-*= *=.9=

5&9-=4+=9-*=3&8(*39=RNA_=TFIIB=&184=(439&(98=49-*7=5479.438=4+=RNA=541=II=&8=<*11=&8=9-*=

:5897*&2=574249*7=DNA=K4897*<&`=Z*11*7=*9=&1_=,**3b=L.:`=B:8-3*11=*9=&1_=,*+*_==TFIIF=

42 F'%30# YT RNA .-*7+#0 1# II 20 ,1!0'.2'-, !7!*#T

A" .2#" $0-+ H &, XVVZR S& ,"'*7 ," R- #021 XVWX

43 &3)= TFIIE= +:79-*7= '.3)= 94= 541>2*7&8*= 43= *.9-*7= 8.)*= 4+= 9-*= (*397&1= (1*+9= .3= <-.(-= 9-*=

9*251&9*= <.11= :19.2&9*1>= 7*8.)*= L:`= Z&<*1= *9= &1_= +33,= O-0:2&= &3)= R4*)*7= +33._=

TFIIH= .39*7&(9= <.9-= DNA= )4<3897*&2= 4+= 97&38(7.59.43= 89&79= W&3,`= S5&3,1*7= *9= &1_=

,**-_=B*+47*=9-*=574249*7=(1*&7&3(*=9-*8*=.39*7&(9.438=2:89='*=1489=94=&114<=9-*=82449-=

7*1*&8*=4+=RNA=541=II_=

B_, =P74249*7=(1*&7&3(*=

O3(*= 9-*= PIC= &88*2'1>= .8= (4251*9*`= TFIIH= (4397418= 9-*= ATP8)*5*3)*39= 97&38.9.43= 4+=

RNA= 541= II= +742= (148*)= 94= 45*3= PIC= .3= <-.(-= 9-*= 2*19*)= 8.3,1*8897&3)*)= DNA= .8=

.38*79*)= .394= 9-*= &(9.;*= 8.9*_= R*(*39= 89:)>= +742= E;&= N4,&1*8= 1&'8= 8-4<= 9-&9= 9-.8=

97&38.9.43=.8=(&9&1>?*)=574'&'1>='>=9-*=XPB=97&3814(&8*=&(9.;.9>=4+=TFIIH=H*`=F&3,=*9=&1_=

,*+-_= A((47).3,= 94= 9-.8= 89:)>`= XPB= )4(0*)= ).7*(91>= 43= 9-*= )4<3897*&2= DNA= 5&9-`=

'*9<**3=(+*=&3)=(,*8'5=548.9.43=7*1&9.;*=94=9-*=TSS=7*8:19.3,=.3=9-*=+472&9.43=4+=++8+/=

'5=':''1*=&74:3)=9-*=TSS=<-.(-=.8=7*6:.7*)=+47=9-*=574):(9.;*=97&38(7.59.43=.3.9.&9.43_==

B_-=P74=.2&1=5&:8.3,=&3)=*143,&9.43=

T-4:,-=+472&9.43=4+=PIC=.8=+47*2489=.25479&3(*=+47=8:((*88+:1=.3.9.&9.43`=&88*2'1>=4+=

PIC= &9= 9-*= 574249*7= )4*8= 349= 3*(*88&7.1>= &88:7*= &'4:9= 9-*= 574):(9.;*= 97&38(7.59.43_=

T7&38(7.598=4+=1*88=9-&3=/=3:(1*49.)*8=&7*=:389&'1*=&3)=-*3(*=7*8:198=.3=&=-.,-=&'479.;*=

.3.9.&9.43_=H4<*;*7`=&=+*=3:(1*49.)*=97&38(7.59=+&;478=9-*=574249*7=*8(&5*=4;*7=&'479.;*=

.3.9.&9.43=&3)=.+=97&38(7.59=1*3,9-=7*&(-*8=&74:3)=,/=3:(1*49.)*8=8:((*88+:1=.3.9.&9.43=.8=

&(-.*;*)_=O3(*=9-.8=,/=3:(1*49.)*=4+=3&8(*39=2RNA=.8=8>39-*8.?*)`=9-*=(&5=897:(9:7*=.8=

&99&(-*)= 94= .98= /*3)_= :-*= 5-485-47>1&9*)= ;*7/= 4+ = CTD= 4+= RNA= 541= II= .8= +:79-*7=

7*(4,3.?*)= '>= 9-*= (&55.3,= *3?>2*= <-.(-= 9-*3= (&9&1>?*8= 9-*= &)).9.43= 4+= &=

2*9->1,:&348.3*= (&5= 94= /= *3)= 4+= 9-*= 3&8(*39= 2<=>= &3)= 574):(9.;*= .3.9.&9.43= .8=

&((4251.8-*)_=T-*=CTD=24).+.(&9.43=4+=RNA=541=II=&3)=9-*=N89*72.3&1=-.8943*=9&.18=4+=

9-*= 3:(1*4842*= &7*= (7:(.&1= +47= &3= &(9.;*= (-742&9.3= *3;.7432*39= 4+= &= 574):(9.;*=

44 .3.9.&9.43=&3)=2*).&9*=9-*=*143,&9.43_=CTD=.8=5-485-47>1&9*)=57.2&7.1>=&9=S*7/='>=C)01=

8:':3.9=4+=TFIIH=):7.3,=97&38(7.59.43=.3.9.&9.43 _= =B*+47*=*39*7.3,=9-*=*143,&9.43`=RNA=

541= II= .8= 5&:8*)= '>= DRB= S*38.9.;.9>= I3):(.3,= F&(947= DSIF= &3)= 3*,&9.;*= *143,&9.43=

+&(947=NELF`=<-.(-='.3)8=94=RNA=541=II=&3)=.3-.'.98=.98=+:3(9.43_=T-.8=3*,&9.;*=*++*(9=

(&3='*=7*1.*;*)='>=9-*=5-485-47>1&9.43=4+=S59/=1&7,*89=8:':3.9=4+=DSIF=&3)=NELF='>=

C)03= &884(.&9*)= <.9-= 58TEF'= Y&2&)&`= Y&2&,:(-.= *9= &1_= ,**0_= A8= 9-*= RNA= 541= II=

574(**)8=94<&7)8=9- *=-=4+=9-*=,*3*`=)*5-485-47>1&9.43=4+=;*7/=4((:78='>=<97+=<.9-=9-*=

8.2:19&3*4:8=.3(7*&8*=.3=S*7,=5-485-47>1&9.43='>=C)03_=RNA=541=II=.8=+:79-*7=&88.89*)=

'>=*143,&9.43=+&(9478=TFIIS`=9-*=ELL=5-485-&9&8*`=*143,.3`=-.8943*=(-&5*743*=(4251*==

FACT`= -.8943*= )*&(*9>1&8*8= HDAC8_= T-*8*= +&(9478= 7*2&.38= &884(.&9*)= <.9-= 9-*=

*143,&9.3,=RNA=541=II=&3)=2&.39&.3=9-*=2=8=3=3:(1*49.)*8=RNAa=DNA=->'7.)=<-.(-=.8=

(7.9.(&1=)*9*72.3&39=4+=9-*=574(*88.;.9>=4+=9-*=RNA=541=II=):7.3,=9-*=*143,&9.43_=

B_.=T*72.3&9.43=&3)=7*.3.9.&9.43=

T*72.3&9.43=.8=9-*=1&89=89*5=.3=9-*=8*7.*8=4+=*;*398=4+=97&38(7.59.43=(>(1*_=T-.8=*;*39=&184=

8*7;*8=&8=&=/:3(9.43=+47=9-*=7*14&).3,=4+=RNA=541=II=94=9-*=574249*7=+47=&349-*7=74:3)=4+=

97&38(7.59.43_=T-*7*=&7*=9<4=034<3=5&9-<&>8=+47=9-*=97&38(7.59.43=9*72.3&9.43b=9-*=541>=

A= )*5*3)*39= 5&9-<&>= &3)= 9-*= S*3+8)*5*3)*39= 5&9-<&>= K:*-3*7`= P*&7843= *9= &1_=

,*++_=T-*=(-4.(*=4+=9-*=5&9-<&>8= )*5*3)8=43=9-*=<=>=-) 8*3)=574(*88.3,=8.,3&18=&3)=

9-*=9*72.3&9.43=+&(9478=9-&9=&7*=57*8*39=&9=9-*=*3)=4+=&=,*3*_=I3=*:0&7>49*8`=2489=4+=9-*=

5749*.3=(4).3,= 2<=>=57*(:78478=(439&.3=541>=>=8.,3&1`=/ 8AAUAAA8 - =<-.(-=.8=9-*3=

+4114<*)='>=A2U=7.(-=8*6:*3(*=&9=9 -*=- 8*3)=4+=9-*=,*3*_=D:7.3,=9-.8=574(*88`=CTD=&(98=

&8=&=8(&++41)=+47=9-*=7*(7:.92*39=4+=9-*=8*;*7&1=+&(9478=7*6:.7*)=+47=9-*=9*72.3&9.43_=P41>=

A= 8.,3&1= .8= 7*(4,3.?*)= '>= (1*&;&,*= &3)= 541>&)*3>1&9.43= 85*(.+.(.9>= +&(947= CPSF=

<-.(-=.39*7&(98=<.9-=9-*=CTD=&3)=.3):(*8=5&:8.3,=4+=*143,&9.3,=RNA=541=II_=A349-*7=

+&(947=(&11*)=(1*&;&,*=89.2:1&947>=+&(947=C89F='.3)8=94=9-*=)4<3897*&2=GU87.(-=7*,.43=

&3)= .39*7&(9.43= '*9<**3= 9-*8*= 9<4= +&(9478= '7.3,8= 9-*= *3)47.'43:(1*41>9.(= (1*&;&,*= 4+=

9-*= 3&8(*39= 97&38(7.59_= T-.8= (1*&;&,*= .8= 9-*3= +4114<*)= '>= 9-*= 541>&)*3>1&9.43= 4+= 9-*=

:5897*&2=(1*&;&,*=574):(9`=)*,7&)&9.43=4+=9-*=)4<3897*&2=(1*&;&,*=574):(9='>=XRN,=

&3)=7*1*&8*=4+=9-*=5&:8*)=RNA=541=II_===

S*3+8)*5*3)*39=5&9-<&>=<&8=+.789=).8(4;*7*)=.3=9-*=>*&89= S_=(*7*;.8.&* =&3)=.39*7*89.3,1>=

34<=.9=-&8='**3=8-4<3=9-&9=9-*=9*72.3&9.43=4+=9-*=8*;*7&1=143,=3438(4).3,=RNA8=<-.(-=

&7*= (1&88.+.*)= &8= (7>59.(= :389&'1*= 97&38(7.598= CUT8= &3)= 89&'1*= :3(-&7&(9*7.?*)=

97&38(7.598=SUT8=<-.(-=)4=349=5488*88=541>=A=:9.1.?*8=9-*=S*3+8)*5*3)*39=5&9-<&>_=

T-*=97&38):(9.43=&3)=9*72.3&9.43=4+=9-*=97&38(7.59=.8=&(-.*;*)='>=&=).89.3(9=8*9=4+=+&(9478=

<-.(-= .3(1:)*= RNA8'.3).3,= 5749*.38= N7)+`= 3:(1*&7= 541>&)*3>1&9*)= RNA8'.3).3,-=

5749*.3=N&'-=&3)=9-*=5:9&9.;*=RNA=&3)=DNA=-*1.(&8*=S*3+_=

I9= <4:1)= 349= '*= <743,= 94= 8&>= 9-&9= 9-*= *3)= .8= &= 3*<= '*,.33.3, _= B*.3,= &9= 9<4= ).89&1=

548.9.438= +742= 43*= &349-*7= 9-*= 97&38(7.59.43&1= 5-*342*343= 9-&9= 9&0*8= 51&(*= &9= 9-*=

'*,.33.3,= &3)= 9-*= *3)= 4+= 9-*= ,*3*= &7*= 4+9*3= 89:).*)= &8= 9<4= 8*5&7&9*= 574(*88*8_=

H4<*;*7=8*;*7&1=89:).*8=-&;*=8-4<3=9-&9=9-*=RNA=541=II=2&(-.3*7>=&9=574249*7=&3)=

9*72.3&947=&7*=*39&3,1*)=94,*9-*7=H&258*>`=S.3,-=*9=&1_=,*++=_=I3)**)`=9-*=7*1*&8*=4+=

9-*= RNA= 541= II= 7*6:.7*8= 9-*= 7*;*78&1= 4+= 9-*= &884(.&9*)= (4;&1*39= 2&708= 43= 9-*= CTD=

7*5*&98= <-.(-= <.11= '7.3,= RNA= 541= II= 94= .98= 47.,.3&1= ->54= 5-485-47>1&9*)= 89&9*= +47= &=

8:'8*6:*39= 74:3)= 4+= 7*.3.9.&9.43_= T-*= 7*1*&8*)= RNA= 541= II= .8= (425*9*39= +47= &= +7*8-=

74:3)= 4+= 97&38(7.59.43_= H4<*;*7`= 842*9.2*8= 8*;*7&1= GTF8= 7*2&.3= &884(.&9*)= 94=

574249*7=&3)=&(9=&8=9-*=8(&++41)=+47=9-*=7*.3.9.&9.43_=T-*=&(9.;&947=)*5*3)*39=.39*7&(9.43=

4+=9-*=574249*7=&3)=9*72.3&947=7*,.43=-&8='**3=8-4<3=94='*=.3;41;*)=.3=97&38(7.59.43=

7*8.3.9.&9.43=;.&=,*3*=1445.3,=E1=K&)*7.`=M*)1*7=*9=&1_=,**3_ =TFIIB=).7*(98=9-*=&88*2'1>=

4+= 8:(-= 7*.3.9.&9.43= 8(&++41)= 94= 9-*= 574249*7= '>= .39*7&(9.3,= <.9-= CPSF= &3)= C89+=

(4251*=*8_=T-.8=.39*7&(9.43=.8=(438*7;*)=.3=2&22&1.&3=8>89*2=&3)=.8=7*,:1&9*)='>=9-*=

5-485-47>1&9.43=4+=9-*=TFIIB=W&3,`=F&.71*>=*9=&1_=,*+*_=A3=*88*39.&1=741*=+47=9-*=TFIIH=

0.3&8*=8:':3.9`=C)01=.3=9-*=+472&9.43=4+=,*3*=14458=-&8=&184=*2*7,*)_=C)01=.8=034<3=

46 94= 7*,:1&9*= 5-485-47>1&9.43= 4+= S*7/= &3)= S*71= 4+= CTD= 4+= RNA= 541= II_= W-.1*= 5-485-48

S*7/= CTD= .8= 7*6:.7*)= +47= 97&38(7.59.43= .3.9.&9.43`= 5-485-48S*71= -&8= '**3= 1.30*)= <.9-=

9*72.3&9.43= &3)= -)= 574(*88.3,= &3)= &184= <.9-= 574249*7 8'4:3)= 5&:8*)= RNA= 541= II_=

I3-.'.9.43=4+=S*71=5-485-47>1&9.43=7*8:19*)=.3=9-*=1488=4+=RNA=541=II=5&:8.3,`='49-=&9=

9-*=574249*7=&3)=9*72.3&9.43=8.9*8=8:,,*89.3,=9-*=.251.(&9.43=4+=C)01=.3=,*3*=1445.3,=

G14;*78C:99*7`= L&74(-*11*= *9= &1_= ,**3_= I3= &)).9.43= 94= TFIIB`= TFIIH= &3)= PC.= 548.9.;*=

(4+&(947= .= &7*= 9-*= 842*= 49-*7= +&(947= 034<3= 94= 574249*= 7*.3.9.&9.43= ;.&= ,*3*= 1445.3,=

9-74:,-=.98=.39*7&(9.43=<.9-=- *3)=4+=9-*=,*3*=S-&3).1>&=&3)=R4'*798=,*+,_==

CT T&# !2'4 2#" 20 ,1!0'.2'-,

D:7.3,=9-*=)*;*1452*39=4+=&3=47,&3.82=9-*7*=&7*=8*;*7&1=5749*.38=<-.(-=&7*=7*6:.7*)=

+47=9-*='&8.(=(*11=2&.39*3&3(*=&3)=9-:8=9-*>=&7*=*=5*(9*)=94='*=*=57*88*)=.3=&11=(*118=4+=

&3=47,&3.82=:3)*7=3472&1=(43).9.438`=.77*85*(9.;*=4+=9.88:*=9>5*`=)*;*1452*39&1=89&,*`=

(*11= (>(1*= 89&9*= &3)= *=9*73&1= 8.,3&1_= T-*8*= ,*3*8= &7*= (&11*)= &8= -4:8*0**5.3,= ,*3*8_=

H4<*;*7`= 9-*7*= &7*= 1&7,*= 3:2'*78= 4+= ,*3*8= <-48*= *=57*88.43= .8= 7*6:.7*)= 431>= &9=

85*(.+.(= 9.2*= &3)= -*3(*= 9-*>= &7*= 7*,:1&9*)= '>= (*79&.3= *3)4,*34:8= 47= *=4,*34:8=

89.2:1:8=8:(-=&8=(*11:1&7=).++*7*39.&9.43=&3)=897*88=7*85438*_=H47243*8=&7*=43*=8:(-=

*=&251*= 4+= &= 89.2:1:8= <-.(-= .39*7&(9= <.9-= 9-*= 85*(.+.(= 3:(1*&7= 7*(*5947= &3)= +:79-*7=

7*,:1&9*=9-*=*=57*88.43=4+=3:(1*&7=7*(*5947=9&7,*9*)=,*3*8_=

C_+=O;*7;.*<=4+=9-*=3:(1*&7=7*(*5947=+&2.1.*8=

N:(1*&7= 7*(*59478= NR8= &7*= 9-*= 2489= &':3)&39= (1&88= 4+= 97&38(7.59.43= +&(9478= 9-&9=

7*,:1&9*= ).;*78*= '.414,.(&1= +:3(9.438= 8:(-= &8= -42*489&8.8`= 7*574):(9.43`= 47=

)*;*1452*39_= T-*>= +:3(9.43= &8= &= 1.,&3)8= &(9.;&9*)= 97&38(7.59.43= +&(9478`= &3)= 9-:8=

574;.)*=&=1.30='*9<**3=9-*=8.,3&1.3,=241*(:1*8=<-.(-=(439741=9-*8*=574(*88*8=&3)=9-*=

97&38(7.59.43&1=7*85438*8_=NR8=8-&7*=&=(42243=897:(9:7&1=47,&3.?&9.43`=:8:&11>=(438.898=

4+= +.;*= +:3(9.43&1= 7*,.438a= 9-*= A2B= 7*,.43= 9-&9= (439&.38= &3= N89*72.3&1= &(9.;&9.43=

47 +:3(9.438+= )42&.3= NTD`= 9-*= (*397&1= C= 7*,.43= 9-&9= (439&.38= &= DNA8'.3).3,= )42&.3=

DBD`=9-*=C89*72.3&1=E=7*,.43=9-&9=(439&.38=&=1.,&3)8'.3).3,=)42&.3=LBD`=&3)=9-*=D=

-.3,*=7*,.43=9-&9=(433*(98=9-*=DBD=&3)=9-*=LBD_=NTD=.8=&=-.,-1>=;&7.&'1*=7*,.43=<-.(-=

(439&.38= &9= 1*&89= 43*= (4389.9:9.43&11>= &(9.;*= 97&38&(9.;&9.43= 7*,.43= AF8+= &3)= 8*;*7&1=

&:943424:8=97&38&(9.;&9.43=)42&.38=AD_=DBD=.8=-.,-1>=(438*7;*)=7*,.43=(439&.3.3,=

9-*=9<4=?.3(=+.3,*78=9-&9=7*(4,3.?*8=85*(.+.(=NR8=7*85438.;*=*1*2*398=43=9-*=7*,:1&947>=

7*,.43=4+=9-*=9&7,*9=,*3*8_=W-*7*&8`=LBD=51&>8=&=(7:(.&1=741*=.3=1.,&3)82*).&9*)=3:(1*&7=

7*(*5947= &(9.;.9>_= B*8.)*8`= .98= 741*= .3= 1.,&3)= 7*(4,3.9.43`= 9-*= LBD= &184= (439&.38= &3=

&(9.;&9.43= +:3(9.438,= AF8,= )42&.3`= <-48*= &(9.43= .8= -.,-1>= )*5*3)*39= 43= 9-*= '4:3)=

1.,&3)_M&3,*18)47+`= T-:22*1= *9= &1_= +33/_= T-*= -.3,*= D`= 94,*9-*7= <.9-= C89*72.3&1= E=

7*,.43`=.8=1*88=(438*7;*)=&3)=8-4<=).89.3(9=897:(9:7&1=+*&9:7*8=&243,=).++*7*39=3:(1*&7=

7*(*59478_=

D*5*3).3,=43=9-*=3&9:7*=4+=9-*=1.,&3)=9-*>='.3)`=9-*=3:(1*&7=7*(*5947=8:5*7+&2.1>=.8=

).;.)*)=.394=9-7**=(1&88*8=.=C1&88=I=.3(1:)*=7*(*59478=+47=89*74.)=-47243*8=.3=9-*=DNA8

'.3).3,=-4248).2*7.(=+472=&3)=4+9*3=14(&9*)=.3=(>94841_=O3(*=9-*>='.3)=94=9-*=1.,&3)=

9-*>= &7*= 97&3814(&9*)= 94= 9-*= 3:(1*:8_= I9= .3(1:)*8= 9-*= ,1:(4(479.(4.)= 7*(*5947= GR`=

*8974,*3=ER=47=574,*89*743*=PR_=..=C1&88=II=7*(*5947='.3)8=24891>=&8=-*9*74).2*78=

<.9-= RXR= 94= DNA= &3)= &7*= :8:&11>= 14(&9*)= .3= 9-*= 3:(1*:8_= T-*>= .3(1:)*= 2&.31>= RXR=

7*(*59478= X= 7*(*5947= <.9-= 7*9.34.(= &(.)`= 9-*= 9->74.)= -47243*= 7*(*5947= TR`= 7*9.34.(=

&(.)=RAR=&3)=;.9&2.3=D=VDR_=...=R*(*59478=4+=(1&88=III=.3(1:)*8=+&2.1>=4+=475-&3=

7*(*59478=+47=<-.(-=1.,&3)8=&7*=89.11=349=;*7>=<*11=034<3_=T-*=2*2'*78=4+=9-.8=,74:5=

.3(1:)*=9-*=1.;*7=7*(*5947=-42414,:*8+=LRH+`=7*;*78*=ERBA8 28 `=(-.(0*3=4;&1':2.3=

:5897*&2=97&38(7.59.43=+&(9478=COUP8TF8`=7*(*59478=SF8+_=

C_,=R*9.34.(=&(.)=7*(*5947=

N:(1*&7=7*9.34.(=&(.)=7*(*59478=RAR8=&7*=97&38(7.59.43&1=7*,:1&9478=<-.(-=(4397418=9-*=

*=57*88.43=4+=85*(.+.(=8:'8*98=4+=,*3*8=.3=&=1.,&3)8)*5*3)*39=2&33*7_=T-*=(4254:3)8=

48 F'%30# ZT S203!230# -$ 0#2',-'! !'" 0#!#.2-0

A" .2#" $0-+ A* T ,-307Q P'1)3,-4 #2 *T XVWY

A RAR1 "#.'!2 "-+ ', -0% ,'8 2'-, 5'2& , 3,1203!230#" NTD ," 25- 5#**V1203!230#" "-+ ',1S !#,20 * DBD ," CV2#0+', * LBDT T&# .&-1.&-07* 2'-, 1'2#1 *-! 2#" ', 2&# NTD ," 2&# LBD 0# 1&-5,T B S203!230 * !& ,%#1 ',"3!#" 3.-, RA ',"',%T T&# !0712 * 1203!230#1 -$ 2&# 3,*'% ,"#" RXR I ," *'% ,"#" RAR L LBD1 0# 1&-5,T H#*'!#1 0# 0#.0#1#,2#" 1 0' -,1 ," * #*#" $0-+ HW 2- HWXT T&# ',"',% "-+ ',1 $-0 !-0#.0#11-01 !- !2'4 2-01 ," $-0 !7!*', H 0# 1&-5,T

49 9-&9=&(9.;&9*=9-*=RAR8=&7*=7*+*77*)=94=&8=7*9.34.)8_=R*9.34.)8=&7*=82&11=8.?*=1.5.)=841:'1*`=

->)745-4'.(= 241*(:1*8= 9-&9= (&3= *&8.1>= (7488= 9-*= 1.5.)8= '.1&>*7= 4+= 9-*= (*11= 2*2'7&3*8_=

R*9.34.)8=.3(1:)*8='49-=(4254:3)8=<-.(-=&7*=897:(9:7&11>=7*1&9*)=94=;.9&2.3=A`=&8=<*11=

&8= (4254:3)8= 9-&9= *=-.'.9= '.414,.(&1= ;.9&2.3= A= &(9.;.9>_= V.9&2.3= A= .8= &3= *88*39.&1=

(42543*39=+47=-:2&3=1.+*=(>(1*`=89&79.3,=+742=*2'7>43.(=)*;*1452*39`=47,&34,*3*8.8=

94= .22:3*= +:3(9.438= &3)= 7*574):(9.43_= I3)**)= )*+.(.*3(>= 4+= V.9&2.3= A= (&3= 1*&)= 94=

3*43&9&1=,74<9-=7*9&7)&9.43=&3)=&=1&7,*=3:2'*7=4+=(43,*3.9&1=2&1+472&9.438=S&2&7:9=

&3)=R4(-*99*8E,1>=,*+,_=N&9:7&1=7*9.34.)8`=8:(-=&8= &11897&38= 7*9.34.(=&(.)=A11=98RA`=&7*=

574):(*)= .3=;.;4= +742=4=.)&9.43=4+=;.9&2.3=A=C-&2'43=,**/_=T-.8=3*<1>=8>39-*8.?*)=

7*9.34.(= &(.)= 9-*3= '.3)8= 94= (>94841.(= (*11:1&7= 7*9.34.(= &(.)8'.3).3,= 5749*.38b= CRABP8I=

&3)= CRABP8II= 94= *39*7= 9-*= 3:(1*:8= D*1;&`= B&89.*= *9= &1_= +333b= B:)-:= &3)= N4>= ,**,_=

S:'8*6:*391>=.9=7*,:1&9*8= 9-*=,*3*=*=57*88.43='>=&(9.;&9.3,=9-*=3:(1*&7=7*(*5947=RAR=

<-.(-=*=.898=.3=9-7**=8:'9>5*8=a= =NR+B+`= NR+B,=&3)= ÒNR+B-=C-&2'43=+330=

*3(4)*)= '>= 9-7**= 8*5&7&9*= ,*3*8_= F47= *&(-= 8:'9>5*8= 9-*7*= &7*= &9= 1*&89= 9<4= .84+4728=

<-.(-=&7*=,*3*7&9*)='>=).++*7*39.&1=574249*7=:8&,*=&3)=&19*73&9.;*=851.(.3,=1*&).3,=94=

9-*=).++*7*3(*=431>=.3=9-*.7=N89*72.3&1=*3)8=G*72&.3`=S9&*18=*9=&1_=,**0=_= I3=;.;4 `=RAR=

97&38):(*= RA= 8.,3&1= &8= -*9*74).2*7= 94,*9-*7= <.9-= RXR= <-.(-= &184= *=.89= &8= 9-7**=

8:'9>5*8a= =NR,B+`= =NR,B,=&3)= Ò=NR,B-=&3)=&7*=.3;41;*)=.3=7*,:1&9.3,=,*3*8=

.251.(&9*)=.3=(*11=).++*7*39.&9.43`=5741.+*7&9.43=&3)=.3=&545948.8_==

RAR8= &3)= RXR8= -&;*= &= <*118)*+.3*)= )42&.3= 47,&3.?&9.43= &3)= 897:(9:7*_= I9= (438.89=

2&.31>=4+=&=;&7.&'1*=N89*72.3&1=)42&.3=NTD=&3)=9<4=-.,-1>=(438*7;*)=)42&.38`=&=

(*397&1= DNA8'.3).3,= )42&.3= DBD= &3)= &= C89*72.3&1= 1.,&3)8'.3).3,= )42&.3= LBD=

'7.),*)='>=&=+1*=.'1*=-.3,*=5*59.)*= F.,:7*=.`=A1=T&34:7>`=P.80:34;=*9=&1_=,*+-= _=

DBD= '.3)8= 94= 9-*= DNA= .3= &= 8*6:*3(*= 85*(.+.(= 2&33*7= &3)= .8= (42548*)= 4+= 9<4= ?.3(8

+.3,*7=)42&.38`=9<4= 8-*1.(*8=&3)=&=COOH89*72.3&1=*=9*38.43=CTEZ*(-*1`=S-*3=*9=&1_=

+33.b= S2&1*= &3)= K&)43&,&= ,**-_= DBD= .3(1:)*8= 8*;*7&1= -.,-1>8(438*7;*)= 8*6:*3(*=

50 *1*2*398`=7*+*77*)=94=&8=P`=D`=T=&3)=A='4=*8=<-.(-=(4397.':9*=94=9-*=85*(.+.(.9>=4+=9-*=

7*85438*=*1*2*398=&3)=94=&=).2*7.?&9.43=.39*7+&(*=<-.(-=.8=.3;41;*)=.3='.3).3,=<.9-=9-*=

DNA= '&(0'43*= G*72&.3`= S9&*18= *9= &1_= ,**0_= RAR8= &3)= RXR8= +472= &3= &8>22*97.(=

-*9*74).2*7=<-.(-='.3)8=94=9-*=RA=7*85438*=*1*2*398=RARE8_T-*=RARE8=&7*=14(&9*)=

.3=9-*=7*,:1&947>=7*,.43=4+=9-*=9&7,*9=,*3*=&3)=&7*=(42548*)=4+=9<4=).7*(9=7*5*&98=4+=

(47*=-*=&2*7.(=249.+=P:G=G2T=TCAM&3,*18)47+=&3)=E;&38=+33/_=A=(1&88.(&1=RARE=

.8= &= /'5885&(*)= ).7*(9= 7*5*&9= 7*+*77*)= 94= &8= DR/`= ':9= RARE8= <.9-= ).7*(9= 7*5*&98=

8*5&7&9*)='>=+'5=DR+=47=,'5=DR,=&7*=&184=+4:3)_=N*;*79-*1*88`=RXR=-424).2*78=

(&3=&184='.3)=94=DR+_==

T-*=LBD=.8=(42548*)=4+=+,=(438*7;*)= 8-*1.(*8=&3)=&= 89:73=<-.(-=.8=8*5&7&9*)='>=9-*=

14458=&3)=+41)*)=.394=&=9-7**=1&>*7*)`=5&7&11*1=-*1.(&1=8&3)<.(-_=T-*=LBD=.8=+:3(9.43&11>=

;*7>= (4251*== &8= .9= (439&.38= 1.,&3)= '.3).3,= 54(0*9= <-.(-= '.3)8= 94= 9-*= 1.,&3)8= &3)= .8=

.3;41;*)= .3= ).2*7.?&9.43= &3)= .39*7&(9.43= <.9-= 8*;*7&1= 49-*7= (487*,:1&9478_= T-*= 1.,&3)=

'.3).3,= 54(0*9= (439&.38= ->)745-4'.(= 7*8.):*8= 2&.31>= .3= -*1.(*8= H-`= H/`= H++= &3)= 8

-&.75.3=B4:7,:*9`=A3)7>=*9=&1_=,***_=T-*=LBD=&184=(439&.38=&=+1*=.'1*=C89*72.3&1=-*1.==

H+,=<-.(-=(&3=(-&3,*=.98=(43+472&9.43=&+9*7=9-*=1.,&3)='.3).3,=)*5*3).3,=43=43*=94=

49-*7= 8:'9>5*8_= M47*4;*7`= (>(1.3= H`= &= 8:':3.9= 4+= CAK= 8:'(4251*== 4+= TFIIH= .8= &184=

034<3= 94= '.3)= 94= 9-*= AF8,= )42&.3= 4+= LBD= &3)= ).7*(98= 9-*= 5-485-47>1&9.43= 4+= AF8+=

)42&.3=Y&2&2494`=I(-.)&=*9=&1_=,**1`=F.,:7*=._==

U31.0*=DBD=&3)=LBD`=NTD=.8=-.,-1>=;&7.&'1*=&3)=.8=349=(438*7;*)=*;*3='*9<**3=9-*=

).++*7*39=8:'9>5*8=&3)=9-*=+4728=4+=RAR8=&3)=RXR8_=S97:(9:7&1=&1,47.9-28=57*).(9.43=

8:,,*89=9-&9=9-*=NTD8=4+=RAR8=&3)=+742=49-*7=NR8=+&2.1>=-&;*=3&9:7&11>=).847)*7*)=

897:(9:7*W&732&70`=T7*:9*7=*9=&1_=,**-=<-.(-=574;.)*8=+1*=.'.1.9>=5*7-&58=7*6:.7*)=+47=

9-*=0.3&8*8=&3)=:'.6:.9.381.,&8*8=D>843=&3)=W7.,-9=,**/_=I3=&)).9.43`=NTD8=4+=RAR8=

&3)=RXR8=&184=(439&.3=5-485-47>1&9.43=8.9*8=<-.(-=&7*=(438*7;*)='*9<**3=9-*=RAR8_==

51 C_-=T7&38(7.59.43=4+=7*9.34.(=&(.)=7*(*5947=9&7,*9=,*3*8=

T-*=2*(-&3.82=4+=97&38(7.59.43&1=7*,:1&9.43=4+=RA89&7,*9=,*3*8=.8=&=8*6:*39.&1=574(*88=

&3)= (4251*9*1>= 7*1.*8= 43= 9-*= '.3).3,= 4+= RAR8= 94= 9-*= RARE8_= RAR8= (&3= (439741=

97&38(7.59.43='49-='>=7*57*88.43=&3)=&(9.;&9.43=<-.(-=)*5*3)8=43=9-*=&884(.&9.43=&3)=

).884(.&9.43= 4+= (*79&.3= (47*57*88478= 47= (4&(9.;&9478= .39*7&(9.3,= <.9-= 9-*= ->)745-4'.(=

8:7+&(*=4+=9-*=LBD=,*3*7&9*)='>=9-*=H-`=H.=&3)=H+,=-*1.(*8_=A((47).3,=94=9-*=(&343.(&1=

97&38&(9.;&9.43=24)*1=+47=RA8=9&7,*9*)=,*3*8=D.1<479-=&3)=C-&2'43=,**+`=9-*=RARE8=

&7*=4((:5.*)='>=&=RAR2RXR=-*9*74).2*7=.3=&3=:31.,&3)*)=89&9*`=<-.(-=2&.39&.38=9-*=

(-742&9.3= .3= &= (43)*38*)= 47= &= 7*57*88*)= 89&9*= F.,:7*= /_= I3= &3= :31.,&3)*)= 89&9*= 9-*=

->)745-4'.(=8:7+&(*=4+=RAR=.39*7&(98=<.9-=9-*=(47*57*88478= 8:(-=&8=3:(1*&7=7*(*5947=

(47*57*8847= NC4R= &3)= 8.1*3(.3,= 2*).&947= 4+= 7*9.34.(= &(.)= &3)= 9->74.)= -47243*=

SMRT _=T-*=RAR=&3)=(47*57*8847=SMRT=.39*7&(9.43=4((:78='*9<**3=9-*=LBD=4+=RAR=

&3)= 9-*= 7*(*5947= .39*7&(9.43= )42&.38= RID= 4+= SMRT_= T-*8*= (47*57*88478= 8*7;*= &8= &=

51&9+472=+47=9-*=7*(7:.92*39=4+=9-*=-.8943*=)*&(*9>1&8*8=HDAC=<-.(-=)*&(*9>1&9*=9-*=

1>8.3*=4+=-.8943*8`=9-:8=2&.39&.3.3,=9-*=7*57*88*)=(-742&9.3=89&9*_=P41>(42'=,74:5=4+=

5749*.38=&7*=&184=034<3=94=.39*7&(9=<.9-=RAR2RXR=-*9*74).2*78=&3)=&(9=&8=&=(4757*8847=

G.11*85.*= &3)= G:)&8= ,**1_= T7&38(7.59.43= .8= 9:73*)= 43= :543= 1.,&3)= &)).9.43= <-.(-=

.3):(*8= 9-*= 7*1*&8*= 4+= 9-*= (47*57*8847= &3)= 8:'8*6:*391>= +&(.1.9&9*= 9-*= '.3).3,= 4+= 9-*=

(4&(9.;&947= 94= 9-*= RAR2RXR= -*9*74).2*7= <-.(-= .3= 9:73= &114<8= 9-*= 7*(7:.92*39= 4+=

8*;*7&1= 49-*7= (4251*=*8= 8:(-= &8= -.8943*= 2*9->1= 97&38+*7&8*8= HMT`= -.8943*= &(*9>1=

97&38+*7&8*8= HAT`= -.8943*= )*2*9->1&8*8= &3)= DNA8)*5*3)*39= ATP&8*8= &3)= 49-*7=

(-742&9.3= 7*24)*1*78= P*7.88.`= J*58*3= *9= &1_= ,*+*_= T-*8*= (4251*=*8= +:79-*7= &114<= 9-*=

-.8943*= 24).+.(&9.438= 1*&).3,= 94= 9-*= (-742&9.3= )*(425&(9.43_= T-*8*= &(9.;&9*)= RAR8=

9-*3=7*(7:.9=9-*=97&38(7.59.43&1=2&(-.3*7>=<-.(-=.3(1:)*8=2*).&947`=RNA=541=II`=GTF8=

&3)= &184= 9-*= NER= +&(9478= 94= &(-.*;*= (-742&9.3= 7*24)*1.3,= = +47= 9-*= 459.2&1= RNA=

8>39-*8.8=L*=M&>`=M49&8F*73&3)*8=*9=&1_=,*+*b=L*=M&>`=F7&).3=*9=&1_=,*+,_==

52 F'%30# [T C-0#%3* 2-0 #6!& ,%# 2 RXR RAR #0-"'+#01T

A I, 2&# 1#,!# -$ *'% ,"Q RAR I RXR #0-"'+#01 -3," 2- DNA 0# 11-!' 2#" 5'2& !-0#.0#11-0 !-+.*#6#1T U.-, *'% ," ',"',%Q 2&# !-0#.0#11-01 "'11-!' 2#Q **-5',% 2&# 0#!03'2+#,2 -$ !- !2'4 2-01 ," * 0%# !-+.*#6#1 5'2& #,87+ 2'! !2'4'2'#1 2& 2 "#!-+. !2 0#.0#11'4# !&0-+ 2',T B W&#, !&0-+ 2', '1 "#!-+. !2#"Q 2&# 20 ,1!0'.2'-, * + !&',#07Q !-,1'12',% -$ 2&# M#"' 2-0Q RNA .-* IIQ 2&# %#,#0 * 20 ,1!0'.2'-, $ !2-01 GTFQ ," 2&# ,3!*# 0 #6!'1'-, 0#. '0 NER $ !2-01Q '1 0#!03'2#" 2- 2&# .0-+-2#0Q 0#13*2',% ', 2&# ','2' 2'-, -$ 20 ,1!0'.2'-,T C T0 ,1!0'.2'-, #,"1 5'2& 2&# 0#!03'2+#,2 -$ ,-,!-,4#,2'-, * !- !2'4 2-01Q 13!& 1 RIPWZVQ 11-!' 2#" 2- * 0%# !-+.*#6#1 5'2& !&0-+ 2',V0#.0#11',% !2'4'27 ," -0 2&0-3%& 2&# "#%0 " 2'-, -$ RAR1 7 2&# 3 '/3'2',V.0-2# 1-+# 1712#+T A" .2#" $0-+ A* T ,-307Q P'1)3,-4 #2 *T XVWYT

H4<*;*7`=9-*=.25479&3(*=4+=NER=+&(9478=&9=574249*7=.8=89.11=349=;*7>=<*11=:3)*78944)_=

I9= 7*2&.38= 94= '*= .3;*89.,&9*)= 9-&9= -4<= 9-*8*= NER= +&(947= (4397.':9*= 94= (-742&9.3=

7*24)*1.3,`= )4= 9-*>= .39*7&(98= <.9-= 9-*= GTF8`= -4<= 9-*>= 7*,:1&9*= 9-*= -.8943*=

24).+.(&9.438=47=9-*=DNA=2*9->1&9.43=43=NR8=9&7,*9*)=,*3*8_==

A9= 9-*= *3)= 4+= 97&38(7.59.43`= 9-*= 1.,&3)*)= RAR8= 7*(7:.9= :3(43;*39.43&1= (47*,:1&9478=

8:(-= &8= RIP0*= 7*(*5947= .39*7&(9.3,= 5749*.3= &3)= PRAME= 57*+*7*39.&11>= *=57*88*)=

&39.,*3=.3=2*1&342&_=T-*8*=(47*,:1&9478=.3-.'.9=9-*=97&38(7.59.43&1=&(9.;.9>='>=+:79-*7=

&114<.3,=9-*=7*(7:.92*39=4+=HDAC8=&3)=P(G=5749*.38=&3)=-*3(*=1.2.9=9-*=RAR=&(9.;.9>=

9-74:,-=&=+:3(9.43&1=+**)'&(0=2*(-&3.82=F.,:7*=/_=I3=&)).9.43=94=).7*(9=*++*(98`=RA=

&184=9&7,*98=3:2'*7=4+=8.,3&1.3,=(&8(&)*8_=RA=&(9.;&9*8=8*;*7&1=0.3&8*=(&8(&)*8=8:(-=&8=

5-2=2.94,*38&(9.;&9*)=0.3&8*=.3=+.'74'1&898`=24:8*=*2'7>4=(&7(.342&=(*118`=2&22&7>=

'7*&89= 9:247= (*118`= &3)= 1*:0*2.&= (*118_= I9= &184= &(9.;&9*8= 5.,2..= *=97&(*11:1&7= 8.,3&1=

7*,:1&9*8= 0.3&8*8= E70= .3= 3*:7438`= S*7941.= (*118= &3)= .3= *2'7>43.(= 89*2= (*118_= I9= &184=

&(9.;&9*8=5-485-4.348.9.)*=-80.3&8*=PI-K=&3)247=5749*.3=0.3&8*=B=PKB2A09=5&9-<&>_=

RAR8=&3)=RXR8=&7*=&184=034<3=&8=5-485-45749*.38=A1=T&34:7>`=P.80:34;=*9=&1_=,*+-_=

RAR8=&7*=5-485-47>1&9*)=&9=8*7.3*=-03=.3=LBD=574'&'1>='>=(8AMP=)*5*3)*39=0.3&8*=47=

MSK+=&3)=&9=8*7.3*=11=.3=NTD='>=()01=8:':3.9=4+=TFIIH_==

B*8.)*8`=5-485-47>1&9.43=4+=RAR8=&3)=RXR8`=8.,3&1.3,=5&9-<&>8=&(9.;&9*)='>=9-*=RA=

&7*=034<3=94=5-485-47>1&9*=49-*7=+&(9478=.3;41;*)=.3=9-*=,*3*=7*,:1&9.43`=<-.(-=2&>=

349='*=9-*=RAR=9&7,*98= ;*7>=<*11=7*;.*<*)=.3=K*7.*1`=S9&7>=*9=&1_=,**,b=R4(-*99*8E,1>=

,*+. `=F.,:7*=0_=A8=&3=*=&251*`=:543=7*(7:. 92*39=94=<><=9&7,*9=574249*78= MSK+=

&3)= 5-485-47>1&9*8= -.8943*8= H-_= B&8*)= 43= 9-*= 89:).*8= 4+= 8*;*7&1= 7*54798`= -.8943*=

5-485-47>1&9.43=(4:1)=(4397.':9*=94=97&38(7.59.43=&8=&=(-742&9.3=2&70=7*85438.'1*=+47`=

.3=(445*7&9.43=<.9-=49-*7=-.8943*= 24).+.(&9.438`=(-742&9.3=7*24)*1.3,=&3)= 574249*7=

7*(7:.92*39=4+=RXR2RAR=-*9*74).2*78=&3)=9-*=97&38(7.59.43&1=2&(-.3*7>=F.,:7*=0_=A9=

9-*= *3)= 4+= 9-*= RA= 8.,3&1`= 5-485-47>1&9.43= 4+= RAR8= (4397418= 9-*= 7*(7:.92*39= 4+=

54 F'%30# \T C0-112 *) #25##, 2&# RAV !2'4 2#" .Y^MAPK . 2&5 7 ," 2&# #6.0#11'-, -$ RAR 2 0%#2 %#,#1T

I, 0#1.-,1# 2- RAQ .Y^MAPK '1 !2'4 2#" Q ," 2&#, 20 ,1*-! 2#1 ',2- 2&# ,3!*#31 ," .&-1.&-07* 2#1 MSKW T A!2'4 2#" MSKW .&-1.&-07* 2#1 &'12-,#1 ! ," RAR I 2 1#0',# *-! 2#" ', 2&# LBD "T S3 1#/3#,2 2- !-,$-0+ 2'-, * !& ,%#1Q 2&# !7!*', H 13 3,'2 -$ 2&# CAK 13 !-+.*#6 -$ TFIIH '1 0#!03'2#" 2- , "( !#,2 "-+ ', #Q **-5',% 2&# $-0+ 2'-, -$ RAR I TFIIH !-+.*#6 ," 2&# .&-1.&-07* 2'-, -$ 2&# NTD 7 2&# !")] )', 1# $T I, 2&# ! 1# -$ 2&# RAR L 13 27.#Q .&-1.&-07* 2'-, -$ 2&# NTD .0-+-2#1 2&# "'11-!' 2'-, -$ !-0#%3* 2-01Q 13!& 1 4',#6',! %T F', **7Q .&-1.&-07* 2#" RAR I '1 0#!03'2#" 2- 0#1.-,1# #*#+#,21 *-! 2#" ', 2&# .0-+-2#0 -$ 2 0%#2 %#,#1 &

A" .2#" $0-+ A* T ,-307Q P'1)3,-4 #2 *T XVWY

:'.6:.9.385749*&842*=8>89*2_=I9=(&3='*=2*).&9*)=9-74:,-=8*;*7&1=+&(9478=8:(-=&8=8:(-=

P.3+ `=SRC8-_=A9=9-*=*3)=RA=(&3=&1 84=&(9.;&9*=HH><2_=:-*7*=&7*=*;.)*3(*8=<-.(-=&184=

7*9.3418=&(9.;&9*=JAK88STAT=5&9-<&>=94=7*,:1&9*=9-*=*=57*88.43=4+=9&7,*9=,*3*8=8:(-=&8=

9-*=8:557*8847=4+=(>940.3*=8.,3&1.3,=-=SOCS-=&3)=PPAR= ʈ8

===

56 DT C&0-+ 2', ," %#,# 0#%3* 2'-,

I3=&=(*11=,*3*9.(=.3+472&9.43=.8=*3(4)*)='>=DNA`=<-.(-=.8=5&(0&,*)=.394=-.*7&7(-.(&11>=

47,&3.?*)= (4251*== 897:(9:7*= (&11*)= (-742&9.3_= = C-742&9.3= .8= (42548*)= 4+= DNA`=

-.8943*=&3)=3438-.8943*=5749*.38_=T-*=,*342*=:3)*7,4*8=8*;*7&1=1*;*1=4+=(425&(9.43=94=

+41)=9<4=2*9*78=4+=DNA=.394=&=3:(1*:8=<.9-=&=).&2*9*7=4+=&74:3)=8.==2.(742*9*78_=I9=

2&>=8**2=(4397&).(947>=9-&9=5749*.38=&7*=&))*)=94=DNA=94=2&0*=.9=247*=(425&(9=':9=

(-742&9.3= (&3= '*= 5&(0&,*)= .394= &= 2:(-= 82&11*7= ;41:2*= 9-&3= DNA= &143*_= T-.8= .8=

&(-.*;*)= &8= 9-*= -.8943*8= &7*= 548.9.;*1>= (-&7,*)= &3)= DNA= .8= 3*,&9.;*1>= (-&7,*)= 9-:8=

9-.8=*1*(97489&9.(=.39*7&(9.43=574;.)*8=*3*7,>=94=+41)=9-*=DNA=<-.(-=(&3=9-*3=+.9=.394=9-*=

3:(1*:8_=T-*=+.789=1*;*1=4+=(425&(9.43=(438.898=4+=DNA=(4.1*)=&74:3)=&=(47*=4+=-.8943*=

5749*.38=(7*&9.3,=&=+:3)&2*39&1=:3.9=(&11*)=3:(1*4842*_=N:(1*4842*=.8=(42548*)=4+=&3=

4(9&2*7=4+=9-*=+4:7=(47*=-.8943*8=H-`=H.`=H,A`=H,B=&74:3)=<-.(-=+.1='&8*=5&.78=4+=

DNA=&7*=<7&55*)_=T-*=5&(0&,.3,=4+=DNA=.394=3:(1*4842*8=8-479*38=9-*=+.'*7=1*3,9-=

&'4:9= 8*;*3+41)= <-.(-= 7*8*2'1*8= 897.3,= 4+= '*&)8_= T-*= DNA= .8= 9-*3= 8:'/*(9*)= 94= &=

8*(43)= )*,7**= 4+= +41).3,= <-.(-= +:79-*7= (425&(9= 9-*= (-742&9.3= .394= -*= 32= 841*34.)=

897:(9:7*= W44)(4(0= &3)= D.2.974;= ,**+_= H.8943*= H+= <-.(-= +4728= 9-*= 1.30*7= .8= ;*7>=

.25479&39=+47=89&'.1.?.3,=9-*=-.,-*7=47)*7=(-742&9.3=897:(9:7*=F.,:7*=1_=F:3)&2*39&1=

574(*88*8=8:(-=&8=97&38(7.59.43`=7*5&.7=&3)=7*51.(&9.43=4((:7=<.9-=9-*=8*5&7&9.43=4+=9<4=

DNA=897&3)8=&3)=-*3(*=-.3)*7*)='>=3:(1*4842*8`=(-742&9.3=+41).3,=&3)=(425&(9.43_=

T-*7*+47*`= .9= '*(42*8= *88*39.&1= +47= 9-*= (*11= 94= 24).+>= 47= 7*24;*= 9-*8*= 3:(1*4842*8=

97&38.*391>= 94= &114<= 97&38(7.59.43= &3)= 7*51.(&9.43= 94= 574,7*88_= D*5*3).3,= 43= 9-*=

(425&(9.43= 1*;*1= (-742&9.3= (&3= '*= (&9*,47.?*)= &8= *:(-742&9.3= &3)= -*9*74(-742&9.3_=

T-*=*:(-742&9.3=.8=9>5.(&11>=*37.(-*)=.3=&(*9>1&9*)=-.8943*8=+47=*=&251*=&3)=H-K3A(`=

H.K+0A(= &3)= &184= 8-4<8= 9-*= -.8943*= H-K.= 2*9->1&9.43= H-K.M*= G7:389*.3= +331b=

B&33.89*7= &3)= K4:?&7.)*8= ,*++_= I389*&)`= -*9*74(-742&9.3= .8= )*+.3*)= &8= &= 247*=

(43)*38*)= 897:(9:7*`= .3&((*88.'1*= 94= 97&38(7.59.43= +&(9478_= I9= .8= (-&7&(9*7.?*)= '>= ->548

57 F'%30# ]T N3!*#-1-+# -0% ,'8 2'-,T

T&# ,3!*#-1-+# '1 2&# 1'! 3,'2 -$ !&0-+ 2',Q ," !-,1'121 -$ .0-2#', -!2 +#0 !-,2 ',',% 25- +-*#!3*#1 # !& -$ &'12-,#1 HXAQ HXBQ HYQ ," HZQ 0-3," 5&'!& WZ] 1# . '01 -$ ,3!*# 0 DNA '1 50 ..#"T C-302#17 &22.S 1! T-0%

58 -.8943*= &(*9>1&9.43= &3)= 2*9->1&9.43= 4+= -.8943*8= 842*= 85*(.+.(= *,= H-K3M*= R*&`=

E.8*3-&'*7=*9=&1_=,***b=J*3:<*.3=&3)=A11.8=,**+=

D_+=E5.,*3*9.(=24).+.(&9.43=4+=(-742&9.3=

E5.,*3*9.(8= .8= )*+.3*)= &8= -*7.9&'1*= (-&3,*8= .3= ,*3*= *=57*88.43= 9-&9= 4((:7= <.9-4:9= &=

(-&3,*= .3= DNA= 8*6:*3(*_= E5.,*3*9.(8= .8= 7*(4,3.?*)= &8= 0*>= )*9*72.3.3,= +&(947= .3=

3472&1= )*;*1452*39= &3)= ).++*7*39.&9.43`= &3)= *5.,*3*9.(= &'3472&1.9.*8= &7*= 7*1*;&39= .3=

2&3>= ).8*&8*8`= .3(1:).3,= ;&7.4:8= 9>5*8= 4+= 3*:7414,.(&1= ).847)*78= &3)= (&3(*78_= K*>=

(42543*398= .3= 9-*= 574(*88*8= 4+= *5.,*3*9.(= 97&38(7.59.43= 7*,:1&9.43= &7*= DNA=

2*9->1&9.43`=-.8943*=24).+.(&9.438=&3)=;&7.&398`=3438-.8943*=(-742&9.3=5749*.38`=82&11=

.39*7+*7.3,= RNA= 8.RNA= &3)= 2.(74= RNA= 2.RNA_= P489897&381&9.43&1= 24).+.(&9.438=

PTM8=4+=-.8943*8=&3)=DNA=2*9->1&9.43=&7*=).8(:88*)=.3=)*9&.18=.3=9-.8=2&3:8(7.59_==

WTW P-12V20 ,1* 2'-, * +-"'$'! 2'-, -$ &'12-,#1

T-*= 97&38.9.43= +742= 97&38(7.59.43&11>= 8.1*39= -*9*74(-742&9.3= 94= ,*3*= *=57*88.3,=

*:(-742&9.3= .8= 2*).&9*)= '>= ).89.3(9= -.8943*= ;&7.&398= &3)= 9-*= 548997&381&9.43&1=

24).+.(&9.438= 4+= -.8943*8= &9= 9-*.7= N89*72.3&1= 9&.18_= T-*8*= (4;&1*39= 24).+.(&9.438= 4+=

-.8943*8= &7*= 7*+*77*)= 94= &8= -.8943*= (4)* _= T-*8*= 24).+.(&9.438= .3(1:)*= &(*9>1&9.43= 4+=

1>8.3*8`= 2*9->1&9.43= 4+= 1>8.3*8= &3)= &7,.3.3*8`= 5-485-47>1&9.43= 4+= 8*7.3*8= &3)=

9-7*43.3*8`=ADP87.'48>1&9.43=4+=,1:9&2.(=&(.)8`=&3)=:'.6:.9.3>1&9.43=&3)=SUMO>1&9.43=

4+= 1>8.3*= 7*8.):*8= S42*= .25479&39= -.8943*= PTM8= &7*= 8-4<3= .3= +.,:7*= 2_= T-*8*=

24).+.(&9.438=&7*=(4397411*)='>='&1&3(*=4+=9-*=*3?>2&9.(=&(9.;.9.*8=4+=;&7.4:8=5749*.38=

<-.(-= &))= <7.9*78= 47= 7*24;*= *7&8*78= &= 85*(.+.(= 456= R:9-*3':7,`= A11.8= *9= &1_=

,**1b= T-425843`= G:55>= *9= &1_= ,*+-`= <-.1*= 7*&)*78= 7*(4,3.?*8= 85*(.+.(= 24).+.(&9.438=

7*8:19.3,= .3= 9-*= 7*(7:.92*39= 4+= 9-*= 97&38(7.59.43&1= 2&(-.3*7>_= R*,:1&9.43= 4+=

97&38(7.59.43='>=*&(-=-.8943*=24).+.(&9.43=.8=).8(:88*)=.3=247*=)*9&.1='*14<_=

59 F'%30# ^T S!&#+ 2'! ! 02--, -$ !-0# &'12-,# PTM1 ," 2&#'0 .-2#,2' * $3,!2'-,

C-302#17 &22.S 1! T-0%

60 STST T R#%3* 2'-, 7 &'12-,# *71',# !#27* 2'-, ," "# !#27* 2'-,T

=T-*= +.789= (4;&1*39= 24).+.(&9.43= ).8(4;*7*)= <&8= 9-*= &(*9>1&9.43= 4+= 1>8.3*1>8= 47= K=

A11+7*>= &3)= M.780>= +30.`= 7*,:1&9*)= '>= <7.9*78= 4+= &(*9>1&9.43`= 9-*= -.8943*=

&(*9>1*97&38+*7&8*8= HAT8= &3)= 9-*= *7&8*78= 4+= &(*9>1&9.43`= 9-*= -.8943*= )*&(*9>1&8*8=

HDAC8_= T-*= HAT8= :9.1.?*= &(*9>1= C4A= &8= (4+&(947= &3)= (&9&1>?*8= 9-*= 97&38+*7= 4+= &3=

&(*9>1= ,74:5= 94= 9-*= 8&2.34= ,74:5= 4+= 1>8.3*= 8.)*= (-&.38= &3)= -*3(*= 3*:97&1.?*8= 9-*=

548.9.;*=(-&7,*=4+=L>8=K`=1*&).3,=94=9-*=<*&0*3.3,=4+=9-*=DNA8-.8943*=.39*7&(9.43=&3)=

8:'8*6:*39= &(9.;&9.43= 4+= 97&38(7.59.43_= I3= &)).9.43`= &(*9>1= 1>8.3*= 7*(7:.98= 49-*7=

(-742&9.3= 24).+.*78= (439&.3.3,= &= '7424)42&.3= 9-&9= 7*(4,3.?*8= &3= &(*9>1= 1>8.3*= 94=

&(9.;&9*=97&38(7.59.43=D-&11:.3`=C&71843=*9=&1_=+333_=M&/47=&(*9>1&9.43=8.9*8=43=-.8943*=

H-=.3(1:)*=K3`=K+.`=K+2`=K,-=&3)=K,1`=&3)=43=H.=.3(1:)*=K/`=K2`=&3)=K+,=K4:?&7.)*8=

,**1b=B&33.89*7=&3)=K4:?&7.)*8=,*++_=A11=&(*9>1&9.43=2&708=&7*=*88*39.&11>=(477*1&9*)=

<.9-=97&38(7.59.43&1=&(9.;&9.43=<-.(-=.8=14(&1.?*)=94=TSS=&3)247=*3-&3(*78=4+=549*39.&11>=

&(9.;*1>= 97&38(7.'*)= ,*3*8_= = I3)**)`= 9-*= 97&38(7.59.43&11>= &(9.;*= 7*,.438= &7*= 7.(-= .3=

&(*9>1&9*)= -.8943*8`= <-.1*= 9-*.7= ->548&(*9>1&9.43= .8= +4:3)= .3= -*9*74(-742&9.3_= T-*=

HAT8= &7*= :8:&11>= (1&88.+.*)= .394= 9<4= ,74:58a= T>5*= A= &3)= 9>5*= B_= T-*8*= .3(1:)*=

*3?>2*8=8:(-=&8=GCN/`=PCAF=47=HAT+`=CBP25-**=*9(_=T-*8*=*3?>2*8=&7*=4+9*3=+4:3)=

&8= &= 5&79= 4+= 2:19.85749*.3= (4251*== .39*7&(9.3,= <.9-= 8*;*7&1= 97&38(7.59.43= +&(9478= &3)=

-*3(*=&++*(9=9-*=,*3*=7*,:1&9.43_=F47=*=&251*`=GCN/2PCAF=HAT8=&7*=7*85438.'1*=H-K3=

&(*9>1&9.43`=&3)=CPB25-**=&7*=7*85438.'1*=+47=H-K+22,1=.3=3:(1*&7=7*(*59478=2*).&9*)=

97&38(7.59.43=J.3`=Y:=*9=&1_=,*++_=

T-*=HDAC8=<4708=.3=(4397&89=94=9-*=HAT8=&3)=7*;*78*=9-*=&(*9>1&9.43=4+=-.8943*8='>=

7*8947.3,='&(0=9-*=548.9.;*=(-&7,*=43=9-*=1>8.3*=7*8.):*8_=HDAC8=&7*=&184=(1&88.+.*)=.394=

.=(1&88*8=)*8.,3&9*)=&8=HDAC=I`=II`=II=&3)=IV_=C1&88=I`=.3(1:)*8=HDAC+`=,`=-=&3)=2b=(1&88=

II`=.3(1:)*8=HDAC=.`=/`=0`=1`=3=&3)=+*b=(1&88=IV=&8= HDAC++=&3)=(1&88=III=(439&.38=9-*=

NAD(= )*5*3)*39= S.7,81.0*= )*&(*9>1&8*8= 47= 8.79:.38_= E=(*59= HDAC= 2`= 7*89= &11= HDAC8=

61 *=.89= .3= -.,-= 241*(:1&7= <*.,-9= 2:19.85749*.3= (4251*=*8_= HDAC8= &(9.;.9>= .8= 9.,-91>=

7*,:1&9*)=<.9-=9-*=49-*7=5749*.3=5&793*78=&3)=-.8943*=PTM8_=D>8+:3(9.43=4+=9-*8*=HAT8=

&3)= HDAC8= *3?>2*8= .8= 4+9*3= &884(.&9*)= <.9-= ).8*&8*8`= 7&3,.3,= +742=

3*:74)*,*3*7&9.;*=).847)*78=94=(&3(*7=R49-'&79=&3)=S97&-1=,*+._==

STST T R#%3* 2'-, 7 &'12-,# *71',# 0%',',# +#2&7* 2'-, ," "#+#2&7* 2'-,

S.2.1&7= 94= &(*9>1&9.43= 8*;*7&1= 1>8.3*= 7*8.):*8= 4+= -.8943*8= (&3= &184= '*= 2434`= ).= 47= 97.8

2*9->1&9*)_=T-*=1*;*1=&3)=9-*=14(&9.43=4+=-.8943*=24).+.(&9.43=+:79-*7=&))=(4251*=.9>=

.3= ,*3*= 7*,:1&9.43= ):7.3,= ).++*7*39= 574(*88*8= 8:(-= &8= 97&38(7.59.43`= DNA= 7*5&.7`=

7*51.(&9.43= &3)= 7*(42'.3&9.43_= U31.0*= 9-*= &(*9>1&9.43= &3)= 5-485-47>1&9.43`= -.8943*=

2*9->1&9.43=)4*8=349=&19*7=9-*=(-&7,*=4+=9-*=-.8943*=5749*.38b=-4<*;*7=.9=)4*8=&++*(9=9-*=

->)745-4'.(= &3)= 89*7.(= 5745*79.*8_= H.8943*= 2*9->1&9.43= (&3= '*= .251.(&9*)= '49-= .3=

97&38(7.59.43&1= &(9.;&9.43= &3)= 7*57*88.43`= .3= (4397&89= 94= 9-*= &(*9>1&9.43= <-.(-= .8=

&884(.&9*)=<.9-=&(9.;&9.43=431>_=H.8943*=H-=(&3='*=2*9->1&9*)=&9=;&7.4:8=548.9.438=8:(-=

&8= 2*9->1&9.43= 4+= K.= &3)= K-0= <-.(-= .8= &884(.&9*)= <.9-= &(9.;&9.43= H-K.2*= &3)=

H-K-02*= &3)=&9=K3=&3)=K,1= <-.(-=.8= 1.30*)=<.9-=7*57*88.43=H-K3=&3)=H-K,1= 4+=

97&38(7.59.43=Z*393*7=&3)=H*3.04++=,*+-_=H4<*;*7`=842*=4+=9-*=)*;*1452*39&1=,*3*8=

7*8.)*8= .3= '.;&1*39= (-742&9.3= 7*,.438= &3)= (439&.38= '49-= 7*57*88.;*= H-K,12*-= &3)=

&(9.;*=H-K.2*-=2&708_=T-*=*3?>2*8=9-&9=(&9&1>?*=9-*=97&38+*7=4+=2*9->1=,74:5=+742=S8

&)*348>12*9-.43.3*= ;<6= 94= &= 1>8.3*8= 8&2.34= ,74:5= &7*= 034<3= &8= -.8943*=

2*9->197&38+*7&8*8_= T-*= +.789= -.8943*= 1>8.3*= 2*9->197&38+*7&8*= HKMT= .)*39.+.*)= <&8=

SUV-3H+=&3)=.9=9&7,*98=H-K3=2*9->1&9.43=R*&`=E.8*3-&'*7=*9=&1_=,***_=M*9->1&9.43=4+=

H-K.= .8= (&9&1>?*)= '>= SET= )42&.3= 4+= KMT,A= K885*(.+.(= 2*9->197&38+*7&8*= ,A`=

(422431>=(&11*)=MLL`=<-*7*&8=H-K,1=.8=2*9->1&9*)='>=PRC,=541>(42'=7*57*88.;*=

(4251*==,_=R*,:1&9.43=4+=97&38(7.59.43&1=&(9.;&9.43=47=7*57*88.43='>=H-K.=47=H-K,1=.8=

89.11= 4'8(:7*_= I9= -&8= '**3= 8-4<3= 9-&9= H-K.= 2*9->1&9.43= 7*(7:.98= 9-*= BAF= (-742&9.3=

7*24)*1.3,= (4251*== ;.&= .98= (-7424)42&.3= 94= &(9.;&9*= 97&38(7.59.43`= 8.2.1&71>=

62 97.2*9->1&9*)=H-K,1=7*(7:.98=PRC8=(4251*== ;.&= 9-*=(-7424)42&.38(439&.3.3,=5749*.3=

CBX+=(-7424'4==-42414,=+= &3)=.3):(*8= 9-*=(425&(9.43=4+=(-742&9.3`= 7*8:19.3,=.3=

97&38(7.59.43&1=7*57*88.43=W>84(0&`=S<.,:9=*9=&1_=,**0b=S.243=&3)=K.3,8943=,*+-_=L.0*=

&)).9.43`=7*24;&1=4+=-.8943*8=2*9->1&9.43=.8=&184=&=-.,-1>=7*,:1&9*)=*;*39=&3)=(&9&1>?*)=

'>=8*;*7&1=-.8943*=L>8885*(.+.(=)*2*9->1&8*8=KDM8_=H-K.=.8=)*2*9->1&9*)='>=KDM+=

(422431>=034<3=&8=LSD+=&3)=KDM/B=JARID+`=<-.1*=H-K,1=.8=)*2*9->1&9*)='>=

KDM0A=UTX=&3)=KDM0B=JMJD-B1&(0`=V&3=R*(-*2=*9=&1_=,*+,_=

I3= &)).9.43= 94= 1>8.3*`= &7,.3.3*= 7*8.):*8= 4+= -.8943*8= (&3= &184= '*= 2*9->1&9*)_= A7,.3.3*=

2*9->1&9.43=.8=(&9&1>?*)='>=&=,74:5=4+=5749*.38=034<3=&8=A7,.3.3*=2*9->197&38+*7&8*8=

PRMT8_=T-*=PRMT8=97&38+*7=&=2*9->1=,74:5=+742=SAM=94=9-*= 8,:&3.).34=,74:5=4+=

&7,.3.3*=<.9-.3=&=;&7.*9>=4+=8:'897&9*8_=

STST!T R#%3* 2'-, 7 -2� &'12-,# +-"'$'! 2'-,1

T-*=).(9.43&7>=4+=9-*=-.8943*=PTM8=-&8=*=5&3)*)=;&891>=8.3(*=.9=<&8=+.789=.)*39.+.*)='>=

V.3(*39=A11+7*>_=B*8.)*8`=<*11=034<3=&(*9>1&9.43=&3)=2*9->1&9.43=4+=9-*=-.8943*8=9-*7*=

&7*= 8*;*7&1= 49-*7= 24).+.(&9.438= <-.(-= .3(1:)* = 5-485-47>1&9.43`= :'.6:.9>1&9.43`=

,1>(48>1&9.43`=ADP87.'48>1&9.43`=(&7'43>1&9.43`=SUMO>1&9.43`=(.97:11.3&9.43=&3)=2&3>=

49-*78_=T-*=1&9*7=2*39.43*)=PTM8=&7*=14<=.3=&':3)&3(*=&8=(425&7*)=94=9-*=247*=<*118

89:).*8=24).+.(&9.438=8:,,*89.3,=9-&9=9-*.7=741*8=.3=5->8.(&1=&3)=+:3(9.43&1=(748889&108=

2&>='*=8:'91*_=H.8943*=5-485-47>1&9.43=<-.(-=4((:78=43=8*7.3*=H-S+* =&3)=9-7*43.3* =

7*8.):*8= (&3= .3+1:*3(*= 97&38(7.59.43`= (-7424842*= (43)*38&9.43`= DNA= 7*5&.7= &3)=

&545948.8_= T-*= 1*;*1= 4+= 9-*= 24).+.(&9.43= .8= (4397411*)= '>= 9-*= 0.3&8*8= &3)= 9-*=

5-485-&9&8*8= 9-&9= &))= &3)= 7*24;*= 9-*= 5-485-&9*= ,74:5= +742= ATP= 94= 9-*= ->)74=>1=

,74:5= 4+= 9-*= 9&7,*9= &2.348&(.)= 8.)*= (-&.3_= U'.6:.9.3&9.43= .8= &349-*7= -.8943*=

24).+.(&9.43= <-.(-= 7*8:198= .3= &= 2:(-= 1&7,*7= (4;&1*39= 24).+.(&9.438= (425&7*)= 94= 9-*=

7*1&9.;*1>= 82&11= 241*(:1&7= (-&3,*= 94= &2.34= &(.)8= 8.)*= (-&.38_= U'.6:.9.3= +:3(9.438= '>=

&99&(-.3,= .98*1+= 94= 9-*= 1>8.3*= ;.&= 8*6:*39.&1= &(9.43= 4+= 9-*= 9-7**= *3?>2*8`= ._*_= E+8

63 &(9.;&9.3,`= E,8(43/:,&9.3,=&3)=E-81.,&9.3,=*3?>2*8_= U'.6:.9.3&9.43=.8=-.,-1>=)>3&2.(=

&3)=9-*=)*,7**=4+=:'.6:.9.3&9.43=(&3=;&7>=)*5*3).3,=43=9-*=8:'897&9*=85*(.+.(.9>_=R*(*39=

&);&3(*8= -&;*= )*+.3*)= (7.9.(&1= 741*8= 4+= -.8943*= :'.6:.9.3&9.43= .3= 97&38(7.59.43&1=

7*,:1&9.43=&3)=DNA=7*5&.7_=T-*=<7.9*78`=*7&8*78`=&3)=7*&)*78=4+=-.8943*=:'.6:.9.3&9.43=

-&;*=&184='**3=1.30*)=94=(&3(*7=)*;*1452*39_=M4348:'.6:.9.3&9.43=4+=K++3=43=H,A=.8=

034<3= 94= '*= .3;41;*)= .3= ,*3*= 8.1*3(.3,= <-.1*= 24348= :'.6:.9.3&9.43= 4+= K+,-= 43= H,B=

51&>8=&=(7:(.&1=741*=.3=97&38(7.59.43&1=.3.9.&9.43=&3)=*143,&9.43_=I3=&)).9.43=94=H,A=&3)=

H,B`= (47*= -.8943*8= H-`= H.`= &3)= 1.30*7= -.8943*= H+= -&;*= &184= '**3= 7*5479*)= 94= '*=

24).+.*)= '>= :'.6:.9.3_= F47= *=&251*`= H-= &3)= H.= <*7*= 541>:'.6:.9.3&9*)= .3= ;.;4= '>=

CUL. :DDB :RBX+= :'.6:.9.3= 1.,&8*= (4251*== &+9*7= UV= .77&).&9.43= C&4= &3)= Y&3= ,*+,_=

SUMO>1&9.43= .8= &= 24).+.(&9.43= 8.2.1&7= 94= :'.6:.9.3&9.43= &3)= .3;41;*8= 9-*= -.8943*8=

1>8.3*8=;.&=E+`=E,=&3)=E-=*3?>2*8_=I9=(&3='*=)*9*(9*)=.3=&11=9-*=+4:7=(47*=-.8943*8=&3)=

+:3(9.438= 8**2.3,1>= .3= 45548.9*= 94= 9-*= &(*9>1&9.43= &3)= :'.6:.9.3&9.43_= G1:9&2&9*= &3)=

9-*= &7,.3.3*= 7*8.):*8= 4+= -.8943*8= &7*= &184= 8:'/*(9*)= 94= *.9-*7= 2434= 47= 541>8ADP=

7.'48>1&9.43_=I9=.8=2*).&9*)='>=541>8ADP=7.'48*=541>2*7&8*=PARP=+&2.1>=4+=5749*.38=

&3)=7*;*78*)='>=9-*=541>8ADP87.'48*8,1>(4->)741&8*=PARG=+&2.1>=4+=5749*.38_=W.9-=

9-*=034<1*),*=4+=&11=9-*8*=-.8943*8=PTM8=.9=-&8='*(42*=.3(7*&8.3,1>=&55&7*39=9-&9=9-*=

(-742&9.38= &884(.&9*)= +&(9478= -&7'478= 2:19.51*= -.8943*= '.3).3,= )42&.38_= T-.8= +:79-*7=

574;.)*8=&=;&7.*)=&3)=*=(.9.3,=574'&'.1.9>=+47=2:19.;&1*39=-.8943*=*3,&,*2*398=<-.(-=

&))8=&=1&>*7=4+=85*(.+.(.9>=94=-.8943*=PTM=7*(4,3.9.43_===

WTX DNA +#2&7* 2'-,

DNA=2*9->1&9.43=.8=&=(42243=*5.,*3*9.(=2&70=<-.(-=.8=(438*7;*)=.3=2&3>=*:0&7>49*8=

&3)=5740&7>49*8_=I3=9-*=14<*7=+4728=&3)=51&398`=2*9->1&9.43=(&3=4((:7=.3=9-*=(439*=9=4+=

&)*3.3*=&3)=(>948.3*`=-4<*;*7=.3=*:0&7>49*8=.9=4((:78=57.2&7.1>=&9=9-*=/8548.9.43=4+=9-*=

(>948.3*=7*8.):*8=/2C=.3=9-*=(439*=9=4+=C5G=).3:(1*49.)*8_=T-*=2&.3=+:3(9.43=4+=DNA=

2*9->1&9.43= .3= '&(9*7.&= .8= 94= 574;.)*= &= 5749*(9.43= 2*(-&3.82`= 9-74:,-= <-.(-= .9=

64 ).8(7.2.3&9*8='*9<**3=9-*=*3)4,*34:8=&3)=+47*.,3=DNA=.3974):(9.43=H*2&;&9->=&3)=

N&,&7&/&= +33/_= I3= 2&22&18= 9-*= DNA= 2*9->1&9.43= 5&99*73= .8= *89&'1.8-*)= ):7.3,=

*2'7>43.(= )*;*1452*39= '>= )*= 34;4= DNA= 2*9->197&38+*7&8*= D329`= D329-&= &3)=

D329-'=O0&34`=B*11=*9=&1_=+333b=L.=&3)=Z-&3,=,*+._=F:79-*7`=9-.8=2*9->1&9.43=5&99*73=

.8= 2&.39&.3*)= 9-74:,-= 9-*= D329+82*).&9*)= (45>.3,= 2*(-&3.82= ):7.3,= 9-*= (*11=

).;.8.43_=T-*=7*,.438=4+=9-*=,*342*=<.9-=-.,-*7=3:2'*7=4+=2*9->1&9*)=(>948.3*=:8:&11>=

1*&)=94=9-*=7*):(9.43=4+=,*3*=*=57*88.43`=84= (&11*)=,*3*=8.1*3(.3,_=M*9->1&9.43=8*7;*8=

57.2&7.1>= &8= &= 51&9+472= +47= 9-*= 7*(7:.92*39= 4+= 2&3>= *3?>2*8_= I3)**)`= 9-*= (-&3,*= .8=

7*(4,3.?*)= '>= MBP= 2*9->1= '.3).3,= 5749*.3`= 8:(-= &8= M*CP,`= MBP,= MBP-= 47= <-.(-=

<.11='.3)=&9=9-*=2*9->1&9*)=C5G`=&3)=9-:8=(&:8*=9-*=7*(7:.92*39=4+=-.8943*=24).+>.3,=

*3?>2*8`= 8:(-= &8= -.8943*= )*&(*9>1&8*8= HDAC= N&3`= N,= *9= &1_= +332= 47=

2*9->197&38+*7&8*8= F:08`= H:7)= *9= &1_= ,**-_= T-*8*= *3?>2*8= (&3= &19*7= 9-*= 897:(9:7*= 4+=

(-742&9.3=-*9*74(-742&9.3=94=(43841.)&9*=9-*=7*57*88.43=4+=97&38(7.59.43_=

T-*= &'8*3(*= 4+= DNA= 2*9->1&9.43= .8= 57*7*6:.8.9*= +47= 9-*= &(9.;*= 97&38(7.59.43_= C5G=

).3:(1*49.)*8= &7*= ).897.':9*)= :3*;*31>= 9-74:,-4:9= 9-*= ,*342*_= I3= ,*3*7&1`= C5G8= &7*=

:3)*787*57*8*39*)= .3= 2&22&18`= 1.0*1>= ):*= 94= 9-*= 2:9&,*3.(= 5745*79.*8= 4+= /2C_=

H4<*;*7`=842*=7*,.438=4+=,*342*=-&;*=-.,-=)*38.9>=4+=C5G=).3:(1*49.)*8=<-.(-=&7*=

7*+*77*)= &8= C5G= .81&3)8= &3)= 9-*8*= .81&3)8= &7*= DNA= 2*9->1&9.43= +7**_= C*79&.3=

97&38(7.59.43= +&(9478= 8:(-= &8= C+5+= &3)= K)2,'= <-.(-= (439&.38= CXXC= )42&.3= '.3)8=

85*(.+.(&11>= 94= 9-*= :32*9->1&9*)= C5G8= <.9-.3= 9-*8*= .81&3)8= D*&943= &3)= B.7)= ,*++=

574;.).3,= &3= :3)*789&3).3,= &'4:9= 9-*= (4397.':9.43= 4+= :32*9->1&9*)= DNA= .3=

97&38(7.59.43&1=7*,:1&9.43_=I3=+&(9`=0* :1*=4+=&3349&9*)=,*3*=574249*78=&7*=&884(.&9*)=

<.9-=&=C5G=.81&3)`=.3(1:).3,=2489=4+=9-*=-4:8*0**5.3,=,*3*8`=)*;*1452*39&1=,*3*8=&8=

<*11=&8=9.88:*885*(.+.(=,*3*8_=

65 F'%30# _T DNA "#+#2&7* 2'-, P 2&5 7T

C72-1',# C '1 !-,4#02#" 2- [V+#2&7*!72-1',# [+C 7 !2'-, -$ #,"-%#,-31 DNA +#2&7*20 ,1$#0 1#1 %0##, . 2&5 7T S#4#0 * +#!& ,'1+1 $-0 DNA "#+#2&7* 2'-, & 4# ##, .0-.-1#"T H-0'8-,2 * 00-51 0#.0#1#,2 -6'" 2'-,V 1#" . 2&5 71 .#0$-0+#" 7 T#2 .0-2#',1S +#2&7* %0-3. -$ [+C '1 !-,1#!32'4#*7 -6'"'8#" 2- &7"0-67+#2&7*Q $-0+7* ," ! 0 -67* %0-3.1 $-0+',% [V&7"0-67+#2&7*!72-1',# &+CQ [V$-0+7*!72-1',# $C ," [V ! 0 -67!72-1',# ! CQ 0#1.#!2'4#*7T B#,2 .* ', 00-51 1&-5 "# +', 2'-,V 1#" . 2&5 71 5�# &+C '1 "# +', 2#" 2- [V&7"0-67+#2&7*30 !'* &+U ', 2&# .0#1#,!# -$ AID APOBEC $ +'*7 "# +', 1#1Q ," "'0#!2 1# #6!'1'-, 0#. '0 BER . 2&5 71 ',4-*4',% TDGQ MBDZ ," SMUGW %*7!-17* 1#1Q 5&'!& ** *# " 2- 20 ,1'#,2 $-0+ 2'-, -$ .70'+'"','! AP 1'2#1 ', DNAT D 1&#" 00-51 "#,-2# 2&# ,#5*7 "'1!-4#0#" &7"0-67+#2&7* 2'-, ," "#&7"0-67+#2&7* 2'-, 0# !2'-,1 .#0$-0+#" 7 !72-1',#V[ +#2&7*20 ,1$#0 1#1 ',V4'20- ," .32 2'4# #,87+#1 "#$-0+7* 1# ," "#! 0 -67* 1# 5&'!& !-3*" "'0#!2*7 0#+-4# 2&# $-0+7* ," ! 0 -67* %0-3.1 $0-+ $C ," ! CQ 0#1.#!2'4#*7T A" .2#" $0-+ K0'3)'#,#Q L'32)#4'!'32# #2 *T XVWX

66 A19-4:,-=9-.8=*5.,*3*9.(=2&70=(&3='*=97&382.99*)=94=8*;*7&1=,*3*7&9.438`=':9=9-*>=&7*=

349= 5*72&3*39_= I3)**)`= 89:).*8= -&;*= 8-4<3= 9-&9= 2*9->1&9.43= 5&99*73= (&3= '*= &19*7*)=

9-74:,-4:9= 9-*= 1.+*= .3= 7*85438*= 94= 9-*= *3;.7432*39&1= (-&3,*8= 47= ):*= 94= 5&9-414,.(&1=

574(*88*8=8:(-=&8=43(4,*3.(=97&38+472&9.43=47=(*11:1&7=&,*.3,_=DNA=2*9->1&9.43=2&708=

(&3= '*= 7*24;*)= 47= 7*574,7&22*)= ):7.3,= *&71>= )*;*1452*39= '>= 9<4= <&>8_= I9= (&3= '*=

*.9-*7= 7*24;*)= 5&88.;*1>= &8= ):7.3,= (*11= ).;.8.43= '>= .3-.'.9.43= 4+= 9-*= 2&.39*3&3(*=

2*9->197&38+*7&8*`= D329+= 47= '>= &3= &(9.;*= )*2*9->1&9.43= 2*(-&3.82= <-.(-= .3;41;*8=

7*24;&1= 4+= 9-*= 2*9->18,74:5= +742= /2C= '>= &= +&2.1>= 4+= DNA= ->)74=>1&8*8= (&11*)= T*9=

5749*.38=9*3=*1*;*3=97&3814(&9.43`=9-&9=*=.89=.3=9-7**=.84+4728a=TET=+`=,=&3)=-_=P&88.;*=

2*9->1&9.43= .8= <*11= :3)*78944)= &3)= &((*59*)= <-.1*= 9-*= 241*(:1&7= 2*(-&3.82= '*-.3)=

&(9.;*=DNA=)*2*9->1&9.43=.8='*,.33.3,=94='*=:3)*78944)=&3)=9-*7*=&55*&78=2:19.51*=

24)*8= 9-74:,-= <-.(-= .9= (&3= 4((:7_= T-*= 2*(-&3.828= '>= <-.(-= &(9.;*= DNA=

)*2*9->1&9.43=4((:78=(&3='*='74&)1>=(1&88.+.*)=:3)*7=9<4=(&9*,47.*8=._*_=4=.)&9.;*=&3)=

7*5&.7='&8*)=DNA=)*2*9->1&9.43= F.,:7*3`=K7.:0.*3*`=L.:90*;.(.:9*=*9=&1_=,*+, _=T-*=

4=.)&9.;*=2*(-&3.82=.3(1:)*8=9-*=*3?>2&9.(=4=.)&9.43=/2C=94=9-*=/8->)74=>2*9->18 , 8

)*4=>(>9.).3*= /-2C`= <-.(-= (&3= '*= +:79-*7= 4=.).?*)= 94= /8+472>(>94(.8*= /+C= 47= /8

(&7'4=>8(>948.3*= /(&C= T&-.1.&3.`= K4-= *9= &1_= ,**3b= I94`= S-*3= *9= &1_= ,*++b= M&.9.= &3)=

D74-&9=,*++_=T-*=97&38.9.43=+742=/+C=&3)=/(&C=94=(>948.3*=.8=)43*='>=9->2.).3*=DNA=

,1>(48>1&8*`= TDG_= I3= 5&79.(:1&7= TET+= &3)= TET-= *=-.'.9= &= CXXC= )42&.3`= &= '.3).3,=

)42&.3=C5G`=<-.(-=9-*>=:9.1.?*=+47=9-*=(43;*78.43=4+= /2C=94=9-*=&19*73&9.;*=+472=4+=

(>948.3*_= T-*= 49-*7= 2*(-&3.828= <-.(-= .3;41;*= DNA= 7*5&.7= -&8= '**3= 574548*)= &8= &3=

&19*73&9.;*= 94= 9-*= 4=.)&9.;*= )*2*9->1&9.43_= T-*8*= .3(1:)*8= '&8*= *=(.8.43= 7*5&.7= BER=

9-74:,-=).7*(9=*=(.8.43=4+=/2C`=)*&2.3&9.43=4+=/2C=94=T=+4114<*)='>=BER=&3)=9-74:,-=

3:(1*49.)*= *=(.8.43= 7*5&.7= NER_= T-*= )*&2.3&9.43= '&8*)= 5&9-<&>`= .3= 5&79.(:1&7= :8*8=

AID= (>948.3*= )*&2.3&8*8= &(9.;&9.43= .3):(*)= (>9.).3*= )*&2.3&8*2= APOBEC= <-.(-=

)*&2.3&9*= -2C= 94= /8->)74=>2*9->1:7&(.1= -2U_= T-.8= 89*5= .8= +4114<*)= '>= 9-*= TDG`=

67 MBD.=&3)=SMUG+=,1>(48>1&8*8`=<-.(-=&11=1*&)=94=97&38.*39=+472&9.43=4+=&5>7.2.).3.(=

AP=8.9*8=.3=DNA=&3)=).7*(9='&8*=*=(.8.43=7*5&.7=BER=5&9-<&>8=F.,:7*=3_=

T-*= DNA= )&2&,* :&884(.&9*)= 5749*.3= G&))./&= <&8= &184= )*8(7.'*)= 94= 5&79.(.5&9*= .3=

8*1*(9.;*= )*2*9->1&9.43= &9= 9-*= 574249*78= 4+= 9&7,*9= ,*3*8= 9-74:,-= 7*(7:.92*39= 4+= 9-*=

DNA= 7*5&.7=2&(-.3*7>= B&77*94`=S(-&+*7= *9=&1_=,**1b=S(-2.9?`=S(-2.99=*9=&1_=,**3`=':9=

-4<=.9=<&8=9&7,*9*)=94=85*(.+.(=8.9*8=7*2&.3*)=:3034<3_=R*(*391>`=S(-C+*7=*9=&1_=8-4<*)=

9-*= .251.(&9.43= 4+= 9-*= 9:247= 8:557*8847= I3,+ ;<-.(-= (439&.38= &= PHD= +.3,*7= )42&.3=

9-&9= 85*(.+.(&11>= '.3)8= H-K.2*- ;.3= DNA= )*2*9->1&9.43= '>= 7*(7:.9.3,= G&))./&= 94=

H-K.2*-=8.9*8=S(-C+*7`=K&7&:1&34;=*9=&1_=,*+-_=I3=&)).9.43=NER=+&(9478b=XPG=&3)=XPF=

-&;*= &184= '**3= 8-4<3= 94= '*= .3;41;*)= .3= DNA= )*2*9->1&9.43= L*= M&>`= F7&).3= *9= &1_=

,*+,_=

D_,=C-742&9.3=7*&77&3,*2*39=

R*,:1&9.43= 4+= ,*3*= *=57*88.43= (&3= 4((:7= 14(&11>= 47= 4;*7= &= 1&7,*= ,*342.(= ).89&3(*= ;.&=

;&7.4:8=7*,:1&947>=*1*2*398=14(&9*)=+&7=:5897*&2=47=)4<3897*&2=4+=9-*=,*3*=&3)=(&3=

&184= (439741= *=57*88.43= 4+= 9-*= ,*3*8= <-.(-= &7*= 57*8*39= 43= 49-*7= (-7424842*8_= S:(-=

143,87&3,*=(439741=4+=,*3*=*=57*88.43=4((:78=9-74:,-=&=574(*88=7*+*77*)=&8=(-742&9.3=

1445.3,_=T-*=+.789=*=&251*=4+=9-*=(-742&9.3=14458=<&8=8-4<3='>=9-*=.39*7&(9.43=4+=14(:8=

(439741= 7*,.43= LCR= 4+= 9-*= 8,14'.3= ,*3*= <.9-= 9-*= *2'7>43.(= &3)= 9-*= &):19= 9>5*= 8

,14'.3= 574249*78= &9= 9-*= &557457.&9*= 9.2*= 4+= )*;*1452*39= C&79*7`= C-&0&14;&= *9= &1_=

,**,_= C-742&9.3= 14458= -&;*= '**3= &184= )*8(7.'*)= .3= 3:2*74:8= ,*3*8`= &8= &= ,*3*7&1=

47,&3.?&9.43= 4+= 9-*= (-742&9.3= +.'*7`= &884(.&9*)= <.9-= 9-*= &(9.;&9.43= 4+= 97&38(7.59.43=

S5.1.&3&0.8=&3)=F1&;*11=,**.=V*73.22*3`=D*=G4''.=*9=&1_=,**1_=D.++*7*39=7*,.438=4+=

9-*= ,*3*8= -&;*= '**3= 8-4<3= 94= '*= .3;41;*)= .3= +472.3,= (-742&9.3= 14458_= I9= (4:1)= '*=

'*9<**3=*3-&3(*78=&3)=9-*=574249*7=K7.;*,&=&3)=D*&3=,*+,=47='*9<**3=9-*=574249*7=

68 &3)= 9*72.3&947= ,*3*8= L&.3*`= S.3,-= *9= &1_= ,**3_= D>3&2.(= 574249*7 :9*72.3&947= 14458=

-&;*= &184= '**3= )*8(7.'*)= +47= 9-*= '7*&89= (&3(*7= BRCA+= ,*3*= T&38W43,`= F7*3(-= *9= &1_=

,**2=&3)=&9=9-*=,*3*=*3(4).3,=9-*= .22:3*8-.89414,.(&1=2&70*7=CD02=.3=2&22&1.&3=

(*118=OR*.11>=&3)=G7*&;*8=,**1_=T-*=&'4;*=9<4=89:).*8`='>=L.&3*=&3)=T&8W&3,=8-4<8=

9-&9=9-*=574249*7=&3)=9*72.3&947=.39*7&(9.43=5*78.89=+4114<.3,=&=(>(1*=4+=97&38(7.59.43&1=

&(9.;&9.43=&3)=7*57*88.43=&3)=9-.8=5-*342*343=.8=7*+*77*)=94=&8=97&38(7.59.43=2*247>_=

H*3(*`=-.,-*7=47)*7=(-742&9.3=47,&3.?&9.43=(&3=(4397.':9*=94=5->8.414,.(&1=741*8=8:(-=

97&38(7.59.43=2*247>=+&(.1.9&9.3,=9-*=7&5.)=7*8*=57*88.43=4+=9-*=,*3*_=

C-742&9.3= 14458= &7*= 89&'.1.?*)= '>= 3:2'*7= 4+= 5749*.38= 8:(-= &8= 9.88:*= 85*(.+.(=

97&38(7.59.43=+&(9478=EKLF`=GAT8+=&3)=L)'+=&9=9-*=2:7.3*= 8,14'.3=14(:8_=O3*=4+=9-*=

2489=89:).*)=5749*.38=.3=(-742&9.3=1445=+472&9.43=.8=CCCTC8='.3).3,=+&(947`=CTCF_=I9=

.8= :'.6:.94:8= &3)= -&8= ;*7>= ).;*78*= +:3(9.43`= .3(1:).3,= *3-&3(*78'14(0.3,`= X8

(-7424842*= .3&(9.;&9.43`= ,*342*= .257.39.3,`= ,*3*= &(9.;&9.43= 47= 7*57*88.43_= G*342*=

<.)*=89:).*8=8-4<8=9-&9=CTCF='.3)8=94=9*38=4+=9-4:8&3)8=4+=,*342.(=8.9*8=&3)=&12489=

+.***= ,*342.(= 7*,.438= &7*= +1&30*)= '>= CTCF= 43= '49-= 8.)*8= 7*+*77*)= &8= CTCF85&.78

)*+.3*)=)42&.38=CPD=4+=&;*7&,*=8.?*=,+*=0'_=T-*=CTCF=-&8='**3=+4:3)=94='*=1.30*)=

94= 9-*= 2*9->1&9*)= DNA= 8*6:*3(*= W&3,`= M&:7&34= *9= &1_= ,*+,_= I9= (&3= '.3)= 94=

2*9->1&9*)= DNA= 8*6:*3(*8= .38;.974 = ':9= 57*+*7*39.&11>= '.3)8= 94= 9-*= :32*9->1&9*)=

8*6:*3(*8=&8=4'8*7;*)=.3=9-*=H+3 :I,+,=14(:8_=CTCF=8.9*8=&7*=&884(.&9*)=<.9-=+7*6:*391>=

+1&30.3,=9-*=1&2.3&8&884(.&9*)=)42&.38=LAD8=&3)=+4:3)=*37.(-*)='*9<**3=9-*=&(9.;*=

(-742&9.3= -.,-= .3= H,K/A(= &3)= .3&(9.;*= (-742&9.3= )42&.38= -.,-= .3= H-K,12*-_=

F:79-*7247*`=CTCF=(&3=+472=(4251*==<.9-=PARP+=&3)=DNMT+=&3)=9-:8=.3+1:*3(*=9-*=

DNA=2*9->1&9.43_=CTCF=&(9.;&9*8=PARP+`=<-.(-=9-*3=(&3=&))=ADP :7.'48*=,74:58=94=

DNMT+=94=.3&(9.;&9*=9-.8=*3?>2*`=<.9-=2&.39*3&3(*=4+=2*9->18+7**=C5G8=&8=9-*=7*8:19=

Y:`= G.3/&1&= *9= &1_= ,**._= A349-*7= <*11= 89:).*)= 24)*1= +47= ,*3*= *=57*88.43= <-.(-= .8=

2*).&9*)='>=(-742&9.3=1445.3,=.3;41;*8=9-*=P41>(42'=,74:58=P(G=&3)=9-*=97.9-47&==

,74:5= 97=G= 7*,:1&947>= 8>89*2_= T-*>= <*7*= .3.9.&11>= ).8(4;*7*)= .3= D74845-.1&= &3)= &7*=

034<3= 94= 2&.39&.3= 9-*= 7*57*88*)= 89&9*= 4+= 9-*= 9&7,*9*)= ,*3*_= P41>(42'= 7*57*88.;*=

*1*2*398= PRE8= (439&.3.3,= F&'1= *1*2*398= (4397418= 9-*= '.9-47&== ,*3*= (1:89*7= .3=

D74845-.1& ='>=(439&(9.3,=.98=574249*7_==

T-*= 2489= <.)*1>= :8*)= 2*9-4)= 94= )*9*72.3*= 9-*= ,*342.(= 14458= .8= (-7424842*=

(43+472&9.43=(&59:7*=-C`=47.,.3&11>=)*;*145*)='>=J4'=D*00*7=.3=,**,=D*00*7`=R.55*=

*9= &1_= ,**,_= S*;*7&1= -C= '&8*)= &5574&(-*8= <-.(-= .3(1:)*= .C`= /C`= H.8C= &3)= C-IA8PET=

&114<8= ,*342*8<.)*= .)*39.+.(&9.43= &3)= &3&1>8.8= 4+= 8*6:*3(*8= .3;41;*)= .3= 9-*= 9-7**=

).2*38.43&1=47,&3.?&9.43=4+=9-*=+:11=,*342*=M439&;43=&3)=D:'4:1*=,*+,_=

70 II_ T-*=TFIIH=&3)=NER=+&(9478=.3=97&38(7.59.43

AT TFIIHS A +3*2'$3,!2'-, * C-+.*#6

TFIIH=<&8=+.789=(-&7&(9*7.?*)=&8=&=,*3*7&1=97&38(7.59.43=+&(947=4+=RNA=541=II=.3=+323_=I9=

<&8=.3.9.&11>=5:7.+.*)=+742=9-*=7&9=1.;*7=C43&<&>=&3)=C43&<&>=+323`=+742=H*L&=(*118=

&8='&8.(=97&38(7.59.43=+&(947=,=G*7&7)`=F.8(-*7=*9=&1_=+33+_=F742=>*&89`=.9=<&8=.841&9*)=&8=

RNA=541=II=97&38(7.59.43=+&(947='=F*&;*7`=G.1*&).=*9=&1_=+33+='>=(43;*39.43&1=(41:23=

(-742&94,7&5->=&3)=1&9*7=5:7.+.*)='>=&=8.3,1*=89*5=.22:34&++.3.9>=5:7.+.(&9.43=2*9-4)=

K*78-3&7`=W:=*9=&1_=+332_=A+9*7=9-*=-42414,>=4+=&11=9-*8*=+&(9478= <&8=.)*39.+.*)`=9-*=

:3.;*78&1=342*3(1&9:7*=+47=97&38(7.59.43=+&(9478=<&8=574548*)=&3)=.9=<&8=)*8.,3&9*)=&8=

TFIIH_=O7.,.3&11>=TFIIH=<&8=9-4:,-9=94='*=*=(1:8.;*1>=&='&8&1=97&38(7.59.43&1=+&(947=':9=

1&9*7=.9=<&8=+4:3)=94='*=.3;41;*)=.3=DNA=7*5&.7=&3)=5488.'1>=.3=(*11=(>(1*=7*,:1&9.43_==

A_+=C42548.9.43=4+=TFIIH=

T7&38(7.59.43=+&(947=II=H=.8=&=2:19.+:3(9.43&1=(4251*==4+=5749*.38=<-.(-=(438.898=4+=9<4=

8:'8(4251*=*8a=&=(47*=(4251*==&3)=&=C)0=&(9.;&9.3,=0.3&8*=CAK=S**=F.,:7*8=2`=T&'1*=

._=T-*=(47*=(4251*==(438.898=4+=8.==8:':3.98a= X*74)*72&=5.,2*3948:2 =B=XPBb=523`=50,`=

5/,`=5..`=5-.=&3)=97.(-49-.4)>89745->=A=TTD8Ab=52=S*7.?&<&`=C43&<&>=*9=&1_=+33-_==

CAK=.8=(42548*)=4+=9-*=9-7**=8:':3.98a=CDK1=5.*`=(>(1.3=5-.=&3)= 2S3&,*=&=97.48 =

MAT+b=5-,_=T-*=(47*=&3)=9-*=CAK=&7*=-*1)=94,*9-*7='>=9-*=X *74)*72&=5.,2*3948:2 =D=

XPDb=52*`=8:':3.9_=E1*(9743=2.(748(45>=8-4<8=9-&9=TFIIH=.8=47,&3.?*)=.394=&=7.3,=1.0*=

(47*=897:(9:7*=+742=<-.(-=,14':1&7=CAK=8:'8(4251*==57497:)*8=4:9=S(-:19?`=F7.'4:7,=

*9=&1_=,***=F.,:7*=+*`=T&'1*=/_=TFIIH=-&8=.25479&391>=9-7**=*3?>2&9.(=&(9.;.9.*8a=DNA8

)*5*3)*39=ATP&8*=XPB=&3)=XPD`=ATP=)*5*3)*39=-*1.(&8*=XPD=&3)=XPB`=&3)=CTD=

0.3&8*= C)01_= I3= &)).9.43`= .9= &184= (439&.38= U'.6:.9.3= 1.,&8*= &(9.;.9>= ).851&>*)= '>= 5..=

&3)= MAT+= C425*= &3)= E,1>= ,*+,_= A11= 9-*= (42543*398= 4+= TFIIH= &7*= 349= +4:3)= .3=

(4251*==+472_=CAK=(&3='*=+4:3)=.3)*5*3)*391>=R488.,341`=K41'8C-*>3*1=*9=&1_=+331_=

71 A B

F'%30# WVT S203!230# -$ TFIIH

A S!&#+ 2'! 0#.0#1#,2 2'-, 5'2& !-0# ', 0#" ," CAK ', *-5Q XPD ', %0##,T B E*#!20-, +'!0-1!-.7 4'#5 A" .2#" $0-+ C-+.# ," E%*7 XVWX

72 S%GRHQI]CV6 AssQH1: VR $VJV 1H H%I:J V:s F%JH 1QJ TFIIH R1sV:sVs CQ`V PB SsC ; R ;#$%&:(V TTD :JR PCLCS ] T`G S `%H %`:C `%JH 1QJ5 1J V`:H s 11 . `:JsH`1] 1QJ :JR NER `:H Q`s

] T`G RV$%C: Vs .V ATP:sV :H 101 7 Q` PB ] SsC E %G1_%1 1J C1$:sV ^1J 7V:s _ ] T`G S `%H %`:C `%JH 1QJ :JR 1J V`:H s 11 . ] ] LTTDRA T`G RV$%C: Vs .V ATP:sV :H 101 7 Q` TTD PB PD PD R:R !"#RV]VJRVJ # ; R;#.VC1H:1V# TTD5 P :JR PLCS :H 101 7 :JR C1J@s .V HQ`V Q CAK CAK CDK K1J K1J:sV MQR%C: Vs .V CDK @1J:sV C7HC1J H CHC :H 101 7 MAT S :G1C1

T *# WT TFIIH !-+.-,#,21 ," 2&#'0 $3,!2'-,1

A" .2#" $0-+ C-+.# ," E%*7 TRST

73 R*2&70&'1>`= 9-*= +7**= CAK= &3)= 9-*= CAK= .39*7&(9.3,= <.9-= 9-*= (47*= TFIIH= -&;*=

8.,3.+.(&391>=).++*7*39=8:'897&9*=85*(.+.(.9>_=F7**=CAK=&(98=&8=C)08&(9.;&9.3,=0.3&8*=&3)=

(&3=5-485-47>1&9*8=3:2'*7=4+=8:'897&9*8=<-.(-=.3(1:)*=C)0+`=C)0,`=C)0.`=&3)=C)00=

.3;41;*)=.3=9-*=(*11=(>(1*=574,7*88.43_=W-*3=.9=.8=.3=(4251*==<.9-=TFIIH=.9=57*+*7*39.&11>=

5-485-47>1&9*8=9-*=CTD=4+=RNA=541=II_=I3=&)).9.43=94=9-&9`=TFIIH=&184=5-485-47>1&9*8=

9-*= NR8= ):7.3,= 97&38(7.59.43_= T-*= CAK= .8= 7*1*&8*)= '>= 9-*= (47*= ):7.3,= 9-*= NER= &3)=

7*&55*&78=&+9*7=DNA=7*5&.7=+47=9-*=7*8:259.43=4+=9-*=97&38(7.59.43=C4.3`=O08*3>(-=*9=

&1_=,**2_=522TTD8A=8:':3.9=4+=9-*=(47*=&184=*=.898=.3=9-*=(*11=.3=).2*7.(=+472=8*5&7&9*1>=

+742=TFIIH_=====

WTW XPB

XPB=ERCC-b=S81,b=R&),/=.8=9-*=1&7,*89=2*2'*7=4+=TFIIH_=T-*=ERCC-=,*3*=*=(.8.43=

7*5&.7=(74888(4251*2*39.3,=,74:5=-=<&8=.3.9.&11>=(-&7&(9*7.?*)=&8=&=,*3*=(477*(9.3,=&=

DNA= 7*5&.7= )*+.(.*3(>= .3= XPB= (*118= +742= 5&9.*39= 8:++*7.3,= +742= &= 8*;*7*= ,*3*9.(=

8>3)742*= X*74)*72&=5.,2*3948:2 =W**)&`=;&3=H&2=*9=&1_=+33*=W**)&`=;&3=H&2=*9=

&1_=+33*_=T-*=,*3*=<&8=(143*)=&+9*7=97&38+*(9.43=4+=-:2&3=(-7424842&1=DNA=94=9-*=

UV88*38.9.;*`= .3(.8.438)*+*(9.;*= C-.3*8*= -&289*7= 4;&7>= CHO= 2:9&39= ,18+_= I9= <&8=

4'8*7;*)`=9-&9=ERCC-=85*(.+.(&11>=(477*(98=9-*=)*+*(98=.3=&3=*&71>=89*5=4+=DNA=*=(.8.43=

7*5&.7= 5&9-<&>= 4+= UV88*38.9.;*= 74)*39= 2:9&398= 4+= (4251*2*39&9.43= ,74:5= -_=

S97:(9:7&11>`=ERCC-=.8=12,=&2.34=&(.)8=5749*.3=5488*88.3,=5:9&9.;*=3:(1*49.)*='.3).3,=

)42&.3`=(-742&9.3='.3).3,=)42&.3`=-*1.=89:738-*1.==DNA='.3).3,=)42&.3=&3)=8*;*3=

(438*(:9.;*= 249.+8_= XPB= '*143,8= 94= 9-*= SF,= 8:5*7+&2.1>= 4+= ATP8)*5*3)*39= DNA= 47=

RNA= -*1.(&8*8= W**)&`= ;&3= H&2= *9= &1_= +33*_= I9= .8= *88*39.&1= +47= '49-= .3.9.&9.43= 4+=

97&38(7.59.43= '>= RNA= 541>2*7&8*= II= &3)= 3:(1*49.)*= *=(.8.43= 7*5&.7_= I98= +:3(9.43= .8=

*;41:9.43&7.1>=(438*7;*)=&243,=*:0&7>49*8_=

C7>89&1= 897:(9:7*= 4+= XPB= -42414,= +742= A7(-&*4,14':8= +:1,.):8 = A+XPB= 7*;*&1*)= 9-&9=

XPB=(438.898=4+=9<4=R*(8A=1.0*=-*1.(&8*=)42&.38=HD+&3)=HD,=(433*(9*)='>=&=+1*=.'1*=

-.3,*`=&=DNA=)&2&,*=7*(4,3.9.43=)42&.3=DRD`=&=:3.6:*=RED=249.+=&3)=&=+1*=.'1*=

9-:2'=249.+=T-M`=F&3`=A7;&.=*9=&1_=,**0_=T-*=548.9.;*1>=(-&7,*)=T-:2'81.0*=)42&.3=

&3)= &3= RED= 249.+= -&;*= '**3= 8-4<3= 94= '*= .251.(&9*)= .3= XPB= 85*(.+.(= &(9.;.9.*8=

O08*3>(-`=B*73&7)*8=)*=J*8:8=*9=&1_=,**3_=HD+=&3)=HD,=&7*=5&(0&,*)=45548.9*=94=*&(-=

49-*7=&3)=+472=&=(1*+9=9-&9='7.3,8=I`=II`=V=&3)=VI=F.,:7*=3&=94=+472=&=(42548.9*=ATP8

'.3).3,= 8.9*= F.,:7*= ++`= F:88= &3)= T&.3*7= ,*++ _= H42414,:*= 4+= -:2&3= XPB= (&3= '*=

+4:3)= .3= &= 3:2'*7= 4+= '&(9*7.&= ._*_`= M>(4'&(9*7.:2= 9:'*7(:148.8 = &3)= K.3*4(4((:8=

7&).4941*7&38= B.8<&8`= P*74= *9= &1_= ,**3_= S:1+414':8= 841+&9&7.(:8 = &3)= 7*1&9*)= &7(-&*&= -&;*=

9<4=-42414,:*8=4+=9-*=XPB=5749*.3`=XPB+=S84*3/3=&3)=XPB,=S84*.1-=<-.(-='.3)8=94=

8.3,1*=897&3)*)=DNA=&3)=-&;*=DNA8)*5*3)*39=ATP&8*=&(9.;.9>`=':9=3*.9-*7=4+=9-*2=

<470=&8=-*1.(&8*=&,&.389=&=7&3,*=4+=)&2&,*)=&3)=:3)&2&,*)=DNA=8:'897&9*8= .38;.974 _=

T-*=*3?>2&9.(=&(9.;.9>=4+=XPB=&(9.;.9>=.8=-.,-1>=7*,:1&9*)='>=49-*7=TFIIH=8:':3.98_=5/,=

.39*7&(98=<.9-=XPB=).7*(91>=&3)= 89.2:1&9*8=.98=ATP&8*=&(9.;.9>= M*7.34`=M&))*3=*9=&1_=

+33-b= J&<-&7.`= L&.3*= *9= &1_= ,**,= <-.1*`= 522TTD8A= 89.2:1&9*8= .3).7*(91>= '>= .39*7&(9.3,=

<.9-=5/,_=I3=&)).9.43`=9-*=NER=)&2&,*=7*(4,3.9.43=+&(947=XPC8HR,-B=&184=89.2:1&9*8=

9-*= ATP&8*= &(9.;.9>= 4+= XPB= &3)= +&(.1.9&9*8= DNA= 45*3.3,= &74:3)= 9-*= 1*8.43= G*= &3)=

R4*)*7=+33._==

M:9&9.438= .3= XPB= ,*3*= 1*&)= 94= 9-*= 7&7*= -:2&3= ,*3*9.(= ).847)*78b= X*74)*72&=

5.,2*3948:2= XP`= (40&>3*= 8>3)742*= CS= &3)= 97.(-49-.4)>8974->= TTD= P-*349>5.(=

(-&7&(9*78=&7*=).8(:88*)=.3=C-&59*7=III_=U5=94=34<`=431>=8.==2:9&9.438=.3=8.==+&2.1.*8=

&7*=7*5479*)=S->0.3)`=K.2=*9=&1_=+33/_=T-*=9<4=2:9&9.438=.3=N89*72.3&1=)42&.3`=F33S=

&3)=T++3P=&7*= 9-*=2489=89:).*)=S**=R*8:19=8*(9.43`= M&3:8(7.59=F.,:7*=+_= T-*8*=9<4=

2:9&9.438=-&;*='**3=8-4<3=94=8-4<=1*88=RNA=8>39-*8.8=.3= .38;.974 =97&38(7.59.43=&88&>=

75 A XPB

B XPD

F'%30# WWT L',# 0 1!&#+ 2'!1 -$ XPB ," XPD &#*'! 1#1T

T&# 1#4#, &#*'! 1# +-2'$1 HDW ," HDX 0# !-++-, 2- -2& XPB ," XPDT T&# $-0 &#*'! 1# +-2'$1 ', HDW IQ I Q IIQ III 0# ',"'! 2#" ', 0#" ," 2&0## IVQ VQ VI ', HDX 0# ',"'! 2#" ', %0##,Q A D + %# 0#!-%,'2'-, "-+ ',Q DRD "-+ ', NV2#0+', * !*-1# 2- HDW '1 !-*-0#" .',)Q " 0) 0#" ," *3# 0#.0#1#,21 2&# RVEVD ," 2&3+ "-+ ', 0#1.#!2'4#*7Q B F#S "-+ ', ', XPD ', -0 ,%# ," A0!& '1 ', .30.*#T A" .2#" $0-+ F311 ," T ',#0 XVWWT

76 <-.1*= 9<4= 49-*7= 3438*38*= 2:9&9.438= 7*5479*)= '>= O-_= SK= *9_&1= 1*&)= 94= 57*2&9:7*=

9*72.3&9.43=4+=9-*=5749*.3=K&3,`=A:'1*=*9=&1_=+33/_=

WTX XPD

XPD2R&)-2ERCC,=.8=&=2*2'*7=4+=SF,=-*1.(&8*=+&2.1>=&3)=9-*=8*(43)=1&7,*89=2*2'*7=4+=

TFIIH_=I9=.8= !"=)*5*3)*39=/ 8-=-*1.(&8*=4+=!45 IH_=XPD=.8=-.,-1>=(438*7;*)=.3=&7(-*&=

&3)= *:0&7>49*8_= = T-*= (148*89= -42414,:*= 4+= XPD= .8= 9-*= '&(9*7.&1= )&2&,*8.3):(.'1*= G=

D.3=G_=I3=&)).9.43=.9=-&8=8*;*7&1=5&7&14,:*8=8:(-=&8=7*,:1&7=9*142*7*=1*3,9-=RTE1+`=

4&3(43.8=&3*2.&=(4251*2*39&9.43=,74:5= F&3(J=&3)=C-1+_=A11=4+=9-*8*=5749*.38=8-&7*8=

(438*7;*)= F*8S= (1:89*7= '.3).3,= )42&.3= &3)= +:3(9.438= &8= -*1.(&8*8= .3= ).++*7*39=

7*(42'.3&9.43=&3)=7*5&.7=574(*88*8=L.:`=R:)41+=*9=&1_=,**2_=C7>89&1=897:(9:7*=4+=&7(-&*1=

XPD=-&8=7*;*&1*)=9-&9=&5&79=+742=9<4=(&343.(&1=-*1.(&8*=)42&.38b=HD+=&3)=HD,`=XPD=

.8= (42548*)= 4+= .= F*8S= (1:89*7= )42&.38= .3;41;*)= .3= DNA= )&2&,*= 7*(4,3.9.43= &3)= &3=

A7(-=)42&.3=F.,:7*=++_=T4,*9-*7`=9-*=A7(-=&3)=.74388:1+:7=)42&.38=+472=&=(-&33*1=

9-74:,-=<-.(-=8.3,1*8897&3)=DNA=.8=)7&,,*)='>=9-*=&(9.43=4+=9-*=24947=)42&.38=.3=&=

(>(1.(&1=ATP8)*5*3)*39=7*&(9.43_=T-.8=A7(-=)42&.3=-&8='**3=7*(*391>=8-4<3=94=&(9=&8=&=

51&9+472=94=7*(7:.9=9-*=CAK`='49-=.3=97&38(7.59.43=&3)=DNA=7*5&.7=A'):17&-2&3`=I19.8=

*9=&1_=,*+-_=F:79-*7247*=Q.=*9_&1`=-&8=8-4<3=9-&9=24342*7.(=XPD=:3<.3)8=9-*=):51*==

DNA= .3= +8'5= 89*58`= >*9= .9= *=-.'.98= +7*6:*39= '&(089*58= &3)= :3)*7,4*8= (43+472&9.43&1=

97&38.9.438= 2&3.+*89*)= .3= /8'5= '&(0<&7)= &3)= +47<&7)8= 89*58_= T-*8*= &11= 2*(-&3.828=

)*5*3)8=43=9-*=&;&.1&'.1.9>=4+=ATP=&3)=+.3&11>=9-*=8.3,1*8'&8*=5&.7=89*55.3,=.3=+47<&7)=

).7*(9.43=,.;*=7.8*=94=9-*=:3<.3).3,=4+=9-*=):51*==DNA=Q.=,*+-_==

XPD=-&8=;*7>=.25479&39=741*=.3=9-*=897:(9:7&1=47,&3.?&9.43=4+=TFIIH`=&(9.3,=&8=&='7.),*=

'*9<**3= 9-*= (47*= &3)= 9-*= CAK_= I9= .39*7&(98= <.9-= M&9+= 4+= 9-*= CAK= 8:'8(4251*==

L&,7&3,*`=K&5&3.).8=*9=&1_=+332_=T-*=-*1.(&8*=&(9.;.9>=4+=XPD=<-.(-=.8=).85*38&'1*=+47=

97&38(7.59.43=.8=&'841:9*1>=7*6:.7*)=+47=DNA=:3<.3).3,=):7.3,=9-*=NER_=XPD=-*1.(&8*=

&(9.;.9>= .8= +:79-*7= 89.2:1&9*)= '>= 5..= 9-74:,-= .98= &884(.&9.43= <.9-= (&7'4=>189*72.3&1=

77 F'%30# WXT XPD ," '21 ',2#0 !2'-,1

A" .2#" $0-+ P3 M#" G#,# IDS XV\^

78 )42&.3= 4+= XPD= C4.3`= O08*3>(-= *9= &1_= ,**1_= XPD= &184= .39*7&(9= <.9-= 8*;*7&1= 49-*7=

5749*.38=&3)=(&3='*=+4:3)=.3=(4251*==<.9-=49-*7=5749*.3=F.,:7*=+,_=S*;*7&1=2:9&9.438=

.3= 9-*= XPD= ,*3*= &7*= 7*5479*)= 9.11= )&9*= <-.(-= ,.;*8= 7.8*= 94= XP`= TTD`= CS= &3)= XP2CS=

5-*349>5*=M:9&9.43=89:).*)=.3=57*8*39=89:)>=&7*=).8(:88*)=.3=)*9&.1=.3=C-&59*7=III=

WTY .\X

50,2T+'+=.8=9-*=897:(9:7&1=(42543*39=4+=TFIIH=(47*`=-.,-1>=(438*7;*)=):7.3,=*;41:9.43=

&3)=+.789=(143*)=.3=+33,='>=F.8-*7=*9_&1=L&:7*39=F.8(-*7=+33,_=50,2T+'+=+&(.1.9&9*8=9-*=

7*(7:.92*39=4+=TFIIH=94=9-*=97&38(7.59.43=57*.3.9.&9.43=(4251*==9-74:,-=&3=.39*7&(9.43=

'*9<**3= .98= &2.3489*72.3&1= PH= )42&.3= &3)= 9-*= &(.).(= (&7'4=>1= 9*72.3:8= 4+= TFIIE =

TFIIE --0 :.-3=X.&4`=P*&7843=*9=&1_=+33._=T-.8=.39*7&(9.43=2&70*)1>=89.2:1&9*8=TFIIH8

)*5*3)*39=5-485-47>1&9.43=4+=9-*=C89*72.3&1=)42&.3=4+=RNA=541= II_= I3=&)).9.43`=9-*=

50,2T+'+=8:':3.9=-&8='**3=8-4<3=94=.39*7&(9=<.9-=5/-=9-74:,-=.98=&(.).(=97&38&(9.;&9.43=

)42&.38= ,= TAD,_= T-*= .39*7&(9.43= '*9<**3= 5/-= TAD,= &3)= 50,2T+'+= .8= *3-&3(*)= '>=

5-485-47>1&9.43=4+=S*78.0=&3)=T-78//=4+=5/-_=T-.8=.39*7&(9.43=.8=9-4:,-9=94='*=.25479&39=

+47=7*,:1&9.43=4+=8*1*(9*)=5/-87*,:1&9*)=,*3*8=9-&9=7*6:.7*=5/-=TAD,`=8:(-=&8=MDM,`=

PUMA`= WAF+`= &3)= BAX+= Z:7.9&= &3)= M*7.34= ,**-_= NMR= 89:).*8= -&;*= 8-4<3= 9-&9=

9-*=&(.).(= TAD= 4+= EBNA,= E589*.3 :B&77= ;.7:8= 3:(1*&7= &39.,*3= ,= 7*8.):*8= .-+8.21=

.39*7&(9= <.9-= 9-*= T+'+250,= 8:':3.9= 4+= TFIIH= &3)= .8= 7*6:.7*)= +47= 9-*= 97&38&(9.;&9.43=

C-&'49`=R&.41&=*9=&1_=,*+._ P4.39=2:9&9.438=.3=9-*=&(.).(=TAD=4+=VP+0=9-&9=7*):(*=.98=

97&38&(9.;&9.43=&(9.;.9>=&184=<*&0*3=.98='.3).3,=94=TFIIH=<-.1*=2:9&9.43=.3=TAD=4+=E,F8

+=1*&)8=94=9-*=1488=4+=TFIIH='.3).3,=;.&=50,=&3)=-*3(*=7*8:198=.3=0*80/=7*):(9.43=.3=

97&38&(9.;&9.43_=I3=&)).9.43`=50,2T+'+=).7*(91>=.39*7&(98=<.9-=9->74.)=-47243*=7*(*5947`=

*3-&3(.3,= T-82*).&9*)= 97&38(7.59.43= B:71*>= &3)= R4*)*7= +330_= I3= DNA= 7*5&.7`=

50,2T+'+= .39*7&(98= <.9-= )&2&,*= 7*(4,3.9.43= (4251*== XPC2R&).`= &8= <*11= &8= <.9-=

897:(9:7*885*(.+.(=*3)43:(1*&8*=XPG2R&),= 9-&9=(1*&;*8=>? =&9=9-*=-=*3)=4+= 9-*=1*8.43`=

I>*7`=R*&,&3=*9=&1_=+330b=Y404.`=M&8:9&3.=*9=&1_=,***_=I3=5&79.(:1&7`=R&).='.3)8=<.9-=9-*=

79 PH=)42&.3=4+=50,2T+'+=94=+472=R&).8T+'+PH=(4251*==<-.(-=-&8=.39*7+&(*=8.2.1&7=&8=94=

43*=:8*)='>=R&),=94='.3)=T+'+PH_=S4`=R&).=(425*9*8=<.9-=R&),=+47='.3).3,=94=9-*=T+'+=

8:':3.9= 4+= TFIIH= .3= NER= L&+7&3(*8V&3&88*`= A78*3*&:19= *9= &1_= ,*+-_= T-*7*= &7*= 34=

)*8(7.'*)=2:9&9.438=.3=-:2&3=50,=9-&9=1*&)8=94=&3>=DNA=7*5&.7=).847)*78`=-4<*;*7=9-*=

(477*1&9.43= '*9<**3= 50,= 8.3,1*= 3:(1*49.)*= 541>2475-.828= SNP= &3)= 9-*= 1:3,= (&3(*7=

7.80=-&;*='**3=7*5479*)=W:`=L.:=*9=&1_=,**3_=

WTZ .[X

5/,2T+',= .8= &= 3438*3?>2&9.(= (42543*39= 4+= TFIIH= &3)= +.789= (485:7.+.*)= &3)= (48

57*(.5.9&9*)=&8=+.+9-=8:':3.9=<.9-=*.,-9=49-*7=TFIIH=8:':3.98=X.*`=K40:'4=*9=&1_=+330_=

5/,2T+',= 5749*.3= 5->8.(&11>= .39*7&(98= <.9-= XPB= &3)= 89.2:1&9*8= .98= ATP&8*= &(9.;.9>=

):7.3,=97&38(7.59.43=&3)=DNA=7*5&.7_=T-*=(4251*==5-.25..250,=T+'.2S81+2T+'+=)4*8=

349= .39*7&(9= <.9-= XPB= .3= 9-*= &'8*3(*= 4+= 5/,= T+',= &3)= 9-*= )*51*9.43= 4+= 5/,= 1*&)8= 94=

)*+*(9= .3= DNA= 45*3.3,= ):7.3,= 97&38(7.59.43= .3.9.&9.43= O75-&3.)*8`= L&,7&3,*= *9= &1_=

+330b=F*&;*7`=H*37>=*9=&1_=+331b=J&<-&7.`=L&.3*=*9=&1_=,**,_=T.11=)&9*`=34=-:2&3=,*3*9.(=

).847)*78= &7*= 034<3= ):*= 94= 9-*= 2:9&9.43= .3= 5/,= ,*3*_= H4<*;*7`= 2:9&9.43= .3=

5/,2 D25/,= .3= D74845-.1& =.8=7*5479*)=<-.(-=.8=034<3=94=(&:8*=3*:7414,.(&1=)*+*(98`=UV8

8*38.9.;.9>= &3)= +*&9:7*8= 8.2.1&7= 94= TTD= 5&9.*398= F7*,484`= L&.3*= *9= &1_= ,**1_= T-*8*=

2:9&9.43=247*=1.0*1>=.25&.7*)=5/,8522TTD8A=.39*7&(9.43`=&8=4;*7*=57*88.43=4+=522TTD8

A=*3-&3(*8=UV8.77&).&9.43=7*8.89&3(*=&3)=8:557*88*8=TFIIH=2:9&9.438=.3=&= D74845-.1&=

TTD=24)*1=A,:.1&78F:*39*8`=F7*,484=*9=&1_=,**2_=F:79-*7247*`=5/,=-&8='**3=8-4<3=94=

'*=&=97&38.*39=(42543*39=4+=9-*=(&9&1>9.(=8.9*=4+=-:2&3=2.94(-43)7.&1=RNA=541>2*7&8*=

2.94RNAP=&3)=&184=.251.(&9*)=.3=574249*7=2*19.3,=S414,:'`=L.943.3=*9=&1_=,**3_==

WT[ .ZZ

T-*=,*3*=*3(4).3,=5..=.8=&=-:2&3=(4:39*75&79=4+=S81+=.3=>*&89=&3)=-&8=&=RING=+.3,*7=

)42&.3= <-.(-= .8= -.,-1>= (438*7;*)= &3)= .39*7&(98= <.9-= XPD= :543= DNA= '.3).3,_= T-.8=

'.3).3,=89.2:1&9*8=9-*=-*1.(&8*=&(9.;.9>=4+=XPD= .38;.974 =F147*8`=H&=*9=&1_=+33*b=T&3&0&`=

80 W&9&3&'*=*9=&1_=,**3_=T-*=NMR=897:(9:7*=4+=5..=RING=+.3,*7=)42&.3=7*8.):*8=-,+=94=

-3/=8-4<8=9<4=?.3(='.3).3,=8.9*8=(447).3&9*)='>=*.,-9=(>89*.3*=7*8.):*8=K*11*3'*7,*7`=

D42.3,:*?=*9=&1_=,**/_=I39*7*89.3,1>=9-*8*=(438*7;*)=(>89*.3*=7*8.):*8=&7*=&184=+4:3)=

.3= 9-*= RING= +.3,*7= )42&.3= 4+= S81+= &3)= &7*= (7.9.(&1= +47= 9-*= E-= 1.,&8*= &(9.;.9>= .3= >*&89_=

M:9&9.43=.3=9-*=+.789=9<4=(>89*.3*=7*8.):*8=.3=>*&89=C.*-A=&3)=C.*0A=&'41.8-*8=S81+=

*3?>2&9.(=&(9.;.9>=.3=&3= .38;.974 =541>:'.6:.9.3&9.43=&88&>=5*7+472*)=.3=9-*=57*8*3(*=4+=

E+= &3)= E,= U'(.= *3?>2*8= &3)= +:79-*7= 7*):(*)= 9-*= >*&89= 8:7;.;&1= 7&9*= +4114<.3,= UV=

.77&).&9.43= 47= 2*9->1= 2*9-&3*8:1+43&9*= MMS= 97*&92*39_= T-*= E-= :'.6:.9.3= 1.,&8*=

&(9.;.9>=.8=+:79-*7=*3-&3(*)='>=&349-*7=8:':3.9=5-.=4+=TFIIH_=A2.34=&(.)=7*8.):*8=+742=

00=94=,**=&7*=.3;41;*)=.3=9-*=.39*7&(9.43=<.9-=XPD`=8.3(*=2:9&9.438='*9<**3=9-.8=7*,.43=

-&;*='**3=8-4<3=94=)*(7*&8*=9-*=-*1.(&8*=&(9.;.9>=&3)=+:79-*7=1*&)8=94=9-*=)*+*(9==.3=9-*=

+.789=89*5=4+=9-*=97&38(7.59.43=7*&(9.43`=._*_=9-*=+.789=5-485-4).*89*7='43)=+472&9.43=&3)=

574249*7= (1*&7&3(*= S*74?`= P*7*?= *9= &1_= ,***_= T-*= 5..= 2RNA= -&8= '**3= 8-4<3= 94= '*=

7*,:1&9*)= '>= 2.(74RNA`= 2.R8,1&_= M47*4;*7`= 2.R8,1&= )*89&'.1.?*8= 9-*= 5..= 8:':3.9= 4+=

9-*= TFIIH= (4251*== ):7.3,= 9-*= G,8M= 5-&8*`= 9-:8= 24):1&9.3,= 9-*= 97&38(7.59.43&1=

8-:9)4<3=4'8*7;*)=):7.3,=9-.8=97&38.9.43=P479&1=,*++_=5..=,*3*=-&8='**3=)*8(7.'*)=.3=

(439*=9=94=9-*=85.3&1=(47)=2:8(:1&7=&9745-.*8_=5..=,*3*=.8=5&79=4+=9-*=/**=0'=.3;*79*)=

):51.(&9.43=43=(-7424842*=/6+-_=O3*=(45>=4+=5..=,*3*=.8=<.9-.3=9-*=9*142*7.(=7*,.43=

<-.1*= 9-*= 49-*7= 7*8.)*8= .3= 9-*= (*39742*7.(= 7*,.43_= D*1*9.43= 4+= 9-.8= ,*3*= .3= 9*142*7.(=

7*,.43=842*9.2*8=&((425&3.*8=)*1*9.43=4+=9-*=3*.,-'47.3,=SMN+=,*3*=7*8:19.3,=.3=9-*=

85.3&1= 2:8(:1&7= &9745->= SMA= 5-*349>5*= ':9= .9= .8= :3(1*&7= .+= )*1*9.43= 4+= 5..= ,*3*=

(4397.':9*8=94=9-*=SMA=5-*349>5*=A7(-&2'&:19`=P&3=*9=&1_=+332_=

WT\ .YZ

5-.=4+=-:2&3=TFIIH=.8=-42414,4:8=94=T+'.=4+=>*&89=&3)=(439&.38=&=?.3(=+.3,*7=)42&.3=

8.2.1&7=94=5..=Y43&-&`=T8:(-.>&=*9=&1_=+331_=I9=-&8='**3=8-4<3=94='*=.25479&39=+47='49-=

97&38(7.59.43= &3)= DNA= 7*5&.7`= 9-4:,-= .9= .8= 349= 034<3= +47= &3>= *3?>2&9.(= &(9.;.9>=

81 F*&;*7`= H*37>= *9= &1_= +331_= R*(*391>`= 9-*= (7>89&1= 897:(9:7*= 4+= 9-*= 5-.= 8:':3.9= 4+= 9-*=

TFIIH=(4251*==+742=9-*=*:0&7>49.(=9-*7245-.1.(=+:3,:8= C-&*942.:2=9-*7245-.1:2 =-&8=

'**3=7*;*&1*)=S(-2.99`=K:5*7=*9=&1_=,*+._=I9=8-4<8=9-&9=5-.=(439&.38=&= ;43=W.11*'7&3)=

F&(947= A= ;WA= 1.0*= +41)= <-.(-= .8= ,*3*7&11>= 034<3= 94= '*= .3;41;*)= .3= 5749*.385749*.3=

.39*7&(9.438_=W.9-.3=TFIIH=5-.=89743,1>=.39*7&(98=<.9-=5..`=&=548.9.;*=7*,:1&947=4+=9-*=

-*1.(&8*=XPD_==

WT] C")]

C)021MO+/2K.3=,2=.8=&=(&9&1>9.(=8:':3.9=4+=CAK=&3)=.8=&=2*2'*7=4+=S*7.3*29-7*43.3*=

5749*.3=0.3&8*=+&2.1>_=CDK=1=(&3=*=*(:9*=):&1=+:3(9.43='>=7*(4,3.?.3,=).++*7*39=(1&88*8=

4+=8:'897&9*_=I3=2*9&?4&38`=CDK1=<&8=.3.9.&11>=.)*39.+.*)=&8=9-*=CDK8&(9.;&9.3,=0.3&8*=

CAK`=<-.(-=5-485-47>1&9*8=(*118(>(1*=CDK8=<.9-.3=9-*=&(9.;&9.43=8*,2*39=T81445_==I9=

+4728= &= -*9*7497.2*7.(= (4251*== <.9-= 9-*= 7.3,= +.3,*7= 5749*.3= M&9+= <.9-.3= 9-*= 2:19.8

(4251*==TFIIH=&3)=&184=5-485-47>1&9*8=9-*=CTD=4+=9-*=1&7,*89=8:':3.9=4+=RNA=541=II_=

T-*= ).++*7*3(*= '*9<**3= CTD81.0*= &3)= T81445= 8:'897&9*8= .8= &184= 8**3= .3= 9-*= &'.1.9>= 4+=

C)01=94=5-485-47>1&9*=5*59.)*8=.3=841:9.43_=CTD=5*59.)*8=&7*=*++*(9.;*=8:'897&9*8= +47=

C)01`= <-*7*&8= T81445= 8:'897&9*8= &7*= 349_= H:2&3= C)01= (&3= 5-485-47>1&9*8= 9-*= S*7= /=

&3)=.9=<&8=+.789=8-4<3='>=R4>=*9=&1_=9-&9=2&22&1.&3=C)01=(&3=5-485-47>1&9*8=&=5*59.)*=

<.9-= A1&3.3*,= 8:'89.9:9.43`= <-.1*= .9= (&3349= 5-485-47>1&9*8= &= 8.2.1&7= 5*59.)*= <.9-=

A1&3.3*= R4>`= A)&2(?*<80.= *9= &1_= +33._= C)01= 0.3&8*= &(9.;.9>= .8= 7*6:.7*)= +47= 9-*=

97&38(7.59.43= .3.9.&9.43= A04:1.9(-*;`= M&0*1&= *9= &1_= +33/= &3)= 9-.8= 0.3&8*= &(9.;.9>= .8=

89.2:1&9*)= '>= 9-*= 2*).&947= (4251*== 9-&9= '.3)8= 94= 9-*= :35-485-47>1&9*)= +472= 4+= 9-*=

CTD= &3)= -*158= 94= 7*(7:.9= RNA= 541= II= 94= 9-*= 57*.3.9.&9.43= (4251*== PIC_=

P-485-47>1&9.43= 4+= S*7/= &3)= S*71= 7*8.):*8= .3= 9-*= CTD= ).87:598= 9-.8= .39*7&(9.43=

5*72.99.3,= 574249*7= *8(&5*= &3)= *397>= .394= 9-*= *143,&9.43= 5-&8*= 4+= 9-*= 97&38(7.59.43=

(>(1*=K.2`=S:-=*9=&1_=,**3_=H4<*;*7`=7*(*391>=.9=-&8='**3=8-4<3=.3=>*&89=9-&9=9-*=K.3,2=

C)K1= -42414,:*= .3= >*&89`= .8= .25479&39= +47= 574249*7= *8(&5*= ':9= 349= +47= *143,&9.43_=

82 P-485-47>1&9.43= 4+= CTD= (&:8*8= ).884(.&9.43= 4+= 2*).&947= &3)= 8:'8*6:*391>= 574249*7=

*8(&5*`= <-*7*&8= .98= )*51*9.43= )7&2&9.(&11>= .3(7*&8*8= 2*).&947= 4((:5&3(>= &9= 9-*= (47*=

574249*7= W43,`= J.3= *9= &1_= ,*+._ = A19-4:,-= C)01= .8= 034<3= 94= (439741= 9-*= 47,&3.82&1=

;.&'.1.9>`=).88*(9.3,=.98=5->8.414,.(&1=741*=.3=97&38(7.59.43=&3)=(*11=(>(1*=(439741=8**28=94=

'*=94:,-_=F47=*=&251*`=1488=4+=C)01=*++*(98='49-=97&38(7.59.43=&3)=2.948.8=.3= C_=*1*,&38 =

L:8*`=S5&3,1*7=*9=&1_=,*++`=<-.1*=&):198= D74845-.1& =<.9-=-424?>,4:8=2:9&9.43=.3=C)01=

&7*= ;.&'1*= +47= 4;*7= .*= )&>8= &9= 9-*= 34385*72.88.;*= 9*25*7&9:7*= &7,:.3,= &,&.389= &3=

*88*39.&1= 741*= .3= 97&38(7.59.43_= I9= -&8= '**3= 8-4<3= 9-&9= 9-*= *++*(9= 43= RNA= 541= II=

97&38(7.59.43=.8=2.3.8(:1*=*;*3=9-4:,-=2.(*=-424?>,4:8=+47=&=C)01=)*1*9.43=).*=*&71>=

.3=*2'7>4,*3*8.8=G&3:?&=,*+,_=T.88:*=85*(.+.(=034(04:98=4+=C)01=.3=&):19=2.(*=(&:8*=

9-*= 1488= 4+= 5741.+*7&9.3,= (*118= ':9= 1.991*= (-&3,*= .3= 34385741.+*7&9.3,= (*118_= T-.8= 7*8:19=

&7,:*8= 9-&9= TFIIH= 0.3&8*= .8= 349= 7*6:.7*)= +47= 97&38(7.59.43= .3= 548982.949.(= (*118_= A11=

94,*9-*7=9-*8*=89:).*8=.3).(&9*=9-&9=C)01=.8=349=*88*39.&1=+47=97&38(7.59.43=.3=&11=9-*=(*118=

.8= 4'8*7;*)_= I3= 8:55479= 4+= 9-.8= ->549-*8.8= <*= (&3= &88:2*= 9-&9= 9-*7*= 2.,-9= '*= 49-*7=

CDK8=<-.(-=(&3=574;.)*=9-*=2.88.3,=CTD=0.3&8*=&(9.;.9>_=

WT^ C7*', H

C>(1.38=&7*=9-*=(438*7;*)=5749*.38=&3)=&':3)&39=):7.3,=9-*=(*11=(>(1*_=T-*>=+:3(9.43=&8=

9-*=7*,:1&9478=4+=CDK8=&3)=+:3(9.438=):7.3,=9<4=574(*88*8`=97&38(7.59.43=&3)=(*11=(>(1*_=

C>(1.3= H= +4728= &= (4251*== <.9-= CDK1= 0.3&8*= &3)= 7.3,= +.3,*7= 5749*.3= MAT+_= C>(1.38

CDK= &884(.&9.43= *3&'1*8= 8:'897&9*= 5-485-47>1&9.43_= F:79-*7247*`= (>(1.3= H= -&8= &3=

*88*39.&1= +:3(9.43= .3= 574249.3,= 9-*= 8*1+87*3*<&1= 4+= 9-*= 51:7.549*39= 89*2= (*118= 4+=

'1&894(>89=89&,*=*2'7>48=8:,,*89.3,=9-*=741*=4+=(>(1.3=H=.3=2&.39&.3.3,=ES=(*11=.)*39.9>=

&3)= .3= *&71>= *2'7>43.(= )*;*1452*39= P&9*1= &3)= S.243= ,*+*_= A184= 9-*= TFIIH= 0.3&8*=

&(9.;.9>=.8=&++*(9*)='>=9-*=.39*7&(9.43=4+=9-*=U+=83RNA=&3)=9-*=(>(1.3=H=B:7&94<80.`=

H&-3=*9=&1_=+323_=C>(1.3=H=.8=&184=5-485-47>1&9*)='>=9-*=C)02=8:':3.9=4+=9-*=2*).&947=

83 (4251*== A04:1.9(-*;`= C-:.04;= *9= &1_= ,***_= T-.8= 5-485-47>1&9.43= 7*57*88*8= '49-= 9-*=

&'.1.9>=4+=TFIIH=94=&(9.;&9*=97&38(7.59.43=&3)=.98=CTD=0.3&8*=&(9.;.9>_==

WT_ M 2W

!-*=@ !+2!+'-=5749*.3=89&3)8=+47= 2S3&,*=A=974.8 =.8=&=9-.7)=(42543*39=4+=F G_=!-.8=

.8= &= RING= +.3,*7= (439&.3.3,= 5749*.3= &3)= 51&>8= &= 897:(9:7&1= 741*= .3= 89&'.1.?.3,= 9-*=

(4251*=_=T-*=(47*=4+=9-*=241*(:1*=(438.898=4+=9<4=7*5*&98=(439&.3.3,=+.;*=-*1.(*8=*&(-=

&3)= +472.3,= 9-*= (&343.(&1= (>(1.3= +41)8= 8.2.1&7= 94= TFIIB_= O:9= 4+= 9-*8*= +.;*= 9<4= -*1.(*8=

)*+.3*= 85*(.+.(.9>= 4+= 9-*= (>(1.3= H= 241*(:1*_= T-*8*= 9<4= 143,= -*1.(*8= *=9*3)8= 9-*= (>(1.3=

+41)= &9= .98= N8= &3)= C89*72.3.= &3)= 5&(0= 94,*9-*7= &,&.389= 9-*= +.789= 7*5*&9= 43= 9-*= 8.)*=

45548.9*=94=9-*=0.3&8*_=D*1*9.43=2:9&398=8-4<=9-&9=9-*8*=9*72.3&1=-*1.(*8=&7*=7*6:.7*)=

+47=&=+:3(9.43&11>=&(9.;*=(>(1.3=H=K7&*2*7`=R&3&114=*9=&1_=,**+_=T-*=(4.1*)8(4.1=&3)=9-*=

->)745-4'.(= )42&.38= 4+= MAT+= .39*7&(9= <.9-= 9-*= N89*72.3&1= )42&.3= 4+= XPD= &3)=

57*;*39=.98=-*1.(&8*=&(9.;.9>`=&19-4:,-=9-.8=.3-.'.9.43=.3=9-*=-*1.(&8*=&(9.;.9>=.8=4;*7(42*=

'>=XPD=<-*3=5..='.3)8=94=.9=S&3)74(0=&3)=E,1>=,**+_==

WTWV .^ TTDVA

522TTD8A2= T+'/= .8= &= 1+= &2.34= &(.)8= 82&11`= = 5749*.3= ':.19= &74:3)= &3= &39.5&7&11*1= 8

8-**9= 9-&9= +4728= &= -424).2*7= <.9-= &3= *=9*3)*)= .39*7+&(*= R4*)*7= +330_= I9= 57*8*398=

:3:8:&1= 897*9(-*8= 4+= (438*7;*)= &3)= ->)745-4'.(= 7*8.):*8= &3)= .8= &12489= (438*7;*)=

&243,=&11=*:0&7>49.(=,*342*8=+742=-:2&3=94=>*&89_=I3.9.&11>`=.9=<&8=8-4<3=94='*=&= '43&=

+.)* =(42543*39=4+=RNA=541=II=57*.3.9.&9.43=(4251*==&3)=&=(42543*39=4+=TFIIH=<-.(-=.8=

7*6:.7*)=+47=*++.(.*39=97&38(7.59.43='49-= .3=;.974 =&3)= .3=;.;4 =R&3.8-`=H&-3=*9=&1_=,**._=

L&9*7= .9= <&8= 8-4<3= 94= '*= &= DNA= 7*5&.7= 85*(.+.(= +&(947= C-&4`= G&)'4.8= *9= &1_= +330_=

F:79-*7`=.9=<&8=8-4<3=94='*=&=NER8)*).(&9*)=8:':3.9=4+=TFIIH='>=G.,1.&8M&7.=*9=&1_=.3=

+.'74'1&89=(*118=G.,1.&8M&7.`=M.6:*1=*9=&1_=,**0_=I9=89.2:1&9*8=9-*=ATP&8*=&(9.;.9>=4+=XPB=

94,*9-*7= <.9-= 9-*= 5/,= &3)= NER= +&(947= XPC8HR,-B_= I9= .39*7&(98= <.9-= 9-*= ->)745-4'.(=

8:7+&(*= 4+= 5/,= &3)= 8**2.3,1>= 5749*(98= 5/,= +742= 841;*398_= S97:(9:7&1= 89:).*8= 43= >*&89=

522TTD8A=&3)=5/,=-&;*=8-4<3=9-&9=9-*=C89*72.3&1=4+=5/,=&3(-478=522TTD8A=&3)=+4728=

&=-*9*74).2*7=C4.3`=B*7,2&33=*9=&1_=+333b=K&.34;`=C:7&=*9=&1_=,*+*_=T-*=522TTDA :5/,=

.39*7&(9.43= <&8= +:79-*7= (43+.72*)= .38;.;4= N433*0*38`= C&'&394:8= *9= &1_= ,*+-_= T-.8=

.39*7&(9.43= 8:'8*6:*391>= &114<8= 9-*= NER= +&(9478= 94= 14(&1.?*= 94= 9-*= )&2&,*= T-*.1`=

N433*0*38=*9=&1_=,*++_==

M:9&9.43= .3= 522TTD8A= 8:':3.9= ,.;*8= 7.8*= 94= 7&7*= +472= 4+= TTD= 5-*349>5*_= T-*= +.789=

7*5479=4+=&=TTD=5&9.*398=):*=94=2:9&9.43=.3=TTD=<&8=2&)*=143,='&(0=.3=+33-=&3)=.9=<&8=

9-4:,-9= 94= '*= TTD= 5-*349>5*= ):*= 94= &= 3*<= *=(.8.4387*5&.7= (4251*2*39&9.43= ,74:5=

S9*+&3.3.`= V*72*:1*3= *9= &1_= +33-_= A+9*7= 8*;*7&1= >*&78= .9= <&8= 8-4<3= 9-&9= 9-*= 5749*.3=

(&:8.3,=9-*=7&7*=+472=4+=TTD=.8=.3)**)=522TTD8A=S-.1&9.+&7)`=C43&<&>=*9=&1_=,**-_=

85 BT NER F !2-01

T-*=NER=+&(9478=&7*=9-*=,74:5=4+=5749*.38=<-.(-=&7*=034<3=94='*=.3;41;*)=349=431>=.3=

NER=5&9-<&>8=':9=&184=.3=8*;*7&1=DNA=7*5&.7=5&9-<&>8_=M:9&9.43=.3=+-=,*3*8=4:9=4+=

-*`= <-.(-= (&77>= 4:9= 9-*= 3:(1*49.)*= *=(.8.43= 7*5&.7= 574(*88`= (&:8*8= -:2&3= ,*3*9.(=

).847)*78= 1.0*= X*74)*72&= 5.,2*3948:2 `= C4(0&>3*= 8>3)742*= &3)= 97.(-49-.4)>89745->_=

T-*8*=5749*.38=.3(1:)*=9-*==*74)*72&=5.,2*3948:2=(4251*2*39&9.43=,74:5=5749*.38b=

9-*=XP=5749*.38=XPA= :=XPG`=XP=5749*.3=;&7.&39=XPV`=522TTD8A`=CSA=&3)=CSB_=B&8*)=

43= 9-*.7= +:3(9.43&1= &85*(98= .3= 9-*= NER= 574(*88`= XP= 5749*.38= .251.(&9*)= .3= NER=

5&9-<&>8=(&3='*='74&)1>=).;.)*)=.394=9-7**=,74:58_=XPA`=XPC`=XPE`=CSA=&3)=CSB=&7*=

9-*=.3.9.&1=5749*.38=9-&9=&7*=7*6:.7*)=+47=8*38.3,=DNA=)&2&,*=&3)=.3.9.&9.3,=9-*=7*5&.7=

574(*88_=XPB=&3)=XPD=&7*=7*85438.'1*=+47=9-*=45*3.3,=4+=9-*=DNA=897&3)=8:774:3).3,=

DNA=1*8.438=):7.3,=NER_=XPG=&3)=XPF=&7*=9-*=*3)43:(1*&8*8=9-&9=5*7+472=9-*=):&1=

.3(.8.438=94=7*1*&8*=9-*=)&2&,*)=897&3)=&3)=&114<=7*8>39-*8.8=:8.3,=9-*=343)&2&,*)=

897&3)=&8=&=9*251&9*=9-*.7=741*=.3=NER=5&9-<&>8=.8=)*8(7.'*)=.3=)*9&.1=.3=3*=9=8*(9.43_=

T-*=8*6:*39.&1=&88*2'1>=4+=9-*8*=5749*.38=43=9-*=)&2&,*)=DNA=<&8=;*7>=<*11=89:).*)=

'>=R.*)1=*9=&1=R.*)1=,**-_=T-*.7=741*8=.3=NER=5&9-<&>=-&;*='**3=;*7>=<*11=&((*59*)_=

H4<*;*7`=9-*8*=5749*.38=&7*=349=431>=7*6:.7*)=.3=DNA=7*5&.7`=9-*>=-&;*=.3)**)=2&3>=

4;*71&55.3,=741*8=.3=DNA=2*9&'41.(=574(*88*8`=(*11=(>(1*=7*,:1&9.43=&3)=97&38(7.59.43=

T&'1*= 0_=T-*.7= *=.89*3(*= .3= 97&38(7.59.43= <&8= 5:9=+47<&7)= 57*(.8*1>= .3= ,**3= B&77*94`=

S(-&+*7=*9=&1_=,**1b=S(-2.9?`=S(-2.99=*9=&1_=,**3b=L*=M&>`=M49&8F*73&3)*8=*9=&1_=,*+*=&3)=

9-*.7=&);*39=.3=97&38(7.59.43=<&8=8-4<3=94='*=&=5&7&).,2=8-.+9=.3=9-*=:3)*789&3).3,=4+=

9-*= -:2&3= ,*3*9.(= ).847)*78= 1.0*= XP`= CS= &3)= TTD_= T-*8*= 89:).*8= -&;*= (1*&71>= 8-4<3=

9-&9= 9-*= NER= +&(9478= &7*= 7*(7:.9*)= 8*6:*39.&11>= 43= 9-*= 574249*7= 4+= &(9.;*= ,*3*_= XPC=

8**28=94='*=9-*=+.789=NER=+&(947=94='.3)=94=&(9.;*=574249*7=8.2.1&7=94=.98=7*(7:.92*39=.3=

NER= &3)= 9-*3= &114<8= 9-*= 7*(7:.92*39= 4+= 49-*7= NER= +&(9478= 8:(-= &8= CSB`= RPA`= XPA`=

XPG=&3)=XPF=):7.3,=97&38(7.59.43_=D:7.3,=NER`=XPC=+:3(9.43=&8=9-*=)&2&,*=8*3847=

86 &3)=&114<8=TFIIH=94=(42*=&9=9-*=)&2&,*=8.9*=43(*=.9=7*(4,3.?*8=9-*=1*8.43`=+4114<*)='>=

49-*7=NER=+&(9478=<-.(-=(4251*9*=9-*=DNA=7*5&.7=574(*88_=D:7.3,=97&38(7.59.43`=XPG=

&3)=XPF=&7*=8-4<3=94='*=7*6:.7*)=+47=9-*=DNA='7*&0=+472&9.43=&9=9-*=&(9.;*=574249*7`=

+:79-*7= &114<.3,= (-742&9.3= 7*24)*1.3,= 94= 4((:7= &3)= *38:7*= &((:7&9*= 97&38(7.59.43_=

O9-*7= NER= 5749*.38= 8:(-= &8= CSB= &3)= CSA= &7*= &184= 034<3= 94= +:3(9.43= &8= (-742&9.3=

7*24)*1*78= F4:89*7.`= V*72*:1*3= *9= &1_= ,**0_= I3= 5&79.(:1&7= CSB= 7*,:1&9*8= 9-*=

97&38(7.59.43&1= 574,7&2= &+9*7= UV= .77&).&9.43= P74.*99.8D*8S&39.8`= D7&3*= *9= &1_= ,**0_=

H4<*;*7`=9-*7*=.8=&=3**)=94='*=*=5147*)=247*=.3=47)*7=94=+:11>=:3)*789&3)=9-*=741*=4+=&11=

9-*=NER=+&(9478=):7.3,=97&38(7.59.43_=T&'1*=0`=7*57*8*398=9-*=.39*7&(9.3,=5&793*78=4+=&11=

9-*=NER=+&(9478=.3=2&3>=574(*88*8=49-*7=9-&3=97&38(7.59.43=&3)=DNA=7*5&.7_=

87 P`Q V1J F%JH 1QJs IJ V`:H 1J$ ]:` JV`s P`QHVss Q` IJ0QC0VIVJ PA D:I:$V ATR N%HCV:` 1I]Q` Q` PA 1J `Vs]QJsV Q sVJsQ` RPA5 PF5 TFIIH NER AB MQR%C: V .V J%HCV:` 1I]Q` Q` PA AB SH:``QCR ]`Q V1J 1J `:JsH`1] 1QJ :JR HVCC H7HCV DDB DDBRIVR1: VR NER PB ; R ;#$%&# PPAR`VC: VR @1J:sV P.Qs].Q`7C: Vs PB RV]VJRVJ P RVR%HV .V `: V Q` :]Q] Qs1s .VC1H:sV SG MQR%C: Vs .V RV$`:R: 1QJ Q` RAD TFIIE T`:JsH`1] 1QJ PC5 PG T`:JsH`1] 1QJ :JR NER ] 5 ] 5 ] PC D:I:$V ATM CVCC H7HCV HQJ `QC :JR `VIQ0V CVs1QJs sVJsQ` CVJ `1J D:I:$V `VHQ$J1 1QJ SMO5 PB5 ] 5 PE5 NER PG TDG BER S :R ]`Q V:sQIV DV$`:R: 1QJ G7 .V S ]`Q V:sQIV ]Qs HQI]CV6 PD ; R ;$%&# .MMS 5 ]5 MAT NER RV]VJRVJ ] RVR%HV .V `: V Q` :]Q] Qs1s .VC1H:sV MMD C.`QIQsQIV sV$`V$: 1QJ PE D:I:$V HRABL 7`Qs1JV @1J:sV DV$`:R: 1QJ G7 .V S ]`Q V:sQIV ]Qs sVJsQ` C%CRCOP T`:JsH`1] 1QJ:C `V]`Vss1QJ PC NER STAGA 5 CBPL] C.`QI: 1J `VIQRVC1J$ `Q` DNA `V]:1` EF CVCC H7HCV :``Vs PF EJRQR (&)) #5,$-. RA5Ms. M1sI: H. `V]:1` J%HCV:sV RAD L RPA IJ V`RH`Qss C1J@VR `V]:1` PALERCC NER TRF FQ`Is : V`J:`7 HQI]CV6 R%`1J$ NER TFIID RV$%C: Vs VCQIV`V 1J V$`1 7 T`:JsH`1] 1QJ ^:_ PG EJRQR N . BER J%HCV:sV PCNA DNA `V]C1H: 1QJ RNA ]QC II5 RPALPA T`:JsH`1] 1QJ5 NER PB5 ] P R`:I1C7 DNA PCNA5 R:R 5 RV0 ACCQ1 `:JsRCVs1QJ s7J .Vs1s ]:s .V R:I:$V ]QC7IV`:sV R:R RVHQIG1J: 1QJ :JR RQ%GCV s `:JR G`V:@ `V]:1` CSA G1_%1 1J AB TCR :JR `:JsH`1] 1QJ C1$:sV RNA ]QC II5 CSB TCR ]P T`:JsH`1] 1QJ G7 RNA ]QC I :JR II DDB5 C%CA5 G1_%1 1J C1$:sV HQI]CV6 HQI]QJVJ 5 `Vs]QJRs Q RQH5COP TQ]Q1sQIV`:sV I Ass1s 1J RsDNA %J11JR1J$ G7 H% 1J$ QJV s `:JR :JR

88 C: V` `V:JJV:C1J$

CSB DNAR CSA TCR RV]VJRVJ PB :JR PD T`:JsH`1] 1QJ G7 RNA ]QC I5 II :JR NER5 .VC1H:sV :H 101 7 ATP:sV Q` ] :JR ] T`:JsH`1] 1QJ G7 RNA ]QC I5 II :JR NER SILSNF RNA ]QC II T`:JsH`1] 1QJ Q` IRNA `:I1C7 RNA ]QC I5 III T`:JsH`1] 1QJ Q` `RNA OGG IJ BER PARPR M:1J :1J .V V`QH.`QI: 1J :JR `V]:1` Q61R: 10V R:I:$V ] CVCC H7HCV HQJ `QC5 GR:``Vs 5 NER :JR :]Q] Qs1s PG NER5 BER PA NER ] LTTDRA II]C1H: VR 1J PB5 ] NER NER

T *# XT NER $ !2-01 ," 2&#'0 ',2#0 !2'-, . 02,#01 A" .2#" $0-+ --) !& .2#01 7 S&#** M #2 *T TRRZ ," S3, G #2 *T TRR[Q H'0-1# ," M ,*#7 TRRR

CT TFIIHS B0'"%',% 20 ,1!0'.2'-, ," 0#. '0

TFIIH=-&8='**3=+.789=.)*39.+.*)=&3)=(-&7&(9*7.?*)=&8=&3=*88*39.&1=(42543*39=4+=RNA=541=

II=2*).&9*)=97&38(7.59.43=2&(-.3*7>`=C43&<&>=&3)=C43&<&>=+323b=G*7&7)`=F.8(-*7=*9=

&1_=+33+_=L&9*7=.3= ,**,`=.9=<&8= 8-4<3=94='*=.3;41;*)=.3= 9-*=97&38(7.59.43= 4+= 7RNA='>=

RNA= 541= I= H44,897&9*3`= N.,,= *9= &1_= ,**,= &3)= 8:'8*6:*391>= .3= 9-*= 97&38(7.59.43= 4+=

343(4).3,=RNA8='>=RNA=541=III=W7&3,*`=O07*9=*9=&1_=+32._=N43*9-*1*88`=2489=4+=9-*=

89:).*8=43=9-*=741*=4+=TFIIH=.3=97&38(7.59.43=<*7*=+4(:8*)=43=RNA=541=II=8>89*2_=TFIIH=

.8=9-*=1&89=+&(947=7*(7:.9*)=.3=9-*=8*6:*39.&1=&88*2'1>=4+=PIC=&9=&=(47*=574249*7_=O3(*=9-*=

PIC=.8=*89&'1.8-*)`=9-*=ATP&8*=&(9.;.9>=4+=XPB=.8=3*(*88&7>=94=45*3=9-*=DNA=&74:3)=9-*=

97&38(7.59.43= 89&79= 8.9*_= I3= 9-.8= 574(*88`= XPB= 574'&'1>= <&108= 43= 9-*= DNA= -*1.== <-.(-=

,*3*7&9*8= &= 8:5*79<.89`= )4<3897*&2= 4+= &= +.=*)= RNA= 541= II8574249*7= (4251*==

574249.3,=9-*=2*19.3,=4+=DNA_=T-*=&(9.;.9>=4+=XPB=.8=7*,:1&9*)='>=5/,=.3=97&38(7.59.43=

&3)=NER=':9='>=522TTD8A=431>=.3=NER_=XPB=ATP&8*=&(9.;.9>=.8=&184=7*,:1&9*)='>=49-*7=

97&38(7.59.43= +&(9478_= D:7.3,= 9-*= 97&38(7.59.43= 4+= MYC= ,*3*`= XPB= ATP&8*= &(9.;.9>= .8=

89.2:1&9*)= '>= FBP= FUSE8'.3).3,= 5749*.3= &3)= .3-.'.9*)= '>= FIR= FBP= .39*7&(9.3,=

7*57*8847_= O5*3= (4251*== +472&9.43= 8:'8*6:*391>= .3.9.&9*8= 9-*= 97&38(7.59.43= <-.(-= .8=

)*5*3)*39= 43= 9-*= 5-485-47>1&9.43= 4+= CTD= 43= S*7/= '>= C)01_= T-*= CTD= S*7= /=

5-485-47>1&9.43=.8=&184=24):1&9*)='>=9-*=MAT+=&3)=C>(1.3=H=8:':3.98=<.9-.3=TFIIH_=

W-*7*&8`=C>(1.3=H=.8=5-485-47>1&9*)='>=C)02=0.3&8*=4+=9-*=2*).&947=<-.(-=3*,&9.;*1>=

7*,:1&9*=9-*=TFIIH='>=.3-.'.9.3,=9-*=C)01=&(9.;.9>=A04:1.9(-*;`=C-:.04;=*9=&1_=,***_=

D*5*3).3,= 43= .98= 5-485-47>1&9.43= 89&9*`= CTD= &997&(98= 2RNA= &3)= -.8943*= 24).+>.3,=

*3?>2*8_= A9= 9-*= (4251*9.43= 4+= 9-*= 97&38(7.59.43= (>(1*= C)01= &184= 5-485-47>1&9*8= 9-*=

S*71=4+=9-*=RNA=541=II=CTD=D*8(7.'*)=.3=)*9&.1=.3=8*(9.43=I_A_,_+_=B*8.)*8=.98=741*=.3=9-*=

'&8&1= 97&38(7.59.43`= TFIIH= (&3= &184= (4397418= 9-*= 7*,:1&9.43= 4+= 8*;*7&1= = 97&38(7.59.43=

+&(9478`=8:(-=&8=5/-`==E589*.3=B&77=3:(1*&7=&39.,*3=,=EBN,`=-*5&9.9.8=B=;.7:8=HBV=X=

P749*.3= HPX`= H*75*8= 8.251*== ;.7.43= 5749*.3= VP+0= &3)= 3:(1*&7= 7*(*59478= NR8=

M&3,*18)47+`= T-:22*1= *9= &1_= +33/_= P-485-47>1&9.43= 4+= NR8= '>= C)01= 4+= TFIIH= (&3=

4((:7= <.9-.3= 9-*.7= A2B= )42&.3_= T-.8= 5-485-47>1&9.43= (&3= 4((:7= *.9-*7= <.9-4:9= 9-*=

1.,&3)= &8= .3= RAR +`= RAR ʈ`= &3)74,*3= 7*(*59478= AR`= 9-*= 5*74=.842*= 5741.+*7&947=

7*(*594788 `= PPAR ʈ+`= PPAR ʈ,= &3)= 9->74.)= -47243*= 7*(*59478 += 47= .3= 7*85438*= 94=

-47243*=1.0*=4*8974,*3=7*(*59478 =&3)=9->74.)=-47243*=7*(*59478 _=

I3= &)).9.43= 94= .98= 741*= .3= 97&38(7.59.43`= TFIIH= 5&79.(.5&9*8= .3= 9-*= N:(1*49.)*= *=(.8.43=

7*5&.7=NER=574(*88_=O3=9-*='&8.8=4+=9-*=7*(4,3.9.43=574(*):7*`=NER=5&9-<&>=(&3='*=

8:').;.)*)= .394= 9<4= 8:'5&9-<&>8a= 9-*= ,14'&1= ,*342*= 7*5&.7= GGR= &3)= 9-*=

97&38(7.59.43=(4:51*)=7*5&.7=TCR_=I3=GGR=8:'5&9-<&>`=9-*=)&2&,*=.8=8*38*)='>=XPC8

RAD,-= (4251*== &3)= (*397.3= ,= CETN,_= U543= (477*(9= '.3).3,= 4+= 9-*= XPC= 94= 9-*=

)&2&,*)=DNA`=9-*=C89*72.3&1=)42&.3=4+=XPC=+4728=9-*=9-7**8).2*38.43&1=897:(9:7*_=

I3=TCR= 8:'5&9-<&>`= )&2&,*=.8= .3).7*(91>=7*(4,3.?*)= ):7.3,=97&38(7.59=*143,&9.43='>=

9-*=89&11.3,=4+=RNA=541=II=&9=&=1*8.43='>=9-*=&(9.43=4+=C4(0&>3*=8>3)742*=WD=7*5*&9=

5749*.3=CSA=&3)=CSB_=A+9*7=)&2&,*=7*(4,3.9.43`=TFIIH=.8=7*(7:.9*)=94=9-*=1*8.43=.3='49-=

GGR= &3)= TCR= 5&9-<&>8_= I3= GGR`= 50,= 8:':3.9= 4+= TFIIH= .39*7&(98= '49-= <.9-= 9-*= C8

9*72.3&1=&3)=9-*=&2.34=9*72.3&1=)42&.3=4+=XPC`=<-*7*&8=XPB=.39*7&(98=431>=<.9-=9-*=

C89*72.3&1=7*,.43=4+=XPC_=I3=TCR`=9-*=CSB='.3)8=94=9-*=89&11*)=RNA=541=II=&3)=&114<8=

9-*= 7*(7:.92*39= 4+= TFIIH_= = U543= '.3).3,= 4+= TFIIH= 94= 9-*= )&2&,*)= DNA`= 9-*= CAK=

8:'(4251*==).884(.&9*8=+742=9-*=(47*=TFIIH=(4251*==&3)=9-.8=).884(.&9.43=4+=CAK=+742=

(47*=.8=*88*39.&1=+47=9-*=:3<.3).3,=4+=9-*=DNA=M&3,*18)47+`=T-:22*1=*9=&1_=+33/_=T-*=

-*1.(&8*= &(9.;.9>= 4+= TFIIH= +:79-*7= 45*38= 9-*= )4:'1*= -*1.== &74:3)= 9-*= 1*8.43= <-.(-= .8=

+:79-*7=89&'.1.?*)='>=XPA=&3)=9-*=8.3,1*8897&3)*)=DNA='.3).3,=7*51.(&9.43=5749*.3=A=

RPA_=RPA='.3)8=94=9-*=8.3,1*8897&3)*)`=(-*2.(&11>=&19*7*)=3:(1*49.)*8=&3)=(4&98=9-*=

:3)&2&,*)= 897&3)_= XPA= 7*(7:.98= 897:(9:7*= 85*(.+.(= *3)43:(1*&8*`= XPF :ERCC+=

-*9*74).2*7`= <-.(-= (7*&9*= &3= .3(.8.43= / D= 94= 9-*= 1*8.43_= T-.8= (43(42.9&391>= &114<8= 9-*=

7*(7:.92*398= 4+= XPG= <-.(-= (:98= 9-*= )&2&,*)= 897&3)= &9= - D= 94= 9-*= 1*8.43= &114<.3,= 9-*=

*=(.8.43= 4+= 9-*= ,, :-*= 3:(1*49.)* 143,= )&2&,*)= 897&3)_= T-*= 97.2*7.(= 5741.+*7&9.3,= (*11=

3:(1*&7= &39.,*3= PCNA= 7.3,`= <-.(-= .8= ).7*(91>= 14&)*)= &+9*7= 9-*= / D= .3(.8.43= '>= XPF :

ERCC+`=7*(7:.98=DNA=P41= `=DNA=P41= =47=DNA=P41= =+47=,&58+.11.3,=DNA=8>39-*8.8_=

T-*=NER=7*&(9.43=.8=(4251*9*)=43(*=9-*=+.3&1=3.(0=.8=+.11*)='>=DNA=1.,&8*=+FEN+=47=

DNA= 1.,&8*= III= XRCC+= M&79*./3`= L&38= *9= &1_= ,*+._= B*8.)*8= TFIIH= &(9.3,= '49-= .3=

97&38(7.59.43=&3)=NER`=9-*=.3;41;*2*39=4+=NER=+&(9478=.3=97&38(7.59.43=-&;*=&184='**3=

)*'&9*)=8.3(*=143,='&(0=<-*3=DNA87*5&.7=5749*.38b=XPG`=XPF=&3)=XPC=<*7*=+4:3)=94=

.39*7&(9= <.9-= RNA= 541= II= M&1)43&)4`= S-.*0-&99&7= *9= &1_= +330_= L&9*7= 8*;*7&1= 89:).*8=

(&2*=<-.(-=8:,,*89*)=9-*=.3;41;*2*39=4+=9-*8*=NER=+&(9478=.3=97&38(7.59.43_===

91 III_ H:2&3=).847)*78=&884(.&9*)=<.9-=2:9&9.43=.3=TFIIH=&3) NER=+&(9478

X*74)*72&=P.,2*3948:2 =XP`=T7.(-49-.4)>89745->=TTD=&3)=C4(0&>3*=S>3)742*=CS=

&7*=7&7*`=7*(*88.;*=).847)*78=(&:8*)='>=2:9&9.43=.3=TFIIH=&3)=NER=+&(9478_==T4=)&9*`=&=

2:19.9:)*=4+=2:9&9.438=.3=9-*=TFIIH=&3)=NER=+&(9478=-&;*='**3=)*8(7.'*)=<-.(-=,.;*=

7.8*=94=97&38(7.59.43&1=)>87*,:1&9.43=&3)=)*+*(9.;*=NER=5&9-<&>_=A((47).3,=94=9-*=*&71>=

(*11= +:8.43= 89:).*8= 9-*= NER= ).8*&8*8= &7*= (&9*,47.?*)= .394= 2:19.51*= (4251*2*39&9.43=

,74:58= &3)= *&(-= ,74:5= 8:'8*6:*391>= (477*8543)8= 94= 5&9.*398= <.9-= 2:9&9.438= .3=

.3).;.):&1= ,*3*8= D*= W**7)8K&89*1*.3`= K*./?*7= *9= &1_= +31,_= F47= .389&3(*= *.,-9=

(4251*2*39&9.43=,74:58=(42*8=:3)*7=9-*=XP=5-*349>5*=XP8A=94=G=&3)=XP8V`=9<4=.3=

CS= CS8A= &3)= CS8B= &3)= +4:7= .3= TTD= XP8B`= XP8D`= TTD8A`= TTDN+= 5-*349>5*_=

M:9&9.438=.3=9-*=,*3*8=4+=9-*8*=(4251*2*39&9.43=,74:58=(&:8*=&=<.)*=7&3,*=4+=(1.3.(&1=

8>259428`=+742=2.1)=5-4948*38.9.;.9>=94=8*;*7*=80.3=(&3(*78`=)*;*1452*39&1=).847)*78=

&3)= 3*:74)*,*3*7&9.43_= S42*9.2*8= 2:9&9.438= .3= 8&2*= ,*3*= (&3= ,.;*= 7.8*= 94= ).++*7*39=

).8*&8*8= 47= &= ;&7.&9.43= .3= 9-*= )*,7**8= 4+= 8*;*7.9>_= G*3*9.(= 89:).*8= -&;*= 8-4<3= 9-&9=

2:9&9.43=.3=XPB`=XPD=&3)=XPG=,.;*=7.8*=94=XP=47=&=(42'.3&9.43=4+=XP=&3)=CS=7*+*77*)=

&8=XP2CS=.3=9-*=7*89=4+=9-*=9*=9`=TTD=.8=:8:&11>=(&:8*)='>=2:9&9.43=4+=XPD`=7&7*1>=4+=

XPB`= 522TTD8A= &3)= TTDN+_= F.,:7*= +- = 8-4<8= 9-*= 4;*71&55.3,= ,*349>5*85-*349>5*=

(4251*=.9.*8=&243,=).++*7*39=).847)*78=K7&*2*7`=P&9743&8=*9=&1_=,**1b=D.G.4;&33&=&3)=

K7&*2*7=,*+,b=S&7&8.3=,*+,_=

D:7.3,=2>=P-D`=I=5&79.(:1&71>=89:).*)=9-*=XP`=XP2CS=&3)=TTD=5-*349>5*=(&:8*)='>=

).++*7*39= 2:9&9.438= .3= XPB`= XPD= &3)= 522TTD8A= ,*3*= T&'1*= 1_= E&(-= 4+= 9-*=2:9&9.43=

&3)=9-*=5-*349>5*=.8=*=51&.3*)=.3=)*9&.1='*14<_=

92 P.VJQ 7]Vs PB PD TTDRA P R

PLCS F S G D

TTD T P RH LPLR s Q] R

T *# YT M32 2'-, ', XPBQ XPD ," TTDVA %#,# 123"'#" "30',% P&D

F'%30# WYT R#* 2'-,1&'. -$ %#,#2'! "'1-0"#01 2- +-*#!3* 0 "#$#!21T

T&# %#,#2'! "'1# 1#1 0#.0#1#,2#" ', !7 , 0#!2 ,%*#1 ',"3!#" 7 "'$$#0#,2 TFIIH ," NER $ !2-01 0#.0#1#,2#" ', " 0) *3#T D'$$#0#,2 +32 2'-,1 ', -,# %#,# + 7 0#13*2 ', 1#4#0 * "'$$#0#,2 !*','! * $# 230#1 A" .2#" $0-+ K0 #+#0Q P 20-, 1 #2 *T XVV]R D'G'-4 ,, ," K0 #+#0 XVWXR S 0 1', XVWX

93 AT X#0-"#0+ .'%+#,2-13+

X*74)*72&= 5.,2*3948:2 = XP= <&8= +.789= )*8(7.'*)= .3= +21.= '>= H*'7&= &3)= K&548.= H*'7&=

&3)= K&548.= +21._= I3= +22,`= K&548.= (4.3*)= 9-*= 9*72= =*74)*72&= 5.,2*3948:2 = +47= 9-*=

(43).9.43`=7*+*77.3,=94=.98=(-&7&(9*7.89.(=)7>`=5.,2*39*)=80.3=47= 5&7(-2*39=80.3 _=XP=.8=

&=7&7*=&:94842&1=7*(*88.;*=).847)*7`=(-&7&(9*7.?*)='>=5-4948*38.9.;.9>`=57*2&9:7*=80.3=

&,.3,`= 5.,2*39&7>= (-&3,*8`= &3)= 2&1.,3&39= 9:247= )*;*1452*39_= C425&7*)= 94= 9-*=

,*3*7&1= 545:1&9.43`= XP= 5&9.*398= (&3= )*;*145= -:3)7*)8= 4+= 80.3= (&3(*78_= XP= 5&9.*398=

:3)*7=&,*=4+=,*=>*&78=-&;*=&=+*`***8=+41)=.3(7*&8*=.3=9-*=+7*6:*3(>=4+=3438=2*1&342&=

80.3=(&3(*7=NMSC`=,***8=+41)=.3(7*&8*=.3=2*1&342&8_=T-*>=&184=-&;*=+***8+41)=-.,-=

+7*6:*3(>=4+=,*99.3,=(&3(*7=43=8:3=*=548*)=9.88:*8=4+=9-*=*>*=&3)=+**`***8+41)=.3(7*&8*=

43= 9-*= 943,:*= K7&*2*7`= L**= *9= &1_= +33.b= B7&)+47)`= G41)89*.3= *9= &1_= ,*++_= XP= 5&9.*398=

:3)*7= &,*= 4+= ,*= >*&78= -&;*= &3= &5574=.2&9*1>= /*8+41)= .3(7*&8*= .3= (&3(*78= 4+= 9-*= '7&.3=

&3)=49-*7=(*397&1=3*7;4:8=8>89*2=M&3,*18)47+=&3)=E;&38=+33/=._*=&12489=+2=4+=9-*8*=

5&9.*398=:3)*7=&,*=,*=&7*=7*5479*)=94=-&;*=3*:7414,.(&1=8>25942&9414,>=7*+*77*)=&8=

XP8ND=.3=7*89=4+=9-*=9*=9=R&5.3=,*+-_=XP=5&9.*398=<.9-=)*+*(98=.3=(4251*2*39&9.43=

,74:58=A`=B`=D=&3)=G=&7*=;*7>=1.0*1>=94=-&;*='1.89*7.3,=':738=43=2.3.2&1=8:3=*=548:7*b=

<-.1*=9-48*=-&;.3,=)*+*(9=.3=(4251*2*39&9.43=,74:58=C`=E=&3)=;&7.&398V=)4=349=8-4<=

8:(-= 8:3= ':738_= I3= XP8V= .9= .8= 349= 9-*= NER= ':9= 9-*=548987*51.(&9.43= 7*5&.7= .8= )*+*(9.;*=

'*(&:8*=4+=9-*=P41=H=,*3*_=XPA=&3)=XPD=&7*=9-*=2&.3=,*3*8=7*85438.'1*=+47=XP8NDb=

XPG=.8=9-*=.3+7*6:*39=(&:8*=4+=9-*=XP8ND=':9=9-*=5-*349>5*=2.,-9='*=8*;*7*_=H4<*;*7`=

&11=&7*=&9=-.,-=7.80=94=)*;*145=*&71>=438*9=+7*(01.3,`=1*39.,.3*8=&3)=80.3=(&3(*78_==

R02-W = .8= 43*= 4+= 9-*= 2:9&9.438= .3= XPD= ,*3*= <-.(-= I= 89:).*)_= I9= ,.;*8= 7.8*= 94= 9-*= XP=

5-*349>5*=&3)=.8=14(&9*)=.3=C89*72.3&1=7*,.43=4+=9-*=XPD=,*3*_=I9=-&8='**3=)*8(7.'*)=

*=9*38.;*1>=.3=84=2&3>=89:).*8=&8=9-.8=2:9&9.43=.8=&=-498549=+47=9-*=XP=5-*349>5*=T&'1*=

1 &3)= .8= +4:3)= &8= &= -*9*74?>,4:8= 2:9&9.43= .3= 12*= 4+= XP8D= 5&9.*398= T&>147`

B74:,-943= *9= &1_= +331b= R*3&:)= &3)= M47&8= ,***b= K4'&>&8-.`= U(-.>&2&= *9= &1_= ,**,_=

94 C:7.4:81>`=9-*=(1.3.(&1=2&3.+*89&9.438=4+=5&9.*398=&7*=(4254:3)=-*9*74?>,49*8=+47=XPD2=

R02-W`= <-*7*= &= 8*(43)= 2:9&9.43= .3(1:)*8=5&9.*398= <.9-= 47= <.9-4:9= 80.3= (&3(*78= &3)=

5&9.*398=<.9-=47=<.9-4:9=8*;*7*=3*:7414,.(&1=.25&.72*398_=T-*=5&9.*39=XP+-/LO=.8=+08

>*&7841)= 2&1*= <.9-= R02-W= 2:9&9.43= &3)= *=-.'.9*)= 5-4948*38.9.;.9>`= 2:19.51*= (&3(*78=

&3)=2.1)=3*:7414,.(&1=&'3472&1.9.*8_=T-*=5&9.*39=(*11=1.3*=+47=9-*=+.789=9.2*=<&8=:8*)=94=

&3&1>?*=9-*=*++*(9=4+=2:9&9.43=43=9-*=842&9.(=->5*72:9&9.43=<-.(-=<&8=9-4:,-9=94='*=

&++*(9*)='>=.25&.7*)=DNA=7*5&.7=574(*88=1*=M&.7*`=T*>88.*7=*9=&1_=,*+*_=L&9*7=.3=+331`=

T&>147=*9=&1_=5*7+472*)=,*349>5*=5-*349>5*=&3&1>8*8=&3)=8-4<*)=9-&9=.9='*143,8=94=XP8

D=(4251*2*39&9.43=,74:5_=T&>147`=B74:,-943=*9=&1_=+331_==

BT C-!)7 ,# S7,"0-+#

C4(0&>3*= 8>3)742*= <&8= +.789= )*8(7.'*)= '>= &= L43)43= 5->8.(.&3`= E)<&7)= A1+7*)=

C4(0&>3*= .3= +3-0= .3= 9<4= 8.'1.3,8= &8= &= 8>3)742*= 4+= )<&7+.82`= 7*9.3&1= &9745->= &3)=

)*&+3*88_= S.3(*= 9-*3`= 247*= 9-&3= +2*= (&8*8= 4+= C4(0&>3*= 8>3)742*= CS= -&;*= '**3=

7*5479*)=.3=9-*=1.9*7&9:7*_=CS`=&3=&:94842&1=7*(*88.;*=).847)*7`=-&8=34=&55&7*39=,*3)*7=

47= *9-3.(= 57*).1*(9.43_= T-*= (1.3.(&1= 8>259428= 2&>= '*= 57*8*39= &9= '.79-= &3)= 9>5.(&11>=

)*;*145='>=&,*=9<4_=P&9.*398=:8:&11>=)4=349=8:7;.;*=94=&):19-44)_=B*8.)*8`=,74<9-=&3)=

)*;*1452*39&1= )*1&>`= &++*(9*)= (-.1)7*3= -&;*= &= 9>5.(&1= +&(*8`= 45-9-&12.(= &3)= &:).947>=

).847)*78= &3)= 8:388*38.9.;.9>_= CS= 8*;*7*1>= &++*(98= 9-*= 3*7;4:8= 8>89*2`= 7*8:19.3,= .3=

574+4:3)= 3*:74)*;*1452*39&1= .25&.72*39= 8:(-= &8= 2.(74(*5-&1>= &3)= 2*39&1=

7*9&7)&9.43=&3)=)*;*1452*39&1=)*1&>_=T-*7*=&7*=+.;*=034<3=(4251*2*39&9.43=,74:58=.3=

CSa= CSA`= CSB`= CS2XPB`= CS2XPD= &3)= CS2XPG_= M:9&9.438= .3= 9-*= CSA= ,*3*= &7*=

7*85438.'1*=+47=9-*=(1.3.(&1=5-*349>5*=4+=CS=T>5*=I`=9-*=(1&88.(=+472=&3)=&7*=57*8*39=.3=

&5574=.2&9*1>= ,/= 4+= CS= 5&9.*398_= I3= (4397&89= 2:9&9.438= .3= CSB= 2&3.+*89= .3= ;&7.&'1*=

5-*349>5.(=*=57*88.438_=F47=*=&251*`=C*7*'748O(:148F&(.&18S0*1*9&1=8>3)742*=COFS=

47= P*3&8S-40*.78II= 8>3)742*8= &7*= 7*(4,3.?*)= &8= &= +472= 4+= CS= T>5*= II_= CS= T>5*= III=

8>259428= &7*= 2.1)*7= 9-&3= T>5*= I`= 2&3.+*89.3,= 1&9*7= &3)= <.9-= 814<*7= ).8*&8*=

95 574,7*88.43_= A349-*7= ;&7.&39= 4+= CS= .8= X*74)*72&= P.,2*3948:2 8C4(0&>3*= 8>3)742*=

<-.(-=(439&.38=2.=*)=8>259428=4+='49-=9-*=5-*349>5*8=XP8CS=<-.(-=.8=*=51&.3*)=.3=&=

)*9&.1=.3=3*=9=8*(9.43_= =

CSA= &3)= CSB= 5749*.38= .3)**)= .39*7&(9= <.9-= 8*;*7&1= 49-*7= 5749*.38= &3)= &7*= &184=

.251.(&9*)=.3=&=3:2'*7=4+=(*11:1&7=574(*88*8=49-*7=9-&3=DNA=7*5&.7=T&'1*/_=T-*7*=&7*=

34<= 8*;*7&1= *;.)*3(*8= <-.(-= 8-4<8= 9-&9= CSB= .8= .251.(&9*)= .3= 97&38(7.59.43= &3)=

(-742&9.3=7*24)*1.3,=VS1*?8C7:?=&3)=E,1>=,*+-_=O3*=7*(*39=89:)>=.3=+&(9=8-4<8=9-&9=

CSB=+:3(9.438=.3=(4845*7&9.43=<.9-=(8J:3=&3)=7*,:1&9*=9-*=97&38(7.59.43=&3)=(-742&9.3=

897:(9:7*=L&0*`=B4*9*+:*7=*9=&1_=,*+._==

CT X#0-"#0+ P'%+#,2-13+ C-!)7 ,# S7,"0-+#

X*74)*72&= 5.,2*3948:22(4(0&>3*= 8>3)742*= XP2CS= .8= &= 7&7*= 5-*349>5*= <.9-=

(1&88.(&1=(1.3.(&1=+*&9:7*8=4+=XP=&3)=CS=5-*349>5*8=L.3)*3'&:2`=D.(0843=*9=&1_=,**+_=

I3=&)).9.43=94=2*39&1=7*9&7)&9.43`=85&89.(.9>`=->54,43&).82=&3)=8-479=89&9:7*`=&++*(9*)=

5&9.*398=-&;*=9-*=9>5.(&1=+&(.&1=+7*(01.3,=&3)=80.3=(&3(*7=57*).8548.9.43=(-&7&(9*7.89.(=4+=

5&9.*398=<.9-=XP_=T-*.7=,*349>5*=.8=XPB`=8D`=47=8G`=349=CS_==

I3=9-*=(4:78*=4+=2>=9-*8.8`=I=&3&1>?*)=9-*=9<4=).++*7*39=XP2CS=2:9&9.438`=43*=.3=XPB=

&3)=49-*7=.3=XPD_=M:9&9.43=.3=XPB`= F33S =<&8=7*5479*)='>=S(499=*9=&1_=S(499`=I9.3=*9=&1_=

+33-_= T-.8= 2:9&9.43= <&8= )*9*(9*)= .3= 43*= 4+= 9-*= 9<4= 8.'1.3,= 5&9.*398= <.9-= XP2CS=

5-*349>5*8=8-4<.3,=9-*=CS=3*:7414,.(=).8*&8*=&3)=349=9-*=XP=3*:7414,.(=).8*&8*_==A9=

9-*=&,*=4+=0=<**08=-*=)*;*145*)=8*;*7*=8:3':73_=H*=<&10*)=&9=+=>*&7=&3)=8540*=-.8=

+.789=<47)=&9=,=>*&78=4+=&,*_=H*=)*;*145*)=842*=-*&7.3,=).++.(:19.*8=&9=9-*=&,*=4+=+4:7=

>*&78_= H.8= 80.3= <&8= )7>= &3)= 5&7(-2*3981.0*= <.9-= 3:2*74:8= +7*(01*81.0*=

->5*75.,2*39*)=2&(:1*8`=*85*(.&11>=.3=8:38*=548*)=&7*&8=-4<*;*7=-&)=34=*;.)*3(*=4+=

&3>=2&1.,3&3(>_=W.9-=&,*=9-*=5&9.*39=)*;*145*)=8*;*7&1=3*:7414,.(&1=&342&1.*8=8:(-=&8=

.3(7*&8*= .3= ->)74(*5-&1:8= <.9-= 57434:3(*)= &9745->= 4+= (*7*'*11:2= &3)= &3= *31&7,*)=

96 +4:79-= ;*397.(1*`= &3= .3(7*&8*= .3= 9-*= )*38.9>= 4+= 9-*= 8543,.48&`= 8(1*748.8= 4+= 8:9:7*8`=

)*2>*1.3&9.3,= 3*:745&9->= &3)= 2&3>= 247*_= H*= &184= )*;*145*)= 8*;*7&1= 8*=:&1=

&'3472&1.9.*8=<.9-=.3(7*&8.3,=&,*_==

A349-*7=&3&1>?*)=2:9&9.43=<.9-=XP2CS=5-*349>5*=<&8= G0*,D`= &=8:'89.9:9.43=.3=XPD=

,*3*=&3)=9-*=5&9.*39=<&8=(&11*)=XPCS8,_=T-.8=2:9&9.43=<&8=+.789=7*5479*)='>=M48-*11=*9=

&1_`= :3)*7= XP8H= (4251*2*39&9.43= ,74:5_= L&9*7= .9= <&8= 7*&88.,3*)= .3= XP8D=

(4251*2*39&9.43=,74:5=R4''.38=+33+b=V*72*:1*3`=S9*+&3.3.=*9=&1_=+33+_=P&9.*39=XPCS8

,=8-4<*)=8>259428=4+='49-=CS=&3)=XP`=.3(1:)*)=3*:7414,.(&1=8>259428=<-.(-=<&8=

&884(.&9*)= <.9-= CS= 7&9-*7= 9-&3= <.9-= XP_= H*= -&)= *&71>= &(:9*= 8:3= 8*38.9.;.9>`= <&8=

+7*(01*)`= &3)= )*;*145*)= 80.3= (&3(*7= &9= &,*= ,_= H*= &184= -&)= 2*39&1= &3)= ,74<9-=

7*9&7)&9.43`=&3)=).*)=&9=&,*=+-=+742=(&3(*7=T&0&>&2&`=S&1&?&7=*9=&1_=+33/_==

DT T0'!&-2&'-"7120-.&7

TTD=.8=&=7&7*=&:94842&1=7*(*88.;*=).847)*7=+.789=)*8(7.'*)='>=P411.99=*9=&1=P411.99`=J*33*7=

*9=&1_=+302=&3)=9*72*)='>=P7.(*=*9=&1_=P7.(*`=O)42=*9=&1_=+32*_=C1.3.(&1=+*&9:7*8=4+=TTD=

&7*=-.,-1>=;&7.&'1*=.3=*=57*88.43`=)*8(7.'*)='>=9-*=&(743>28`=PIBIDS`=IBIDS=&3)=BIDS=

<-.(-= 7*57*8*39= 9-*= P-4948*38.9.;.9>`= I(9->48.8`= B7.991*= -&.7= &3)= 3&.18`= I39*11*(9:&1=

.25&.72*39`= D*(7*&8*)= +*79.1.9>= &3)= S-479= 89&9:7*= I9.3`= S&7&8.3= *9= &1_= ,**+_=

P-4948*38.9.;*= +472= 4+= TTD= .8= (&:8*)= '>= 9-*= 2:9&9.43= .3= XPB`= XPD= &3)= 522TTD8A=

8:':3.9=4+=TFIIH_=C*11:1&7= 89:).*8= -&;*=8-4<3=9-&9= 9-*= 5-4948*38.9.;*=+472= 3472&11>=

4((:78= '*(&:8*= 4+= )*+*(9.;*= NER= 8.2.1&7= 94= XP= &3)= CS`= -4<*;*7= 34385-4948*38.9.;*=

TTD=).851&>=&=3472&1=NER=(&5&(.9>=S9*+&3.3.`=L&,42&78.3.=*9=&1_=+320_=T*3=5*7(*39&,*=

4+= &11= 9-*= 34385-4948*38.9.;*= +472= 4+= TTD= 5&9.*398= &7*= 034<3= 94= -&;*= 2:9&9.43= .3=

TTDN+=,*3*`=+:3(9.43=4+=<-.(-=.8=349=;*7>=(1*&7=>*9_=A((47).3,=94=&=3*<=C1.3.(48,*3*9.(=

(1&88.+.(&9.43`= TTD= (&3= '*= (1&88.+.*)= .394= 9-7**= (&9*,47.*8= ._*_= .= &= ,74:5= <.9-= DNA=

7*5&.7=&342&1.*8`=..=&=,74:5=<.9-4:9=DNA=7*5&.7=)*+*(9=&3)= <.9-=TTDN+=2:9&9.43`=

&3)= ...= &= ,74:5= <.9-4:9= DNA= 7*5&.7= )*+*(9= &3)= <.9-4:9= .)*39.+.*)= ,*3*9.(= '&8.8=

M47.(*8P.(&7)`= C&7.48A3)7S= *9= &1_= ,**3_= S9:)>= )43*= 43= 9-*= '&8.8= 4+= 9-.8= 3*<=

(1&88.+.(&9.43= 8-4<8= 9-&9= 9-*= +7*6:*3(>= 4+= (43,*3.9&1= .(-9->48.8`= (4114.).43= '&'>= 9>5*=

<&8=8.,3.+.(&391>=-.,-*7=.3=TFIIH=2:9&9*)=,74:5_==

I= &3&1>?*)= +4:7= ).++*7*39= 2:9&9.438= .3= XPB`= XPD= &3)= 522TTD8A= 8:':3.98= 1*&).3,= 94=

TTD=5-*349>5*=T&'1*=1_= T++3P =2:9&9.43=.3=XPB=,*3*=<.9-=TTD=5-*349>5*=<&8=+.789=

7*5479*)= '>= W**)&= *9= &1= W**)&`= E;*34= *9= &1_= +331_= T-*= 5&9.*39= <&8= 2&1*= &3)= <&8=

*=&2.3*)=+.789=&9='.79-=<.9-=(43,*3.9&1=.(-9->48.8=(4114).43='&'>_=T-*=80.3=(43).9.43=

.2574;*)=<.9-.3=-=<**08`=1*&;.3,=&=2.1)=.(-9->48.8=4+=9-*=97:30_=TTD=<&8=8:85*(9*)=&9=

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Manuscript in preparation

102 Involvement of TFIIH in the NER factors mediated chromatin remodeling.

Singh Amita, Le May Nicolas*, Jean-Marc Egly*.

Institut de G n tique et de Biologie Mol culaire et Cellulaire (IGBMC)

Department of Functional Genomics and Cancer CNRS/Inserm/Universit de Strasbourg BP 163, 67404 Illkirch Cedex, CU Strasbourg, Fr.

*To whom correspondence should be addressed:

Le May Nicolas, Jean-Marc Egly.

Institut de G n tique et de Biologie Mol culaire et Cellulaire (IGBMC) 1, rue Laurent Fries 67404 Illkirch, France Tel: +33 (0)3 88 65 34 47 email: [email protected] , [email protected]

103 Abstract:

Fidelity in transcription of the gene requires a congregation of set of proteins around the promoter, upon gene activation. The TFIIH complex is central among these proteins and plays a key role through its enzymatic subunits. Mutations in TFIIH subunits XPB, XPD and p8/TTD-A leads to three distinct autosomal recessive disorders: xeroderma !"#$%&'()#* +,-.* ('#$&!#$(* /(('0!/&$1* 2!&3* 4'05/6%$S(* (6%18'#$* +,-94:.* /%1* trichothiodystrophy (TTD). By studying the different mutation in these three subunits of TFIIH from mentioned genetic disease models, we have shown that each mutation analyzed led to a specific transcriptional dysregulation of the RAR-target gene RAR 2. The architectural and enzymatic integrity of TFIIH condition the appropriate recruitment of TFIIH complex and further the arrival of the Nucleotide Excision Repair (NER) factors. By disturbing their recruitment, mutated TFIIH consequently compromised the chromatin remodeling mediated by NER factors such as histones post-translational modifications (PTMs), DNA breaks induction, DNA demethylation and gene looping. Hence it can be concluded that in addition to its enzymatic activities, TFIIH provide a platform to recruit the NER factors and orchestrates the related functions in transcription. Such varying penetrance among mutants gives rise to a phenotype gradient as observed in TTD, XP or XP/CS patients.

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Introduction

TFIIH, a well-conserved transcription factor during evolution plays a key role in maintaining the genome stability as well as in transferring the genetic information. Mammalian TFIIH includes a core, (containing the six subunits XPB, p62, p52, p44, p34, and p8/TTD-A) bridged by XPD to the cdk-activating kinase module (CAK, composed of the three subunits cdk7, cyclin H, and MAT1) (Figure 1A). In addition to its function in the RNA polymerases transcription, TFIIH is also involved in nucleotide excision repair (NER) pathway, thus illustrating the important interplay existing between both so disparate processes (Feaver, Svejstrup et al. 1993; Schaeffer, Roy et al. 1993; Drapkin, Reardon et al. 1994). NER, that is responsible for the removal of a variety of bulky DNA adducts, such as those induced by UV irradiation, is subdivided into two sub pathways: the Global Genome Repair (GGR) is responsible for the removal of DNA lesions from the whole genome and Transcription Coupled Repair (TCR) is responsible for the accelerated removal of lesions located on the transcribed strand of active genes (Mellon, Spivak et al. 1987). In GGR, the XPC-hHR23B complex recognizes the damage-induced DNA distortion, while in TCR the RNA polymerase II (pol II) stalled in front of a lesion promotes the recruitment of the TCR-specific proteins CSB and CSA (Kamiuchi, Saijo et al. 2002). Both NER sub-pathways then funnel through the action of TFIIH that unwinds the DNA via the ATPases/helicases activities of XPB and XPD regulated by p52/p8-TTD-a and p44 respectively. This event favors the recruitment of XPA and RPA assisting in the expansion of the DNA bubble around the damage and the arrival of XPG and XPF endonucleases. XPG and XPF then generate cuts in the 3 S*/%1*;S*(!1$(*'<* the lesion respectively, thereby removing the damaged oligonucleotide before the re- synthesis machinery fills the DNA gap. In protein coding genes transcription, XPB is involved in the opening of the promoter while the cdk7 kinase of TFIIH phosphorylates the serine 5 of the carboxy-terminal domain (CTD) of pol II as well as the activators. Interestingly, NER factors were found to be associated and participate to the regulation of gene expression (Barreto, Schafer et al. 2007; Schmitz, Schmitt et al. 2009; Le May,

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Mota-Fernandes et al. 2010). It remains to be established whether TFIIH influences the recruitment and the function of the NER factors at the promoter of activated genes to achieve chromatin remodeling required for accurate transcription. The importance of TFIIH and its relationship with the other NER factors is put forward by the existence of human genetic disorders with a broad range of clinical features (Table1). Indeed, mutations in the XPB, XPD and p8/TTD-A subunits of TFIIH originate three distinct autosomal recessive disorders: xeroderma pigmentosum (XP), ('#$&!#$(* /(('0!/&$1* 2!&3* 4'05/6%$S(* (6%18'#$* +,-94:.* /%1* &8!03'&3!'16(&8' 36* (TTD) (de Boer and Hoeijmakers 2000; Kraemer, Patronas et al. 2007; Cleaver, Lam et al. 2009). XP is characterized by numerous skin abnormalities ranging from excessive freckling, ichtyosis to multiple skin cancers, the frequency of which is about 2000-fold greater than in normal individuals. In addition to a hyper-photosensitivity to sunlight, XP patients display a progressive neurological degeneration (Kraemer, Lee et al. 1987; Lehmann, McGibbon et al. 2011). Although XP can be discriminated from CS phenotype there are rare cases of individuals with the combined symptoms of XP and CS (XP/CS). XP/CS patients display a combination of the cutaneous abnormalities from XP with the severe neonatal later onset of neurological and developmental anomalies typical of CS. The typical hallmark of TTD is sulphur-deficient brittle hair, caused by a greatly reduced content of cysteine-rich matrix proteins in the hair shafts. Mental retardation and ichthyosis also characterize TTD patients ( Sarasin A 1992; Itin, Sarasin et al. 2001).Some TTD patients are sensitive to sunlight without any unusual pigmentation changes and severe skin lesions or cancer (Stefanini, Lagomarsini et al. 1993). Accumulating evidences suggest that the clinical features of these three disorders due to mutations in all the NER factors including TFIIH cannot be exclusively explained on the basis of DNA repair defects but also involve transcription deficiencies (Evans, Moggs et al. 1997; Keriel, Stary et al. 2002; Dubaele, Proietti De Santis et al. 2003; Coin, Oksenych et al. 2007) (Table 1). In this study, we intend to understand how mutations in XPB, XPD and p8/TTD-A result in impairment of gene expression. Knowing the close connections between TFIIH and NER factors in the removal of DNA damage

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(Zurita and Merino 2003; Schrer 2008; Compe and Egly 2012), we have investigated the consequences of mutations found on three of the TFIIH subunits on the recruitment of the NER factors and the roles of the later in the various steps that lead to accurate RNA synthesis including histones post-translational modifications (PTMs), DNA breaks, DNA demethylation and gene looping. In addition to improve our understanding of gene expression regulation, such systematic approach could help to determine transcriptional default hallmarks to molecularly define the different genetic disorders.

Results : Mutations in TFIIH subunits compromise the formation of the transactivation complex To further determine the transcriptional defects due to the mutations in XPB, XPD and p8/TTD-A subunits of TFIIH, we analyzed cells derived from XP, XP/CS and TTD patients bearing mutations as indicated (Table 1 and figure 1). To investigate the transcriptional process, we focused on the Retinoic Acid Receptor gene RAR 2 as a model. Few hours post all-trans retinoic acid (t-RA) treatment of the TFIIH mutated cells (Materials and Methods), we observed different patterns in the RAR 2 mRNA synthesis compared to their respective XPBwt, XPDwt and the rescued p8/TTD-Awt cells (Figure 2A1, B1 to J1). The synthesis of RAR 2 mRNA was significantly lower for XPB/F99S and XPB/T119P compared to XPBwt that peaked at 8 h post-treatment (Figure 2A1, B1 and C1). In the four XPD mutated cells, the inductions of RAR 2 were similarly reduced compared, to the cells rescued with an XPDwt (Figure 2F1 to J1). We did not notice any reduction in the RAR 2 mRNA level between p8/TTD-A and its rescued (Figure 2D1 and E1). We next evaluated the dynamic recruitment of pol II partners at the RAR 2 promoter, using chromatin immunoprecipitation (ChIP) overtime. ChIP analyzed by quantitative PCR showed a concomitant recruitment of the retinoic acid receptor (RAR), pol II and the general transcription factor TFIIB at 6/8h post t-RA treatment in XPBwt, XPDwt and p8/TTD-Awt (Figure 2A2, D2 and F2 respectively). At this time, TFIIH was

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also recruited as visualized by the presence of its XPB, XPD, p44 and cdk7 subunits (Figure 2A3, D3 and F3). We also detected the simultaneous presence of the XPA, XPG and XPF NER factors (Figure 2A4, D4 and F4). Our analysis next indicated that each mutation in the XPB, XPD and TTD-A/p8 subunit of TFIIH led to different and specific deregulations in the recruitment of the components of the transactivation complex. In XPB/F99S RAR was detected at early time, whereas pol II and TFIIB accumulated at the promoter after 1h post t-RA induction (Figure 2B2). We also noticed a non-concomitant recruitment of the TFIIH subunits and the NER factors. In XPB/F99S cells, while XPG was recruited at 3h, XPA and XPF were not detected until 12h (Figure 2B4). The XPB/F99S mutation prevents the accurate opening of the promoter and consequently the recruitment of the NER factors within the transcription complex (Coin, Bergmann et al. 1999; Coin, Oksenych et al. 2007). In XPB/T119P cells, RAR, pol II and TFIIB were detected at the promoter around 3h (Figure 2C2). The XPB subunit was found at the promoter at 1h while XPD, p44 and cdk7 subunits were detected later (Figure 2C3). Similarly the XPA arrival at 1h precedes XPG (at 6h), whereas XPF is not detected (Figure 2C4). It therefore appeared that the recruitment and the formation of TFIIH complex could be initiated at the promoter upon gene induction. In p8/TTDA deficient cells, we repeatedly observed a synchronized recruitment of TFIIH subunits and NER factors with the transcriptional machinery at 8h post t-RA treatment that is similar to what was observed with the corresponding rescued wild type cell (Figure 2E2 to E4 and D2 to D4). We next focused on the XPD mutations resulting in three different patient phenotypes (Table 1). In XPD/R112H cells, the transcription and NER factors are recruited in a cyclic and synchronized manner. All these factors were found in a second cycle of recruitment that peaked at 8h, remained present until 12h post t-RA treatment and paralleled the RAR 2 mRNA induction (Figure 2G1 and G2-G4). In the XPD/G602D cells, the recruitment pattern at 1 h and 6 h post tRA-treatment of pol II, TFIIB, TFIIH as well as the NER factors at the promoter was similar to XPDwt cells

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(Figure 2H2-H4). The XPD/G602D mutation that affects the helicase motif V does not seem to disturb the TFIIH architecture (Dubaele, Proietti De Santis et al. 2003). In XPD/R683W, the recruitment of TFIIH and the NER factors was highly deregulated at early time post t-RA treatment (Figure 2I2-I4). At 8h only pol II, TFIIB and XPA were detected (Figure 2I2 and 2I4). In XPD/R722W in which the mutation is located in the C- terminal unfolded domain, we observed the recruitment of both transcription and NER factors at 8h (Figure 2J2-J4); a continuous accumulation of pol II, TFIIB as well as some of TFIIH subunits was also visible at 12h. In these cells, the R683W and R722W mutations in XPD are known to weaken the interaction with the p44 subunit and consequently destabilize the architecture of TFIIH (Botta, Nardo et al. 2002; Keriel, Stary et al. 2002), see discussion. Altogether, our data suggested that XPB and XPD mutations disturbed the RAR 2-gene activation by impeding the formation of the pre-initiation complex at the promoter of activated genes. Although each dysregulation is mutation-specific, we noticed a compromised integrity of TFIIH complex and in some cases the absence of NER factors at the promoter. Such observations suggested a link between the presence of an accurate TFIIH complex at the promoter of activated genes and the concomitant recruitment of the NER factors.

Impaired chromatin remodeling reveals the implication of TFIIH Previous works have underlined the sequential recruitment of the transcription and the NER components at the promoter of activated gene and their role in conditioning the chromatin modifications e.g. histone post-translational modifications (PTMs), DNA breaks and DNA demethylation (Le May, 2010, 2012, Schmitz, 2009, Barreto, 2006): Euchromatin is characterized by acetylated (H3K9-Ac and H4K16-Ac) and methylated (H3K4me3, and H3K79me2) histone H3 and H4 and allows transcription while heterochromatin that inhibits RNA synthesis is characterized by chromatin marks such as di- and trimethylated H3K9 (H3K9me2-3) and H3K27 (H3K27me2) (Bannister and Kouzarides 2011)

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In t-RA treated XPBwt and XPDwt cells, we observed upon ChIP analysis, the characteristic histones PTMs signature (increase of H3K9ac and H3K4me2 concomitantly to a decrease H3K9me2, Figure 3A1 and F1, coincided with the RAR 2 mRNA induction peak) at 8h. In p8/TTD-A wt cells, the H3K9me2 is not detectable (Figure 3D1). The typical histone signature observed in wild type cells at the promoter of activated RAR 2 is not observed in all the mutated cells tested so far (Figures 3B1, C1, E1, G1, H1, I1 and J1) indicating a persistence of active histones PTMs around the RAR 2-promoter. As an example, in XPD/R112H cells in which the transcription and the NER factors were shown to be recruited at the promoter 8h post-treatment, H3K9 was not demethylated, while we noticed a high acetylation of H3K9 and methylation of H3K4 (Figure 3G1). Knowing how crucial was the presence of NER factors, and particularly of XPG and XPF endonucleases in RAR 2 expression , we next evaluated the formation of transient DNA breaks in its promoter surrounding (Ju, Lunyak et al. 2006; Le May, Fradin et al. 2012). Using a BioChIP assay that measures the incorporation of biotinylated dUTP within broken DNA; we observed a concomitant increase of DNA cleavage at both promoter and terminator of all the wild type cells upon t-RA activation (Figure 3A2, D2, F2 and supplemental figure S2). In these cells, we noticed a parallel between the presence of XPG and XPF at both the promoter and the terminator and the DNA breaks. Except in the XPD/R112H and XPD/R722W in which we noticed the recruitment of XPG and XPF together with the presence of some DNA breaks (Figure 3G2 and 3J2), in all the other mutated cell lines we did not observed any significant formation of DNA breaks nearby the RAR 2 promoter (Figure 3B2, E2, H2 and I2). We also noticed some DNA breaks (although to a much lower extent) at the RAR 2 promoters in XPB/T119P (Figure 3C2). Surprisingly, there was no detectable presence of DNA breaks in p8/TTD-A cells (Figure 3E2), whereas all the NER factors are present together with the transcriptional machinery both at promoter and at terminator (Figure3 E2-E4 and Supplemental figure S2). It has to be noticed that the DNA breaks were observed around the terminator of RAR 32 in wild type cells, whereas in all the

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other mutated cell lines, no DNA breaks were detected (Supplemental figure S2). Several studies have documented a relationship between XPG and DNA demethylation upon transcription (Barreto, Schafer et al. 2007; Le May, Fradin et al. 2012) Using an unmethylated DNA immuno-precipitation (unMeDIP) approach, we measured the removal of the 5-methylcytosine (5mC) at RAR 2 promoter. We found that DNA demethylation occurred at the promoter at 8 h post t-RA in the three wild type cells and perfectly paralleled the recruitment of the entire transcription machinery (Figures 3A3, D3 and F3). On the contrary, there was a complete lack of DNA demethylation in all the XPB and XPD cells (Figures 3B3, C3, and G3-J3) except in the p8/TTD-A cells (Figure 3E3 and Supplemental figure 2). The above data strongly supported an involvement of TFIIH in the recruitment of the entire transcription machinery, in the chromatin remodeling including the histone PTMs, the formation of DNA breaks and active DNA demethylation.

Chromatin looping is perturbed without proper assembly of TFIIH and NER factors The detection of the basal transcription machinery together with the NER factors at both RAR 2 promoter and terminator is correlated with the formation of a chromatin loop mediated by the CCCTC- binding factor (CTCF) chromatin organizer (Splinter, Heath et al. 2006; Le May, Fradin et al. 2012). Such gene looping was shown to parallel DNA demethylation and DNA breaks at both regions. Quantitative chromatin conformation capture assays (q3C) were performed to analyze the interactions between -65 kb, promoter (Pro), terminator (Ter), or +323 kb as well as an intronic region (M1) of the RAR 2 locus (Figure 4, upper scheme). Using Ter and M1 as a bait, we observed that Pro could specifically and significantly interact with Ter at 8 h in t-RA-treated wild type cells, (Figure 4A, D and F), paralleling the recruitment of the entire transcriptional apparatus on both regions (Figure 1 and Supplemental Figure S1). By contrast, in all the mutated TFIIH cells, no increase in the interaction/ frequency between Ter and Pro was revealed from t=0 to T= 6/8h post t-RA induction (Figure 4B, C, E, G-J). It is worthwhile to notice that the lack in loop formation between Pro and Ter could be related to defect

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in the recruitment of some of the transcription and NER factors at Pro and Ter in most of the mutated cells (Figure 2 and Supplemental figure S1). In addition, we also observed that in these cells CTCF was either undetectable or delayed in its recruitment at both Pro and Ter of RAR 2 (Figure 2B4, C4, G4, H4, J4). As controls, no specific interactions were observed between the intronic M1 bait and Pro or between all the other analyzed fragments upon t-RA treatment (Supplemental figure S3). By impeding the accurate recruitment of NER factors at the activated RAR 2 promoter, TFIIH further disturbed the gene looping conformation required for optimal gene expression.

Mutations in TFIIH impair some of its enzymatic activities We next addressed the contribution of XPB and XPD activities to the formation of an accurate transcription initiation complex, a prerequisite for optimal RNA synthesis. We first generated recombinant rIIH6 (the core TFIIH containing p62, p52, p44, p34, p8 and either XPBwt or XPBmut), XPDwt or XPD mut and CAK. These rIIH6 sub- complexes were added to an in vitro transcription assay containing, in addition to the adenoviral major late promoter (MLP, run-off of 309nt long), all the basal transcription factors and pol II (Gerard, Fischer et al. 1991) and when indicated, CAK and XPD either alone or in combination. When added to the transcription assay that contains all the factors including the XPD/CAK sub-complex, rIIH6-XPB/F99S exhibits a much weaker basal transcription activity than the wild type and rIIH/XPB/T119P (Figure 5A). Addition of CAK together with XPDwt and rIIH6-XPB/F99S did not improve its transcriptional activity (lanes 5-7) contrary to what occurs with rIIH6-XPBwt and -XPB/F119P that requires absolutely its CAK sub-complex for optimal RNA synthesis (lanes 1-3 and 8-10). Since XPB/F99S mutation weakens the contact with the p52 regulatory subunit within TFIIH (Coin, 2007), it results in a defect in the unwinding of the RAR 2 promoter by XPB and a defect in RNA synthesis. Interestingly, we also noticed that the absence of XPDwt

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results in a very weak RNA synthesis (Figure 5A, lane 4 and Figure 5B, lanes 1 and2). Knowing that the XPD-p44 interaction, conditions the anchoring of CAK to the core TFIIH (Coin, Oksenych et al. 2007), we next investigated the transcription activity of XPD mutated TFIIHs. Addition of CAK and increasing amount of XPDwt to our transcription assay that already contained rIIH6wt stimulated RNA synthesis (lanes 3-4) as also observed in the presence of XPD/R112H or XPD/G602D (lanes 5-8). On the contrary, when either XPD/R683W or XPD/R722W was added instead, there was no significant increase of RNA synthesis (lanes 9-12). In these latter cases, XPD/R683W and XPD/R722W mutations prevented a contact with p44 and consequently impeded the anchoring of CAK to the core TFIIH to exploit its kinase activity towards pol II (Coin, Oksenych et al. 2007). CAK was shown to phosphorylate the serine 5 of the carboxy- terminal domain (CTD) of the largest subunit of pol II, and allow promoter escape (Lu, Zawel et al. 1992; Bensaude, Bonnet et al. 1999). We then investigate the impact of CAK on the phosphorylation status of pol II. pol II phosphorylation status was carried out following a classical run-off transcription experiment (Materials and Methods). Using antibody directed against the CTD, we observed that the hyper-phosphorylated form of pol II (IIo) was prevalent in the presence of XPD wt, XPD/R112H or XPD/G602D (Figure 5C, lanes 2-7) and paralleled the increase in RNA synthesis (Figure 5B, lanes 3-8). On the contrary, in the presence of XPD/R683W and XPD/R722W which were deficient in stimulating RNA synthesis (Figure 5A, lanes 9-12), pol II was not hyper-phosphorylated (Figure 5C, lanes 8-11). Moreover ChIP experiments further demonstrated the presence of phosphorylation of Serine5 of pol II CTD in XPB/T119P, XPD/R112H, XPD/G602D as in the wild type cells, at RAR 2 promoter (Figure 5D). In all the other XPD, XPB cell lines, we did not detect phosphorylated pol II (Figure 4E). Altogether the above data suggest that the gene expression dysregulation observed in some XPB and XPD cells might results at least in part from a defect in phosphorylating pol II at the transcription initiation level.

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Discussion The present study underline the key role of TFIIH in (i) the synchronized recruitment of the NER factors, (ii) the histones modifications, (iii) the DNA breaks and DNA demethylation as well as (iv) gene looping, four crucial steps in transcription. Each mutation that affects one of the TFIIH subunits specifically disturbs the transcription process of activated genes.

TFIIH integrity and enzymatic activities Although in wild type cells, we observed upon t-RA induction a concomitant and synchronized recruitment of all the TFIIH subunits at the RAR 32 promoter (Figure 2), in XPB and XPD cells, TFIIH subunits recruitment is rather dispersed. Indeed, in XPB/F99S cells, XPD subunit is found late at the RAR 32 promoter than the other TFIIH subunits (Figure 2B3), while in XPB/T119P cells, XPB is recruited at 1h while the other TFIIH subunits are detected at 6h (Figure 2C3). Similarly in XPD/R683W only subunits XPD and Cdk7 are the detected at early time, whereas in XPD/R722W cells, the arrival of TFIIH subunits at the promoter occurred over a much larger period of time in an unsynchronized way (Figure 2I3, J3) . In Rift Valley Fever Virus infected cells, p44 as well as XPB subunits of TFIIH were found in the nucleus, while XPD was maintained in the cytoplasm (Le May et al., 2004; Kainulainen et al., 2014). As a consequence we observed a transcriptional defect not only due to the trapping of some of the subunits as a filament but also for the absence of others such as XPD that could not enter in the nucleus. All these observations suggest that the formation of TFIIH occurs at the promoter upon gene induction to initiate RNA synthesis. Any mutation on XPB, XPD and p8/TTD-A that would prevent the accurate recruitment of and/or the interaction network between the TFIIH subunits, would disturb transcription. The XPB/F99S mutation weakens its interaction with p52; consequently p52 could no more up-regulates XPB to unwind the activated

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promoter(Coin, Oksenych et al. 2007). Similarly the XPD/R683W and XPD/R722W mutations that weaken the contact with the p44 subunit and consequently the anchoring of CAK to the core TFIIH are detrimental for both in vivo and in vitro transcription (Figures 2 and 5). In absence of accurate phosphorylation by CAK, pol II cannot be used as a docking site for enzymes required for PTMs of histones such as H3K4me3 and the un-phosphorylated nuclear receptors will be defective in regulating their responsive genes. In these cases, the transcription inhibition results from defect in phosphorylation by CAK with some consequences in chromatin modification (Figure 5) rather than defect in XPD helicase regulation by p44. This demonstrates once more that the XPD helicase activity is not crucial for transcription. It however remains that the gene expression defects could not be exclusively explained by a lack of the enzymatic activities of the mutated TFIIH. Indeed, we observed that the XPB/T119P, XPD/R112H and XPD/G602D mutations did not inhibit the in vitro transcription activity of the corresponding TFIIHs whereas mRNA induction of RAR 32 is defective in vivo (Figure 5 and Figure 2).

TFIIH a platform for recruiting NER factors It seems that the TFIIH mutations might disturb either directly or indirectly the accurate positioning of the various components around the transcription initiation. Indeed close physical connections between TFIIH and transcription and NER factors are required for accurate RNA synthesis. We show here that the XPD/R7222W mutation impedes the recruitment of TFIIB, TFIIE and TFIIF at RAR 32 promoter (Figure 2 & supplemental figure). By stabilizing TFIIH architecture through interaction with XPB and XPD, XPG regulates the expression of certain nuclear receptor responsive genes (Ito, Kuraoka et al. 2007). It is also likely that mutations in TFIIH could disturb its interaction with RPA, XPA (Coin, Oksenych et al. 2007), or XPF (Coin, Auriol et al. 2004) as already highlighted for NER. Here we observed that each TFIIH mutation specifically disturbs the sequential recruitment of the NER factors. The XPB/F99S mutation affects the recruitment of XPA and XPG (Figure 1B4), a point that was also

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observed in NER (Coin, Oksenych et al. 2008) . In XPB/T119P, XPA and XPF recruitment does not parallel the one for TFIIH and XPG (Figure C3 and C4). In XPD/R602D cells, XPF is not detected at the RAR 32 promoter. Altogether, our results strongly suggest a role of TFIIH as a platform to recruit the NER factors and organize the transactivation complex.

TFIIH orchestrates the chromatin remodeling One of the crucial question concerns the interplay between TFIIH and the NER factors in the cascade of events that lead to the formation of a chromatin environment suitable for gene expression. For all the TFIIH mutations we observed a misregulation of H3K4me2, H3K9ac and H3K9me2 histone modification state around the RAR 32 promoter that could consequently prevent the establishment of a permissive environment for transcription. The defective histone modifications would therefore disturb the DNA methylation (D'Alessio, Weaver et al. 2007) and probably DNA breaks formation (Figure 3). Several studies have documented a relationship between DNA demethylation and DNA repair mechanisms upon transcription (Barreto, Schafer et al. 2007; Wossidlo 2010; Le May, Fradin et al. 2012). The DNA breaks surrounding the RAR 32 promoter that are visible in all the wild type cells, were not detected in mutated cells except in the XPD/R112H, XPD/R722W and to a lower extend in XPB/T119P cells (Figure 3). In these cells as well as in wild type ones, XPG and XPF are present at both promoter and terminator of RAR 32 (Figure3) suggesting a relationship between the presence of those endonucleases and DNA breaks formation (Ju, Lunyak et al. 2006; Le May, Fradin et al. 2012). How DNA breaks and DNA demethylation are connected is unclear since in XPD/R112H and XPD/R722W cells there are DNA breaks and no detectable DNA demethylation while in p8/TT-DA we observed the inverse situation (Figure 4). In the p8/TTD-A cells, we did not observe any default in the recruitment of the transcriptional machinery, TFIIH subunits and NER factors, the typical histones PTMs

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signature for active transcription, DNA demethylation. Surprisingly, no DNA breaks induction and gene looping were detected. This allows us to suggest that the DNA methylation is unrelated to DNA breaks formation and such DNA modification is important to avoid temporal aberrant chromatin rearrangement but is not obligatory for mRNA synthesis . Another hypothesis would propose that p8/TDD-A besides mediating the DNA unwinding in NER might also regulate either directly or indirectly some DNA breaks. Altogether our data underline the key role of TFIIH in the transcription process, in which it will combine its primary enzymatic activities together with the recruited NER factors and to further orchestrate the downstream events such as histones PTMs, DNA breaks and DNA methylation. All the cells bearing the mutations on XPB, XPD and p8/TTD-A that derived from XP, XP/CS, TTD patients, present a common transcriptional defect but a specific (and unique) pattern of dysregulation with hallmarks that are not suitable for the gene expression. As a function of the location of the mutation, we observed that the heterochromatin to euchromatin transition is rather incomplete. These subtle transcriptional differences that varies among mutants seems gives rise to a phenotype gradient as observed on these XP, XP/CS, TTD patients, in addition to some specific DNA repair deficiencies that could also have indirect effect in gene expression.

Acknowledgments We thank F. Coin for fruitfull discussion, F. Costanzo for reading the manuscript, C. Braun for helping in in vitro experiments and the Institute of Genetics and Molecular and Cellular Biology Cell Culture Facility. This study was supported by grants from the >)8' $/%*?$($/803*4')%0!@*A1B/%0$1*:0!$%&!(&(C*@SA(('0!/&!'%*1$*@/*?$03$803$*0'%&8$*@$* Cancer (ARC-SL220100601 DEE;* FEGHGIGJKED;* &'* LM>.C* @SA"$%0$* N/&!'%/@$* 1$* Recherche (ANR12.BSV80017-01) La ligue nationale contre le Cancer E Equipe labellis e Ligue F/%1* @SA(('0!/&!'%* %/&!'%/@$* 1$(* #$#P8$(* 1$* @SQ818$* N/&!'%/@* 1)*

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M rite ANMONM). A.S. was supported by a JME (Jean-Marc Egly) Advanced European Research Council grant and by an ARC fellowship for young scientists.

Material and Methods:

Cell Lines, culture conditions and transfection. XPB wt, XPB/F99S and XPB/T119P are SV40-transformed human fibroblasts (XPCS2BASV) expressing His-ERCC3-HA(Winkler, Vermeulen et al. 1998), XPB-T296C (Cl. 14, XPB/F99S) and XPB-A355C (Cl. 5, XPB/T119P) respectively (Riou, Zeng et al. 1999). p8TTDA cells are SV40-transformed human fibroblasts (TTD1Br-SV) bearing the TTDA L21P and R56Stp mutations(Stefanini, Vermeulen et al. 1993). The p8TTDA wt (TTD1Br-SV + TTDA-GFP) cells are rescued TTDBr1 with pEGFP-N1-TTDA vector stably expressing TTDA-GFP(Giglia-Mari, Miquel et al. 2006). XPD wt (GM637) cells are SV40-transformed human fibroblasts from a normal 18-year-old female. XPD/G602D (XPCS2) (Takayama, Salazar et al. 1995), XPD/R112H (TTD8PV) (Stefanini, Giliani et al. 1992), XPD/R683W (XP135LO) (Taylor, Broughton et al. 1997) and XPD/R722W (TTD1BEL) (Broughton, Steingrimsdottir et al. 1994) are human primary fibroblasts. All cells were cultured in appropriate medium. Cells were incubated with red phenol-free medium containing charcoal-treated fetal calf serum (FCS) and 40mg/ml gentamycin. Cells were treated with 10 !M of all-trans retinoic acid (t-RA, MP biomedicals) into the same medium. XPD/R112H, XPD/G602D, XPD/R683W and XPD/R722W cells were transiently transfected 48 h before the t-RA treatment with pEGFP-XPD WT using the X-tremeGENE HP DNA transfection reagent (Roche). Antibodies. Mouse monoclonal antibodies towards XPB (1B3), XPD (2F6), p44 (1H5), RAR (9A6), XPA (1E11), XPG (1B5), RNA pol II (7C2) were from IGBMC antibody facility. Cdk7 (C- 19), TFIIB (C-18), XPF (H-300), Biotin (33) antibodies were obtained from Santa-Cruz Biotechnology. CTCF (ab70303) and RNA pol II ser5P (61085) antibodies were obtained

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from Abcam and Active Motif respectively. H3K4me2 (#9726), H3K9me2 (#9753), H3K9Ac (#9671) antibodies were purchased from Cell signaling technology.

Reverse transcriptase and quantitative PCR. Total RNA was isolated using a GenElute Mammalian Total RNA Miniprep kit (Sigma) and reverse transcribed with SuperScript II reverse transcriptase (Invitrogen). The quantitative PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and the Lightcycler 480 (Roche). The primer sequences for Retinoic Acid Receptor isoform 2 ( RAR 2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes used in real-time qPCR are listed (see Table in supplemental data). RAR 2 mRNA levels were normalized against the GAPDH mRNA.

Chromatin Immunoprecipitation (ChIP) and Biotin-ChIP Cells were cross-linked at room temperature (RT) for 10 min with 1% formaldehyde. Chromatin was prepared and sonicated on ice for 30 min using a Bioruptor (Diagenode) as previously described (Le May et al., 2010). Samples were immunoprecipitated with antibodies at 4 C overnight, and protein G Sepharose beads (Upstate) were added, incubated for 4 hr at 4 C, and sequentially washed. Protein-DNA complexes were eluted, and DNA fragments were purified using QIAquick PCR purification kit (QIAGEN) and analyzed by quantitative PCR using a set of primers targeting the promoter and terminator regions of RAR 32 (see Table 2 in supplemental data). Cross-linked cells prepared as described above were permeabilized with cytonin (Gentaur biologique) for 30 min at RT. After extensive washes with phosphate buffer salt (PBS), terminal deoxynucleotidyl transferase (TdT) reaction was performed using Biotin-16-dUTP (Roche) and 60 units of recombinant enzyme rTdT (Promega). TdT reaction was stopped with specific stop buffer for 15 min at RT. After extensive washes with PBS, the resulting samples were sonicated on ice for 20 min (40 cycles, pulse 10 s, pause 20 s) using a Bioruptor (Diagenode) and immunoprecipitated using anti-Biotin

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antibodies and treated as described in the ChIP assay. The purified DNA fragments were analyzed by quantitative PCR using a similar set of primers described above.

Unmethylated DNA immunoprecipitation Genomic DNA was extracted using GenElute Mammalian Genomic DNA Mini-prep Kit (Sigma). Unmethylation of 5mC on the promoter and terminator regions of RAR 32 was measured by digesting 2 mg genomic DNA with 10 units of MseI (Fermentas) and by using the UnMethylcollector kit (Active Motif). Unmethylated DNA Immunoprecipitation Kit (UnMeDIP) is based on the affinity of the three zinc coordinating CXXC domains that specifically bind nonmethylated CpG sites. The resulting samples were selected using magnetic beads conjugated with CXXC domains, extensively washed, and analyzed by quantitative PCR.

Quantitative chromosome conformation capture (q3C). The quantitative chromosome conformation capture (q3C) assay was performed as previously described (Vernimmen, De Gobbi et al. 2007; Le May, Fradin et al. 2012).

Construction of baculoviruses and purification of complexes Baculoviruses over-expressing XPB, XPD, p62, p52, p44, p34, cdk7, cyclin H, MAT1, and p8 in sf21 cells were produced as previously described (Dubaele et al., 2003; Coin et al., 2007). For expression of recombinant mutated Flag-XPB and Flag-XPD, the cDNA encoding XPB and Flag-XPD cloned in pSK278 and PVL1392 vectors respectively were used for PCR site-directed mutagenesis. The resulting vectors were recombined with baculoviruses DNA (BaculoGold; PharMingen). The recombinant viruses were purified from isolated plaques and viral stocks were prepared by three-step growth amplification. Sf21 insect cells were infected with WT or mutated Flag-XPB, WT or mutated Flag-XPD, p62, p52, p44, p34, Flag-cdk7, cyclin H, Mat1 and p8 baculoviruses allowing the preparation of core-IIH, CAK, and XPD separately. The whole-cell extracts were

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initially incubated 4 h, with agarose beads bound to anti NM2-Flag antibody at 4 C. After washing with a buffer containing, 50 mM Tris/HCl (pH 7.9), 20% glycerol, and 50 mM KCl, rIIH9 complexes were eluted for 8 N12 h with the same buffer containing 0.2 mg/ml of the epitope peptide (Jawhari et al., 2002). For the core-IIH, the eluted fraction was then incubated with anti-p44 (1H5) antibody in a buffer containing 50 mM Tris/HCl (pH 7.9), 20% glycerol, and either 50 or 150 mM KCl. The immunoprecipitated fraction was then either boiled or eluted with a synthetic peptide recognized by Ab-p44. The recombinant TFIIH was made by mixing purified core-IIH, CAK, and XPD allowing the preparation of the different XPD and XPB variants.

In-vitro assays Run-off transcription assays were performed using recombinant TFIIB, TFIIE, TFIIF, TBP, endogenous RNA pol II, and the different TFIIH (rIIHs), as previously described (Gerard et al., 1991). RNA Pol II phosphorylation was carried out as a classical run-off transcription assay as previously described (Ueda et al., 2009). Hypophosphorylated (IIA) and hyper phosphorylated (IIO) forms of RNA Pol II were resolved by SDS/PAGE and detected using the monoclonal antibody (7C2) directed against the CTD.

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Figure 1. Schematic representation of TFIIH: The CAK sub-complex (in blue) is bridged to the core TFIIH (in red) by the XPD helicase (in green). Mutations in the XPB, XPD and p8/TTD-A subunits as well as the XP, XP/CS and/or the TTD are indicated. Black squares indicate the helicase motifs (I, Ia, II, II, IV, V, and VI); NTD: N-Terminal domain.

122 Figure 2

XPB wt XPB/ F99S XPB/ T119P p8/TTD-A wt p8/TTD-A (XP/CS) (TTD) (TTD)

20 A120 B120 C120 D120 E1

wt 10 10 10 10 10 mut

0 0 0 0 0 Fold induction Fold 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

0.4 A20.4 B20.4 C2 1 D2 0.5 E2

0.2 0.2 0.2 0.5 0.25 RAR polII

% Input TFIIB 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

0.4 A30.4 B30.4 C31 D3 0.5 E3 XPD 0.2 0.2 0.2 0.5 0.25 XPB p44 % Input Cdk7 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

0.4 A40.4 B40.4 C41 D40.5 E4 XPA 0.2 0.2 0.2 0.5 0.25 XPG XPF

% Input CTCF 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time(h) Time(h)Time(h) Time(h) Time(h)

XPD wt XPD/ R112H XPD/ G602D XPD/ R683W XPD/ R722W (TTD) (XP/CS) (XP) (TTD) 6 F190 G130 H180 I140 J1 wt 3 45 15 40 20 Trans Non-trans

Fold induction Fold 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

4 F2 4 G2 2 H210 I25 J2

RAR 2 2 5 1 2.5 polII

% Input TFIIB 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

4 F34 G32 H310 I35 J3 XPD

2 2 1 5 2.5 XPB p44 % Input Cdk7 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 68 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

4 F4 6 G4 2 H4 5 I45 J4 XPA XPG 2 3 1 2.5 2.5 XPF % Input CTCF 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time(h) Time(h)Time(h) Time(h) Time(h)

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Figure 2. RAR 32 mRNA expression profile and ChIP on the RAR 32 promoter Relative RAR 32 mRNA expression monitored by qPCR overtime from t-RA-treated XPBwt, XPB/F99S, XPB/T119P, p8/TTD-A wt, p8/TTD-A, XPDwt, XPD/R112H, XPD/G602D, XPD/R683W and XPD/R722W cells (A1-J1). Red curves show the mRNA expression of RAR 32 in the relative XPD mutant cell lines; blue and dotted blue curves show mRNA expression of RAR 32 in wild type cells and transfected cells respectively as indicated in each panel. Error bars represent the standard deviation of three independent experiments. ChIP monitoring the t-RA-dependent recruitment of RAR, pol II, TFIIB (panels A2-J2), XPB, XPD, p44, Cdk7 subunits of TFIIH (panels A3-J3) and XPA, XPG, XPF, CTCF (panels A4-J4) on the RAR 32 promoter; Each series of ChIP is representative of at least two independent experiments as indicated by standard deviation. Values are expressed as percentage of the input.

124 Figure 3

XPB wt XPB/ F99S XPB/ T119P p8/TTD-A wt p8/TTD-A (XP/CS) (TTD) (TTD) 10 A110 B110 C1 10 D110 E1 Histone PTMs H3K9ac 5 5 5 5 5 H3K9me2 H3K4me2

% Input 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

1 A21 B21 C2 1 D21 E2

0.5 0.5 0.5 0.5 0.5 DNA breaks

% Input

0 0 0 0 0 0 8 0 6 0 6 0 8 0 8

20 A320 B320 C3 30 D310 E3

DNA 10 10 10 15 5 demethyl

Fold induction Fold 0 0 0 0 0 0 8 0 6 0 6 0 8 0 8 Time(h) Time(h)Time(h) Time(h) Time(h)

XPD wt XPD/ R112H XPD/ G602D XPD/ R683W XPD/ R722W (TTD) (XP/CS) (XP) (TTD)

15 F115 G1 H1 15 I115 J1 15 Histone PTMs

10 10 10 10 10 H3K9ac H3K9me2 5 5 5 5 5

% Input H3K4me2

0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

1 F22 G21 H2 1 I24 J2

0.5 1 0.5 0.5 2 DNA breaks

% Input

0 0 0 0 0 0 8 0 8 0 6 0 8 0 8

20 F320 G320 H3 20 I320 J3

DNA 10 10 10 10 10 demethyl

Fold induction Fold 0 0 0 0 0 0 8 0 8 0 6 0 8 0 8 Time(h) Time(h)Time(h) Time(h) Time(h)

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Figure 3. Histone PTMs, DNA breaks and active DNA demethylation ChIP monitoring the t-RA dependent occupancy of H3K4me2, H3K9me2 and H3K9ac (panels A1-J1) on the RAR 32 promoter overtime in all the cell lines as mentioned above and figure 2. Each series of ChIP is representative of at least two independent experiments. Detection of DNA breaks at RAR 32 promoter at 0, 3 and 6/8 hour post t- RA treatment depending on the peak formation corresponding to RNA expression profile (A2-J2). DNA breaks are detected through the incorporation of Biotin-dUTP via a terminal deoxynucleotidyl-transferase (TdT) reaction. DNA fragments containing DNA breaks are immuno selected by Biotin antibodies (BioChIP) and analyzed by qPCR. Each series of BioChIP is representative of three independent experiments as indicated by standard deviation, and values are expressed as percentage of the input. UnMedIP was performed using the UnMethyl-Collector kit (Active Motif) at the indicated time (panels A3-J3). Samples containing unmethylated DNA were analyzed by qPCR on the RAR 32 promoter. Each series of UnMedIP is representative of two independent experiments as indicated by standard deviation, and values are expressed as percentage of the input.

126 Figure 4

Ter Pro TATA M1 RAR β2 RAR +323kb -65kb -82 -36 +1 +200kb

XPB wt XPB/ F99S XPB/ T119P p8/TTD-A wt p8/TTD-A (XP/CS) (TTD) (TTD) 0.3 A0.3 B 0.3 C 0.3 D0.3 E -65/Ter 0.2 0.2 0.2 0.2 0.2 PRO/Ter 0.1 0.1 0.1 0.1 0.1 M1/Ter

Interaction frequency 0 0 0 0 0 +323/Ter 0 8 0 6 0 6 0 8 0 8 Time(h) Time(h)Time(h) Time(h) Time(h)

XPD wt XPD/ R112H XPD/ G602D XPD/ R683W XPD/ R722W (TTD) (XP/CS) (XP) (TTD) 0.3 F 0.3 G 0.9 H I 0.3 J 0.3 -65/Ter

0.2 0.2 0.6 0.2 0.2 PRO/Ter M1/Ter 0.1 0.1 0.3 0.1 0.1

Interaction frequency +323/Ter 0 0 0 0 0 8 0 8 0 6 0 0 8 0 8 Time(h) Time(h)Time(h) Time(h) Time(h)

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Figure.4. TFIIH is involved in RAR 32 looping Schematic representation of the quantitative chromatin conformation capture (q3C) (Upper panel). One probe was designed at Ter of RAR 32 to investigate the associations between the different elements including upstream (-65 kb), Pro, intronic (M1), and downstream (+323 kb) regions as indicated by the black arrows. q3C assays were performed using crosslinked and HindIII-digested chromatin from all the cells as indicated above the A-J panels at 0, 3, and 6/8 hr post-t-RA treatment. The bar chart (y axis) shows the enrichment of PCR product (%) normalized to the enrichment within the human XPB (=100%). Each PCR was performed at least three times and averaged as indicated by standard deviation. Signals were normalized to the total amount of DNA used, estimated with an amplicon located within a HindIII fragment in RAR 32 (see the Experimental Procedures).

128 Figure 5

A IIH6 XPBwt + + + + ------IIH6 XPB/F99S -- - - ++ + ------IIH6 XPB/T119P ------+ + + - - - XPD +- + -- + + - ++ + -- CAK -- ++- -+- -+ +- - TFIIH ------+

Transcription 309nt 1 2 3 4 5 6 7 8 9 10 11 12 13

B IIH6 XPBwt + + + + + + ++ + ++ + XPDwt ------XPD/R112H ------XPD/G602D ------XPD/R683W ------XPD/R722W ------+ + + ++ ++ + + ++ Transcription CAK

309nt 1 2 3 4 5 6 7 8 9 10 11 12

C XPDwt ------XPD/R112H ------XPD/G602D ------XPD/R683W ------XPD/R722W ------IIO Phosphorylation IIA 1 2 3 4 5 6 7 8 9 10 11

D XPB wt XPB/ F99S XPB/ T119P p8/TTD-A (XP/CS) (TTD) (TTD) 0.2 D10.2 D20.2 D30.2 D4

0.1 0.1 0.1 0.1

% Input

0 0 0 0 0 3 8 0 3 6 0 3 6 0 3 8 Time(h) Time(h) Time(h) Time(h) XPD wt XPD/ R112H XPD/ G602D XPD/ R683W XPD/ R722W (TTD) (XP/CS) (XP) (TTD) 1.4 D52.4 D61.4 D71.4 D81.4 D9

0.7 1.2 0.7 0.7 0.7

% Input

0 0 0 0 0 0 3 8 0 3 8 0 3 8 0 3 8 0 3 8 Time(h) Time(h) Time(h) Time(h) Time(h)

129

Figure 5. In vitro RNA synthesis and RNA pol II phosphorylation in TFIIH deficient cells In vitro transcription activity of recombinant IIH6 XPBwt, XPB/F99S or XPB/T119P either alone or in combination with XPD and/or CAK (A) and of rIIH6 in addition to either XPD wt, XPD/R112H, XPD/G602D, XPD/R683W or XPD/R722W (B) in the presence or absence of CAK as indicated. Size (309 nt) of the transcript is indicated on the right side of each panel. (C) In vitro phosphorylation of pol II when added in the reconstituted transcription assay as described in panel B. Arrows indicate hypo (IIa) and hyper (IIo) phosphorylated forms of RNA pol II. (D) ChIP monitoring the t-RA dependent occupancy of the serine 5 phosphorylated pol II on the RAR 32 promoter from wild type and mutated cells

130 Figure S1

XPB wt XPB/ F99S XPB/ T119P p8/TTD-A wt p8/TTD-A (XP/CS) (TTD) (TTD)

0.4 A10.4 B10.4 C12 D12 E1

0.2 0.2 0.2 1 1 RAR polII % Input TFIIB 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

0.4 A20.4 B20.4 C2 2 D2 2 E2 XPD 0.2 0.2 0.2 1 1 XPB p44 % Input Cdk7 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

0.4 A30.4 B30.4 C32 D3 2 E3 XPA XPG 0.2 0.2 0.2 1 1 XPF % Input CTCF 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time(h) Time(h)Time(h) Time(h) Time(h)

XPD wt XPD/ R112H XPD/ G602D XPD/ R683W XPD/ R722W (TTD) (XP/CS) (XP) (TTD) 4 F14 G12 H18 I110 J1 RAR 2 2 1 4 5 polII

% Input TFIIB

0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

4 F2 4 G2 2 H28 I210 J2 XPD XPB 2 2 1 4 5 p44 Cdk7

% Input 0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

4 F34 G32 H38 I310 J3 XPA XPG 2 2 1 4 5 XPF

% Input CTCF

0 0 0 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time(h) Time(h)Time(h) Time(h) Time(h)

131

Figure S1. ChIP on the terminator region of RAR !2 ChIP monitoring the t-RA-dependent occupancy of RAR, pol II, TFIIB on the terminator region or RAR !2 during a 12 hour time course in XPBwt, XPB/F99S, XPB/T119P p8/TTD-Awt, p8/TTD-A, XPDwt, XPD/R112H, XPD/G602D, XPD/R683W and XPD/R722W cells (A1- J1) and and TFIIH (XPB, XPD, p44, CDK7) (A2- J2). Presence of NER factors XPA, XPG, XPF and chromatin organizer CTCF on RAR !2 terminator region in XPBwt, XPB/F99S, XPB/T119P p8/TTD-Awt, p8/TTD-A, XPDwt, XPD/R112H, XPD/G602D, XPD/R683W and XPD/R722W cells (A3- J3). Each series of ChIP is representative of at least two independent experiments as indicated by standard deviation, and values are expressed as percentage of the input.

132 Figure S2

DNA break at terminator region

XPB wt XPB/ F99S XPB/ T119P p8/TTD-A wt p8/TTD-A (XP/CS) (TTD) (TTD) 1 A1 B1 C1 D1 E

0.5 0.5 0.5 0.5 0.5

% Input

0 0 0 0 0 0 8 0 6 0 6 0 8 0 8 Time(h) Time(h)Time(h) Time(h) Time(h)

XPD wt XPD/ R112H XPD/ G602D XPD/ R683W XPD/ R722W (TTD) (XP/CS) (XP) (TTD) 1 F1 G1 H 1 I 1 J

0.5 0.5 0.5 0.5 0.5

% Input

0 0 0 0 0 0 8 0 8 0 6 0 8 0 8 Time(h) Time(h)Time(h) Time(h) Time(h)

133

Figure S2: DNA Breaks at terminator region of RAR !2 BioChIP shows the presence of DNA breaks on the terminator region of RAR !2 without t-RA and with t-RA treatment either at 6 hour or at 8 hour depending on the peak formation corresponding to RNA expression profile in XPBwt, XPB/F99S, XPB/T119P p8/TTD-Awt, p8/TTD-A (A to E) and in XPDwt, XPD/R112H, XPD/G602D, XPD/R683W and XPD/R722W cells (F to J).

134 Figure S3

Pro TATA M1 Ter RAR β2 RAR +323kb -65kb -82 -36 +1 +200kb

XPB/ F99S XPB/ T119P p8/TTD-A wt p8/TTD-A XPB wt (XP/CS) (TTD) (TTD)

0.9 A0.9 B C0.9 D E 0.9 0.9 -65/M1 0.6 0.6 0.6 0.6 0.6 PRO/M1

0.3 0.3 0.3 0.3 0.3 Ter/M1

Interaction frequency +323/M1 0 0 0 0 0 0 8 0 6 0 6 0 8 0 8 Time(h) Time(h) Time(h) Time(h) Time(h)

XPD wt XPD/ R112H XPD/ G602D XPD/ R683W XPD/ R722W (TTD) (XP/CS) (XP) (TTD) FG H I J 0.9 0.9 0.9 0.9 0.9 -65/M1 0.6 0.6 0.6 0.6 0.6 PRO/M1

0.3 0.3 0.3 0.3 0.3 Ter/M1

Interaction frequency +323/M1 0 0 0 0 0 0 8 0 8 0 6 0 8 0 8 Time(h) Time(h) Time(h) Time(h) Time(h)

135

Figure S3: Chromatin looping between intronic region M1 and other regions of RAR !2 Chromatin looping was analyzed at upstream/downstream elements of RAR !2 using intronic region M1 bait. q3C assays were performed using crosslinked, HindIII-digested chromatin without t-RA and with t-RA treatment either at 6 hour or at 8 hour in XPBwt, XPB/F99S, XPB/T119P p8/TTD-Awt, p8/TTD-A (A to E) and in XPDwt, XPD/R112H, XPD/G602D, XPD/R683W and XPD/R722W cells (F to J). These independent graphs represent a measure of !"#$ %&&'()%!)'*$ +#!,##*$ !"#$ -.&!/#%0$ %23$ 4+5$ 6/'5$ !#/$ %*7$ !"#$ 7',*&!/#%0 +323 kb regions along the gene using M1 as bait (M1 Probe). Each PCR was performed at least three times and averaged as indicated by standard deviation. Signals were normalized to the total amount of DNA used, estimated with an amplicon located within a HindIII fragment in RAR !2 gene.

136

T:GCV

M% : 1QJs S7JR`QIV CC1J1H:C `V: %`Vs RV`V`VJHVs PBLF S PLCS R MQRV`: V s@1J ].Q QsVJs1 101 75 s@1J H:JHV` SHQ V :C85 ^ _ R MQRV`: V ].7s1H:C :JR IVJ :C `V :`R: 1QJ V`IV%CVJ V :C8^ _ PBLT P TTD R MQRV`: V s@1J ].Q QsVJs1 101 75 s@1J H:JHV` VVR: V :C85 ^ _ R M1CR CV:`J1J$ R1s:G1C1 7

PDLRH TTD R H1$. s@1J ].Q QsVJs1 101 75 JQ s@1J H:JHV` S V`:J1J1 V :C85 ^ _ R S%C`%`RRV`1H1VJ G`1 CV .:1` :JR J:1C R MQRV`: V ].7s1H:C :JR IVJ :C `V :`R: 1QJ PDLG D PLCS R .1$. s@1J ].Q QsVJs1 101 75 s@1J H:JHV` T:@:7:I: V :C85 ^ _ R P`Q$V`Q1R `V: %`Vs PDLR P R H1$. s@1J ].Q QsVJs1 101 75 s@1J H:JHV` T7:CQ` V :C85 ^ _ R MVJ :C `V :`R: 1QJ PDLR TTD R H1$. s@1J ].Q QsVJs1 101 75 JQ s@1J H:JHV` S V`:J1J1 V :C85 ^ _ R S%C`%` RV`1H1VJ G`1 CV .:1` :JR J:1C B`Q%$. QJ V :C85 ^ _ R SV0V`V ].7s1H:C :JR IVJ :C `V :`R: 1QJ ] LLP TTD R MQRV`: V s@1J ].Q QsVJs1 101 75 JQ s@1J H:JHV` S V`:J1J1 V :C85 ^ _ R S ] R DV0VCQ]IVJ :C RVC:75 s.Q` s : %`V G1$C1:RM:`1 V :C85 ^_ R MVJ :C `V :`R: 1QJ

T:GCV8 M% : 1QJs 1J PB5 PD :JR ] RTTDA s%G%J1 s :JR .V HC1J1H:C ].VJQ 7]Vs :ssQH1: VR 11 . .V ]: 1VJ s GV:`1J$ .V s:IV I% : 1QJ8

137 T:GCV

P`1IV`s FQ`1:`R RV0V`sV P`1IV`s `Q` IRNA GAPDH AGCTCACTGGCATGGCCTTC ACGCCTGCTTCACCACCTTC ! CCAGCAAGCCTCCATGTTC TACACGCTCTGCACCTTTAGC

$`1IV`)*`Q`* ! `Q` C.IPLB1QC.IPLJIV .7C P`QIQ V` ^P`Q_ TGGTGATGTCAGACTAGTTGGGTC GCTCACTTCCTACTACTTCTGTCAC TV`I1J: Q` ^TV`_ TGTTTGTGCTCTTTGGGCACT CGGTCGGGCTAGGAAACAAGTAAA

C ]`1IV`s R CCTGGCAATTGAAACATGAAAGT P`Q TCCAAAGATGCCTATTAAGTTGTAAGAG M AGCAGCAAAATGCAGGCTTTA TGACACCAGTGAAAAGGAAGCA TV` AAGATGCAGTTTGAGAGCATC CTGGGCAACATGAAATAAAAGATG CCAAACAATTTTCTTCATGGTCATT ! *]`QIQ V` CAGACTAGTTGGGTCATTTGAAGGT TTGAATTGCCTAATATATGCGAGTGA PB CGGTGAGGTGAGTTTGTGGAAT AGGATCTCTGTTTAATGGAAAAGCTT

C P`QGVs TV` ]`QGV `FAMaTTGCTCTTTCTGATGCTCTCAAA`TAMa M ]`QGV `FAMaCAGTACAGTCAAGGTGGCCCGTCT`TAMa ! *]`QIQ V`*]`QGV `FAMaAGCCCGGGTAGGGTTCACCGAAAG`TAMa PB ]`QGV `FAMaAAGGATGAAGGCGTGATCCGACTCTG`TAMa

T:GCV 8 L1s Q` P`1IV`s

138

References

B:JJ1s V`5 A8J85 :JR KQ%<:`1RVs5 T8 ^_8 RV$%C: 1QJ Q` H.`QI: 1J G7 .1s QJV IQR1`1H: 1QJs8 CVCC RVs 5 R 8 B:``V Q5 G85 SH.:`V`5 A85 M:`.QCR5 J85 S :H.5 D85 S1:I1J: .:J5 S8K85 H:JR:5 85 DQRV`CV1J5 G85 M:C `75 N85 %5 85 L7@Q5 F8 5 V :C8 ^ _8 G:RR : ]`QIQ Vs V]1$VJV 1H $VJV :H 10: 1QJ G7 `V]:1`RIVR1: VR DNA RVIV .7C: 1QJ8 N: %`V 5 R 8 BVJs:%RV5 O85 BQJJV 5 F85 C:ssV5 C85 D%GQ1s5 M8F85 N$%7VJ5 8T85 :JR P:C:JH:RV5 B8 ^ _8 RV$%C: VR ].Qs].Q`7C: 1QJ Q` .V RNA ]QC7IV`:sV II CR VÌ1J:C RQI:1J ^CTD_8 B1QH.VI CVCC B1QC 5 R 8 BQ :5 E85 N:`RQ5 T85 LV.I:JJ5 A8R85 E$C75 J8M85 PVR`1J15 A8M85 :JR S V`:J1J15 M8 ^_8 RVR%HVR CV0VC Q` .V `V]:1`L `:JsH`1] 1QJ `:H Q` TFIIH 1J `1H.Q .1QR7s `Q].78 H%I MQC GVJV 5 R 8 B`Q%$. QJ5 B8C85 S V1J$`1IsRQ 1`5 H85 VGV`5 C8A85 :JR LV.I:J5 A8R8 ^ _8 M% : 1QJs 1J .V 6V`QRVÌ: ]1$IVJ Qs%I $`Q%] D DNA `V]:1`L `:JsH`1] 1QJ $VJV 1J ]: 1VJ s 11 . `1H.Q .1QR7s `Q].78 N: %`V $JV 1Hs 5 R 8 CCV:0V`5 J8E85 L:I5 E8T85 :JR RV0V 5 I8 ^ _8 D1sQ`RV`s Q` J%HCVQ 1RV V6H1s1QJ `V]:1`7 .V $VJV 1H :JR IQCVH%C:` G:s1s Q` .V V`Q$VJV1 78 N: RV0 GVJV 5 R 8 CQ1J5 F85 A%`1QC5 J85 T:]1:s5 A85 CC101Q5 P85 VÌV%CVJ5 85 :JR E$C75 J8M8 ^_8 P.Qs].Q`7C: 1QJ Q` PB .VC1H:sV `V$%C: Vs TFIIH J%HCVQ 1RV V6H1s1QJ `V]:1` :H 101 78 EIGQ J 5 R 8 CQ1J5 F85 BV`$I:JJ5 E85 T`VIV:%RB`:0:`R5 A85 :JR E$C75 J8M8 ^ _8 M% : 1QJs 1J PB :JR PD .VC1H:sVs `Q%JR 1J 6V`QRVÌ: ]1$IVJ Qs%I ]: 1VJ s 1I]:1` .V `:JsH`1] 1QJ `%JH 1QJ Q` TFIIH8 EIGQ J 5 R 8 CQ1J5 F85 [email protected] 85 :JR E$C75 J8M8 ^ _8 D1s 1JH RQCVs `Q` .V PBL] :JR PDL] S%GHQI]CV6Vs Q` TFIIH 1J D:I:$VR DNA O]VJ1J$ R%`1J$ N%HCVQ 1RV E6H1s1QJ RV]:1`8 MQC CVCC 5 R 8 CQ1J5 F85 [email protected] 85 MQH_%V 5 85 G`Q.5 S85 BC: JV`5 C85 :JR E$C75 J8M8 ^ _8 N%HCVQ 1RV V6H1s1QJ `V]:1` R`10VJ G7 .V R1ssQH1: 1QJ Q` CAK ``QI TFIIH8 MQC CVCC 5 R8 CQI]V5 E85 :JR E$C75 J8M8 ^_8 TFIIH7 1.VJ `:JsH`1] 1QJ IV DNA `V]:1`8 N: RV0 MQC CVCC B1QC 5 R 8 DACVss1Q5 A8C85 V:0V`5 I8C8G85 :JR S<7`5 M8 ^ _8 AHV 7C: 1QJRIJR%HVR T`:JsH`1] 1QJ Is RV_%1`VR `Q` AH 10V DNA DVIV .7C: 1QJ 1J MV .7C: 1QJRS1CVJHVR GVJVs8 MQCVH%C:` :JR CVCC%C:` B1QCQ$7 5 R 8 RV BQV`5 J85 :JR HQV1=I:@V`s5 J8H8 ^_8 N%HCVQ 1RV V6H1s1QJ `V]:1` :JR .%I:J s7JR`QIVs8 C:`H1JQ$VJVs1s 5 R 8 D`:]@1J5 R85 RV:`RQJ5 J8T85 AJs:`15 A85 H%:J$5 J8C85 :1VC5 L85 A.J5 K8J85 S:JH:`5 A85 :JR RV1JGV`$5 D8 ^ _8 D%:C `QCV Q` TFIIH 1J DNA V6H1s1QJ `V]:1` :JR 1J `:JsH`1] 1QJ G7 RNA ]QC7IV`:sV II8 N: %`V 5 R 8 D%G:VCV5 S85 P`Q1V 1 DV S:J 1s5 L85 B1VJs QH@5 R8J85 KV`1VC5 A85 S V`:J1J15 M85 :J HQ% VJ5 B85 :JR E$C75 J8M8 ^_8 B:s:C `:JsH`1] 1QJ RV`VH R1sH`1I1J: Vs GV 1VVJ 6V`QRVÌ: ]1$IVJ Qs%I :JR `1H.Q .1QR7s `Q].7 1J PD ]: 1VJ s8 MQC CVCC 5 R 8 E0:Js5 E85 MQ$$s5 J8G85 H1:J$5 J8R85 E$C75 J8M85 :JR QQR5 R8D8 ^ _8 MVH.:J1sI Q` Q]VJ HQI]CV6 :JR R%:C 1JH1s1QJ `QÌ: 1QJ G7 .%I:J J%HCVQ 1RV V6H1s1QJ `V]:1` `:H Q`s8 EIGQ J 5 R 8 FV:0V`5 8J85 S0V=s `%]5 J8Q85 B:`R1VCC5 L85 B:`R1VCC5 A8J85 B%`: Q1s@15 S85 G%C7:s5 K8D85 DQJ:.%V5 T8F85 F`1VRGV`$5 E8C85 :JR KQ`JGV`$5 R8D8 ^ _8 D%:C `QCVs Q` : I%C 1]`Q V1J HQI]CV6 ``QI S8 HV`V01s1:V 1J `:JsH`1] 1QJ :JR DNA `V]:1`8 CVCC 5 R 8 GV`:`R5 M85 F1sH.V`5 L85 MQJHQCC1J5 85 C.1]Q%CV 5 J8M85 C.:IGQJ5 P85 :JR E$C75 J8M8 ^ _8 P%`1`1H: 1QJ :JR 1J V`:H 1QJ ]`Q]V` 1Vs Q` .V .%I:J RNA ]QC7IV`:sV BÎI_ $VJV`:C `:JsH`1] 1QJ `:H Q` BTF8 J B1QC C.VI 5 R 8

139

G1$C1:RM:`15 G85 M1_%VC5 C85 T.V1C5 A8F85 M:`15 P8O85 HQQ$s `: VJ5 D85 N$5 J8M85 D1J:J 5 C85 HQV1=I:@V`s5 J8H85 :JR V`IV%CVJ5 8 ^ _8 D7J:I1H IJ V`:H 1QJ Q` TTDA 11 . TFIIH Is S :G1C1

140 S]C1J V`5 E85 HV: .5 H85 KQQ`VJ5 J85 P:Cs `:5 R8RJ85 KCQ%s5 P85 G`Qs0VCR5 F85 G:C=:` 5 N85 :JR RV L:: 5 8 ^ _8 CTCF IVR1: Vs CQJ$R`:J$V H.`QI: 1J CQQ]1J$ :JR CQH:C .1s QJV IQR1`1H: 1QJ 1J .V R$CQG1J CQH%s8 GVJVs DV0VCQ]IVJ 5 R 8 S V`:J1J15 M85 G1C1:J15 S85 N:`RQ5 T85 M:`1JQJ15 S85 N:<<:`Q5 85 R1<

141 BT E,87+ 2'! !2'4'27 -$ TFIIH

T-:8=+&7=741*=4+=TFIIH=.3=97&38(7.59.43=-&8='**3=8-4<3=.3=8*;*7&1=89:).*8_=T-*=57*;.4:8=

8*(9.43= 4+= 9-*= 7*8:198= &))*)= 842*= 247*= .3+472&9.43= 94= 4:7= 034<1*),*`= 8-4<.3,= 9-&9=

2:9&9.43=.3=43*=47=9-*=49-*7=8:':3.9=4+=TFIIH=.3+1:*3(*)=).++*7*39=*;*398=.3=(-742&9.3=

7*24)*1.3,= ):7.3,= 97&38(7.59.43_= T4= 574;.)*= &3= *=51&3&9.43= 4+= 842*= (1.3.(&1= +*&9:7*8=

4'8*7;*)= <.9-.3= 9-*= XP`= XP2CS= &3)= TTD= 5&9.*398= ):*= 94= 2:9&9.43= .3= XPB`= XPD= &3)=

522TTD8A= 8:':3.98= &3)= 94= +:79-*7= )*+.3*= 9-*.7= 741*= .3= 97&38(7.59.43`= I= -&;*= *=&2.3*)=

9<4= )*+.3*)= *3?>2&9.(= &(9.;.9.*8= ATP&8*= &3)= -*1.(&8*= &3)= -&;*= (-&7&(9*7.?*)= 9-*8*=

5&9-4,*3.(= 2:9&9.438= <.9-= 7*85*(9= 94= 9-*.7= )*+.(.*3(.*8= .3= *&(-_= T-*= C)01= 0.3&8*=

&(9.;.9>= <&8= &17*&)>= 8-4<3= .3= 9-*= 57*;.4:8= 8*(9.43_= S.3(*`= 522TTD8A= 8:':3.9= -&8= 349=

'**3= 034<3= 94= -&;*= &3>= *3?>2&9.(= &(9.;.9>`= 431>= TFIIH= (42548.9.43= &3&1>8.8= .3=

522TTD8A<9=&3)=2:9&39=(*11=1.3*=<&8=5*7+472*)_=

B_+ S:':3.9=(42548.9.43=4+=TFIIH=

T4=574;.)*=+:79-*7=.38.,-98=.394=9-*=741*=4+=XPB=&3)=XPD`=<-41*=(*11=*=97&(9=WCE=<&8=

57*5&7*)= +742= 9-*= XP8B= &3)= XP8D= 5&9.*398= (&77>.3,= *.9-*7= XPB= 47= XPD= 2:9&9.43= &8=

8-4<3= .3= T&'1*= 1= &3)= .3= 9-*= 2&3:8(7.59= 4+= 9-*= 5&5*7_= W*89*73= '149= &3)= C48

.22:3457*(.5.&9.43= C48IP= &3&1>8.8= 7*;*&18= &= 8.2.1&7= (43(*397&9.43= &3)= 8:':3.9=

(42548.9.43= 4+= 9-*= .22:345:7.+.*)= TFIIH= .3= XPB<9`= XPB2F33S`= &3)= XPB2T++3P=

(4251*=*8=F.,:7*=+.`=<*89*73b=1&3*=,8.`=C48IPb=1&3*=/81_=S.2.1&71>`=<*89*73=&3)=C48IP=

<&8= 5*7+472*)= :8.3,= WCE= +742= XPD<9`= XPD2R++,H`= XPD2G0*,D`= XPD2R02-W`=

XPD2R1,,W`= 522TTD8A<9= &3)= 522L,+P8R/0S95= F.,:7*= +/`= <*89*73b= 1&3*= +81`= C48IPb=

1&3*=38+/_=TFIIH=(43(*397&9.43=<&8=4'8*7;*)=94='*=8.,3.+.(&391>=14<=.3=XPD2R02-W=&3)=

XPD2R1,,W`= ':9= .3= XPD2R++,H= &3)= XPD2G0*,D= .9= <&8= (425&7&'1*= 94= XPD<9_=

M47*4;*7=9-*7*=<&8=&=7*2&70&'1*=).89:7'&3(*=.3=9-*=.39*,7.9>=4+=TFIIH=.3=XPD2R02-W=

&3)= XPD2R1,,W= F.,:7*= +/`= 1&3*= +,8+-_= T-*8*= 2:9&9.438= &7*= 14(&9*)= .3= C89*72.3&1=

)42&.3= 4+= XPD= &3)= 1*&)= 94= 9-*= ).884(.&9.43= 4+= 9-*= CAK= <.9-= C47*= TFIIH_= H4<*;*7=

142 F'%30# WZT TFIIH 13 3,'21 ', XPB52 ," XPB +32 ,2 !#** *',#1T

XV A% -$ WCE $0-+ H#* !#**1Q XPB52Q XPB F__SQ ," XPB TWW_P 5#0# 31#" 2- , *78# 2&# TFIIH 13 3,'21 * ,# WVZ 0#1.#!2'4#*7T C-VIP 5 1 .#0$-0+#" 31',% 2&# XVV A% -$ WCE ', XPB52Q XP F__SQ ," XPB TWW_P * ,# [V] 0#1.#!2'4#*7T

F'%30# W[T TFIIH 13 3,'21 ', XPD52Q XPD +32 ,2Q .^ TTDVA52 ," .^ TTDVA +32 ,2 !#** *',#T

XV A% -$ WCE $0-+ XPD52Q XPD RWWXHQ XPD G\VXDQ XPD R\^YWQ ," XPD R]XXWQ .^ TTDVA52 ," .^ LXWPVR[\S2. 5#0# 31#" 2- , *78# 2&# TFIIH 13 3,'21 * ,# WV] 0#1.#!2'4#*7T C-VIP 5 1 .#0$-0+#" 31',% 2&# XVV A% -$ WCE ', XPD52Q XPD RWWXHQ XPD G\VXDQ XPD R\^YWQ ," XPD R]XXWQ .^ TTDVA52 ," .^ LXWPVR[\S2. * ,# _VW[ 0#1.#!2'4#*7T WCE $0-+ &#* !#**1 1&-51 2&# TFIIH !-,20-* * ,# ^

143 522TTD8A=2:9&9.43=8-4<*)=;*7>=.38.,3.+.(&39=7*):(9.43=.3=9-*=(43(*397&9.43=4+=TFIIH=

F.,:7*=+/`=1&3*=081`=':9=9-*=.39*,7.9>=4+=TFIIH=.3=522L,+P8R/0S95=8**28=94='*=).89:7'*)=

<-*3=(425&7*)=94=9-*=522TTD8A<9=F.,:7*=+/`=1&3*=+.8+/_=I9=.8=.25479&39=94=349*=9-&9=

TFIIH= +742= 522TTD8A<9= &3)= 2:9&39= +.'74'1&89= (*11= 1.3*8= 8-4<*)= 8.2.1&7= 7*(7:.92*39=

5&99*73=4+=&11=9-*=97&38(7.59.43=2&(-.3*7>=.3(1:).3,=TFIIH=8:':3.98=R*8:198=8-4<3=.3=

2&3:8(7.59=4+=9-*=5&5*7_==

B_, ATP&8*=&3)=-*1.(&8*=&(9.;.9>=4+=TFIIH=

T4=4'8*7;*=9-*=.25479&3(*=4+=*3?>2&9.(=&(9.;.9>=.3=97&38(7.59.43&1=)>87*,:1&9.43=.3=&11=

9-*= XPB= &3)= XPD= 2:9&39= (*118`= ATP&8*= &3)= -*1.(&8*= &88&>= <&8= 5*7+472*)_= P:7.+.*)=

*=97&(98=+742='&(:14;.7:88.3+*(9*)=.38*(9=(*118=4;*7*=57*88.3,=*.9-*7=<.1)=9>5*=XPB=47=

2:9&39=XPB`=5/,=&3)=522TTD8A=52=.3=+.,:7*=<*7*=:8*)=.3=&3=.38;.974=ATP&8*=&88&>=94=

2*&8:7*=9-*=ATP&8*=&(9.;.9>=4+=XPB_==

ATP&8*=&(9.;.9>=<&8=*89.2&9*)=&8=&=+:3(9.43=4+=9-*=->)741>?*)=5-485-&9*8=P._=XPB<9=

(1*&71>=8-4<8=&=;*7>=-.,-=ATP&8*=&(9.;.9>=F.,:7*=+0A=1&3*=,8-=&3)=&)).9.43=4+=5/,=&3)=

8:'8*6:*391>= 522TTD8A= +:79-*7= *3-&3(*)= 9-*= 8.,3&1= +47= ATP&8*= &(9.;.9>= F.,:7*= +0_A=

1&3*= 081= &3)= 1&3*= +,8+-`= &184= 8-4<3= '>= 842*= 57*;.4:8= 89:).*8= J&<-&7.`= L&.3*= *9= &1_=

,**,b= C4.3`= O08*3>(-= *9= &1_= ,**1_= W-*3= +4(:8.3,= 43= XPB2F33S= &3)= XPB2T++3P`= &=

8.,3.+.(&39= )*(7*&8*= .3= 9-*= ATP&8*= &(9.;.9>= <&8= 4'8*7;*)= F.,:7*= +0B_= A349-*7=

2:9&9.43= XPB2K-.0R`= .3= ATP&8*= A= W&10*7= 249.+= I`= <-.(-= <&8= 034<3= 94= (4251*9*1>=

).87:59= 9-*= ATP&8*= &(9.;.9>= 4+= XPB= <&8= :8*)= &8= &= (439741_= T-*= ,7&5-= 7*57*8*398= 9-*=

)*38.942*97>=&3&1>8.8=.3=9*728=4+=9-*=5*7(*39&,*=4+=5-485-&9*=7*1*&8*)=P.2= ATP(P. =

+742=9<4=.3)*5*3)*39=*=5*7.2*398_=

P7*;.4:8=89:).*8=-&;*=8-4<3=9-&9=XPD=.8=).85*38&'1*=+47=97&38(7.59.43=':9=7*6:.7*)=+47=

NER= C4.3`= O08*3>(-= *9= &1_= ,**1_= T-*= 7*24;&1= 4+= 1*8.438= .3= NER= )*5*3)8= 43= 9-*=

45*3.3,=4+=9-*=DNA=&74:3)=9-*=)&2&,*)=8.9*_=M:9&9.438=.3=XPD=5749*.38=(&3=).8&'1*=

144 F'%30# W\T ATP 1# !2'4'27 -$ XPB

AT P30'$'#" XPB52 ', ',!0# 1',% +-3,2 5 1 2#12#" ', , ATP 1# 11 7 #'2� *-,# * ,#1 X Y -0 ', 2&# .0#1#,!# -$ [V ," WVV ,% -$ .30'$'#" .[X * ,#1 \ ," ]Q WV ," XV ,% -$ .30'$'#" .^ TTDA * ,#1 WVVWY 13 3,'21 -$ TFIIHT BT S'+'* 0*7Q .30'$'#" XPB52Q XPB F__SQ XPB TWW_P ," XPB KYZ\R 5#0# 2#12#" #'2� *-,# * ,#XQ[Q^ ," WW 0#1.#!2'4#*7 -0 ', .0#1#,!# -$ ',!0# 1',% .[X * ,#1 YVZQ \V]Q _VWVQ WXVWYT T&# %0 .& 0#.0#1#,21 2&# .#0!#,2 %# -$ .&-1.& 2# 0#*# 1#" P' ATP*P' $0-+ 25- ',"#.#,"#,2 #6.#0'+#,21T

145

DNA=45*3.3,='>=.25*).3,='49-=.98=ATP&8*=&3)=H*1.(&8*=&(9.;.9>=.3=NER_=C:7.4:81>` =.38

;.;4 = 89:).*8= +742= 5&9.*398= (*11= 1.3*8= &8= 8-4<3= .3= 9-*= 2&3:8(7.59= 8-4<8= 89743,=

97&38(7.59.43=)>87*,:1&9.43=&7,:.3,=+:79-*7=.3;*89.,&9.43=&'4:9=9-*=.3;41;*2*39=4+=XPD=

):7.3,=97&38(7.59.43=&8=<*11_=I9=&184=7&.8*8=9-*=6:*89.43=.+=ATP&8*=&(9.;.9>=.8=7*6:.7*)=&8=&=

574;.)*7=4+=*3*7,>=+47=.98=-*1.(&8*=&(9.;.9>=):7.3,=574249*7=45*3.3,=47=.9=-&8=).89.3(9=

+:3(9.43_=P:7.+.*)=*=97&(98=+742='&(:14;.7:88.3+*(9*)=.38*(9=(*118=4;*7*=57*88.3,=XPD<9`=

XPD2R++,H`=XPD2G0*,D`=XPD2R02-W=&3)=XPD2R1,,W=<*7*=:8*)=+47=9-*=*89.2&9.43=

4+= 9-*= ATP&8*= &(9.;.9>_= ATP&8*= &(9.;.9.*8= 4+= XPD2G0*,D= &3)= XPD2R02-W= 2:9&398=

F.,:7*=+1`=1&3*=28+-=<*7*=7*):(*)=<-.1*=49-*7=9<4b=XPD2R++,H=&3)=XPD2R1,,W=&7*=

(425&7&'1*=94=9-*=XPD<9=C425&7*=F.,:7*=+1=1&3*=/81=&3)=+.8+0=94=1&3*=,8._=D*85.9*=4+=

9-*=;*7>=89743,=)>87*,:1&9.43=.3=9-*=97&38(7.59.43=&3)=(-742&9.3=7*24)*1.3,=*;*398=.3=

&11=9-*=XPD=2:9&39=5&9.*39=(*11=1.3*8`=9-*=ATP&8*=&(9.;.9>=.8=349=.25*)*)=9-&9=89743,1>_==

N*=9`=I=&3&1>?*)=9-*=-*1.(&8*=&(9.;.9>=4+=XPD=.3=&11=9-*=XPD=2:9&9398_=P:7.+.*)=*=97&(98=

4'9&.3*)= +742= 9-*= '&(:14;.7:8*8= .3+*(9*)= .38*(9= (*118= 4;*7*=57*88.3,= XPD<9`=

XPD2R++,H`= XPD2G0*,D`= XPD2R02-W= &3)= XPD2R1,,W= <*7*= :8*)= .3= .3(7*&8.3,=

&24:39_= 5..= .8= 9-*= 7*,:1&947>= 8:':3.9= 4+= TFIIH`= .9= .39*7&(98= <.9-= XPD= &3)= -*3(*=

*3-&3(*=.98=-*1.(&8*=&(9.;.9>`=&184=8-4<3=57*;.4:81>= D:'&*1*`=P74.*99.=D*=S&39.8=*9=&1_=

,**-b=C4.3`=O08*3>(-=*9=&1_=,**1_=XPD<9=&143*=<.9-4:9=5..=8-4<8=9-*=2.3.2&1=8.,3&1=

<-.(-=.8=3*&71>=*6:.;&1*39=94=9-*=3438)*3&9:7*)=574'*=C425&7*=F.,:7*=+2=L&3*=+,=94=

1&3*= +-_= T-*= -*1.(&8*= &(9.;.9>= .3(7*&8*8= 8.,3.+.(&391>= <-*3= 5..= .39*7&(98= <.9-= XPD<9=

F.,:7*= +2= L&3*= -= ':9= 9-*7*= <&8= 34= -*1.(&8*= &(9.;.9>= 4'8*7;*)= .3= XPD2R++,H`=

XPD2R02-W= &3)= XPD2R1,,W= *=(*59= XPD2G0*,D= <-.(-= 8-4<8= 81.,-91>= 7*):(*)= ':9=

349.(*&'1*=8.,3&1=+47=9-*=).851&(*)=-*1.(&8*=574'*_==

146 F'%30# W]T ATP 1# !2'4'27 -$ XPD

I,!0# 1',% +-3,2 -$ .30'$'#" XPD52Q XPD RWWXHQ XPD G\VXDQ XPD R\^YW ," XPD R]XXW #620 !2 2#12#" ', , ATP 1# 11 7T T&# %0 .& 0#.0#1#,21 2&# .#0!#,2 %# -$ .&-1.& 2# 0#*# 1#" P' ATP*P' $0-+ 25- ',"#.#,"#,2 #6.#0'+#,21T

F'%30# W^T H#*'! 1# !2'4'27 -$ XPD

I,!0# 1',% +-3,2 -$ .30'$'#" XPD52Q XPD RWWXHQ XPD G\VXDQ XPD R\^YW ," XPD R]XXW #620 !2 2#12#" ', H#*'! 1# 11 7T T&# %0 .& 0#.0#1#,21 2&# .#0!#,2 %# -$ "'1.* !#" -*'%-,3!*#-2'"# 7 2&# XPD $0-+ 25- ',"#.#,"#,2 #6.#0'+#,21T

147 B_- =E=5*7.2*39&1=574(*):7*8a=

T-.8=5&79=4+=9-*=*=5*7.2*39&1=574(*):7*=(4;*78=431>=9-*=)*9&.18=4+=9-*=*=5*7.2*398=)43*=

.3=9-.8=8*(9.43=8:(-=&8=<*89*73`=C48IP`=ATP&8*=&88&>=&3)=H*1.(&8*=&88&>_==

W*89*73=&3)=C48IPa=

W-41*=(*11=*=97&(9=<&8=57*5&7*)=:8.3,=RIPA=':++*7=,/=2M=T7.88HC1`=5H=1_0`=+/*=2M=

N&C1`= += N43.)*9= P8.*`= += 84).:2= )*4=>(-41&9*`= *_+= SDS`= &3)= 5749*&8*= .3-.'.947=

2.=9:7*_=I22:3457*(.5.9&9.43=<&8=(&77.*)=4:9=<.9-=,**= ,=4+=WCE`=4;*73.,-9=:8.3,=

&39.'4)>= &,&.389= 50,= S&39&= C7:?= B.49*(-3414,>= &3)= )>3&'*&)8= P749*.38G=

I3;.974,*3_=A+9*7=8*;*7&1=<&8-*8=<.9-=PBST=+X=PBS(=*_*/==9<**3`=9-*=5749*.3=<&8=

*1:9*)= &3)= 14&)*)= 43= +*= SDS2PAGE`= 97&38+*77*)= 94= 3.974(*11:148*= 2*2'7&3*= &3)=

574'*)= <.9-= XPB= +B-`= XPD= ,F0`= 5/,= +D++`= 5..= +H/`= C>(= H= ,D.`= <-.(-= <*7*=

+742= IGBMC= &39.'4)>= +&(.1.9>= &3)= C)01= C8+3= S&39&= C7:?= B.49*(-3414,>_= T:':1.3=

<&8=:8*)=&8=&=14&).3,=(439741_==

ATP&8*=&88&>a=

P749*.3=+7&(9.438=<*7*=.3(:'&9*)=+47=,=-7=&9=-* °C=.3=9-*=57*8*3(*=4+=+= C.= 8-,P =ATP=

1***=C.22241`=H&792&33=A3&1>9.(=.3=&=,*= 1=7*&(9.43=;41:2*=.3=,*=2M=T7.88HC1=5H=

1_3`=.=2M=M,C1,`=+=2M=DTT`=/*=2,221=BSA`=<.9-=+,*=3,=4+=8:5*7(4.1*)=)4:'1*=897&3)=

DNA=5SK_=R*&(9.438=<*7*=89455*)='>=&)).3,=EDTA=94=/*2M=&3)=SDS=94=+=<2<_=

T-*=7*&(9.438=<*7*=9-*3=).1:9*)=/8+41)`=85499*)=4394=541>*9->1*3.2.3*=PEI=TLC=51&9*8=

M*7(0`=7:3=.3=*_/=M=L.C12+=M=+472.(=&(.)`=&3)=&:947&).4,7&5-*)_= =

H*1.(&8*=&88&>a=

H*1.(&8*= 574'*= .8= (43897:(9*)= +742= 9-*= 8.3,1*8897&3)*)= M+-= 5-&,*= <-.(-= .8= &=

->'7.).?*)= 41.,43:(1*49.)*= (4251*2*39&7>= 94= 9-*= 7*,.43= 0,+280,/+_= O3(*= 1&'*1*)= 9-*=

*3)8`=9-*=574'*=.8=51&(*)=43=&=(41:23=4+=S*5-&748*=CL8.B=,*1=+.197&9.43=P-&72&(.&D_=

T-*= 45*3.3,= 4+= DNA= .8= )*243897&9*)= '>= 9-*= ).851&(*2*39= 4+= 9-*= 41.,43:(1*49.)*8=

148

8*5&7&9*)=43=3438)*3&9:7.3,=,*1_=T-*=+7&(9.438=(439&.3.3,=9-*=5:7.+.*)=+&(947=TFIIH=.8=

.3(:'&9*)=./=2.3:9*8=&9=-1JC=.3=9-*=57*8*3(*=4+=-*1.(&8*=574'*==-****8.****=(52=b=+=8=-=

3,=DNA==.3=,*=2M=T7.8=8=HC1`=5H=1_3`=+=2M=DTT`=.=2M=M,C1=,=&3)=.=2M=ATP=`=

(439&.3.3,=+**=:,221=BSA`=94=&=+.3&1=;41:2*=4+=,/= 1=_=T-*=7*&(9.43=.8=89455*)='>=&)).3,=

+*=4+=0*= 1=2M=EDTA`=/*=,1>(*741`=*_1/=SDS=&3)=*_+=='74245-*341='1:*_=S&251*8=

&7*=7:3=43=&=+*=541>&(7>1&2.)*=,*1_=T-*=,*1=<&8=+.=*)=+47=+*=2.3:9*8=.3=+*=&(*9.(=

&(.)`=+*==2*9-&341=&3)=&:947&).4,7&5->=0=94=+,=-4:78=&9=82*=J=C_=

149 D.8(:88.43=

150 D.8(:88.43==

AT TFIIHQ NER $ !2-01 ," !&0-+ 2', 0#+-"#*',%

TFIIH= (&2*= .394= *=.89*3(*= .3= +323`= &8= 43*= 4+= 9-*= GTF8= .251.(&9*)= .3= 97&38(7.59.43= '>=

RNA= 541= II= C43&<&>= &3)= C43&<&>= +323_= T-*= :3)*789&3).3,= 4+= 9-*= TFIIH= '*(&2*=

247*= (&59.;&9.3,= &3)= .397.(&9*= <-*3`= 8*;*7&1= 89:).*8= (43+.72*)= .98= 741*= .3= 9-*=

2&.39&.3.3,= ,*342*= 89&'.1.9>= '>= .98= 5&79.(.5&9.43= .3= DNA= 3:(1*49.)*= *=(.8.43= 7*5&.7=

S(-&*++*7`= R4>= *9= &1_= +33-b= S(-&*++*7`= M43(411.3= *9= &1_= +33._= I3= 97&38(7.59.43`= TFIIH=

+:3(9.438=&8=9-*='&8&1=+&(947=<470.3,=&9=.3.9.&9.43`=574249*7=8(&5*=&3)=*&71>=*143,&9.43=

89*58=&3)=&184=.3=97&38(7.59.43=7*8.3.9.&9.43=&+9*7=RNA=541=II=5&:8.3,_=XPB=&3)=C)01=&7*=

9-*=9<4=*3?>2&9.(=8:':3.98=<-.(-=24):1&9*8=9-*8*=97&38(7.59.43&1=*;*398_=C4397&7>=94=

9-.8`= XPB= &3)= XPD= &7*= 9-*= 9<4= 0*>= -*1.(&8*8= 8:':3.98= <-.(-= 5&79.(.5&9*= .3= NER=

5&9-<&>8_= A= 7*(*39= )*;*1452*39= <-.(-= )*243897&9*= 9-&9= NER= +&(9478= 14(&1.?*= 94= 9-*=

574249*7= 4+= 9-*= &(9.;&9*)= ,*3*= <.9-4:9= &3>= DNA= )&2&,*= &3)= +:79-*7= (4397.':9*= .3=

(-742&9.3= 7*24)*1.3,= *;*398= -&;*= 897*3,9-*3= 9-*= .)*&= 4+= 9-*= .39*751&>= '*9<**3=

97&38(7.59.43= &3)= NER= S(-2.9?`= S(-2.99= *9= &1_= ,**3b= L*= M&>`= M49&8F*73&3)*8= *9= &1_=

,*+*b=L*=M&>`=F7&).3=*9=&1_=,*+,_==

A_+=B.4(-*2.897>=4+=TFIIH=&3)=9-*=4;*71&55.3,=,*3*9.(8=4+=).8*&8*8=

B*.3,= 7.,-9= &9= 9-*= *5.(*39*7= 4+= 9-*= 97&38(7.59.43= &3)= DNA= 7*5&.7`= 9-*= )*9&.1*)=

:3)*789&3).3,=4+=-4<=TFIIH=+:3(9.438=94=(4847).3&9*=9-*8*=9<4=574(*88*8=(4:1)=+:79-*7=

574;.)*=&3=*=51&3&9.43=+47=9-*=5-*349>5*8=4'8*7;*)=.3=9-*=5&9.*398='*&7.3,=2:9&9.43=.3=

XPB`=XPD=&3)=522TTD8A=8:':3.98=4+=TFIIH_=T-*=4;*71&55.3,=(1.3.(&1=+*&9:7*8=4+=9-*8*=

5&9.*398=F.,:7*=+-=+:79-*7=-&22*7=9-*=349.43=4+=.39*77*1&9.438-.5=4+=TFIIH=<.9-=NER=

+&(9478=.3=97&38(7.59.43=&3)=NER_==

151

T-*= 57*8*39= 89:)>= (1*&71>= :3)*71.3*8= 9-*= +:3(9.43&1= 7*1&9.438-.5= '*9<**3= TFIIH= &3)=

NER= +&(947= .3= 97&38(7.59.43_= H*7*= <*= )*243897&9*)= 9-&9= 57*8*3(*= 4+= TFIIH= .8= &3=

*88*39.&1= (7.9*7.43= +47= 9-*= 8>3(-743.?*)= 7*(7:.92*39= 4+= NER= +&(9478_= A3>= 2:9&9.43= .3=

XPB=&3)=XPD=8:':3.98=57*;*398=9-*=5745*7=&884(.&9.43=4+=&11=9-*=TFIIH=(42543*398=&9=

9-*=574249*7=<-.(-=(4:1)=+:79-*7=9-<&79=9-*=97&38(7.59.43&1=3*9<470=)4<3897*&2_=F47=

.389&3(*= XPB2F33S= 2:9&9.43= <*&0*38= .98= .39*7&(9.43= <.9-= 5/,`= <-.(-= 7*):(*8= XPB=

ATP&8*= &(9.;.9>= 7*6:.7*)= ):7.3,= 97&38(7.59.43= R*8:19= B_,`= F.,:7*= +0_= H4<*;*7`=

XPB2T++3P=<-.(-=8-4<8= .38;.974 =97&38(7.59.43=(425&7&'1*=94=XPB<9`=&184=8-4<8=2:(-=

7*):(*)= RNA= 8>39-*8.8= 4+= ! , = M&3:8(7.59= F.,:7*= ,= &3)= F.,:7*= /_= S.2.1&71>`=

XPD2R02-W= &3)= XPD2R1,,W= 2:9&9.438= .25&.7= 9-*= ATP&8*= &3)= -*1.(&8*= &(9.;.9>= 4+=

XPD= R*8:19= B_,`= F.,:7*= +1`= +2_= A19-4:,-`= 9.11= 34<= XPD= -&8= '**3= 8-4<3= 94= '*=

).85*38&'1*=+47=97&38(7.59.43`=034<.3,=9-*=(4251.(&9*)=3*9<470=4+=.39*7&(9.43=4+=TFIIH=

<.9-= 8*;*7&1= 5749*.38`= .9= (&3349= '*= )*3.*)= +47= .3).7*(91>= 7*,:1&9.3,= 9-*= 97&38(7.59.43_=

M47*4;*7`=9-*8*=9<4=XPD=2:9&9.438=&7*=034<3=94=).87:59=9-*=XPD85..=.39*7&(9.43=&3)=

(438*6:*391>= (&:8*= 9-*= ).884(.&9.43= 4+= CAK= +742= 9-*= (47*_= A8= &= 7*8:19= 4+= 9-.8=

).884(.&9.43=9-*=5-485-47>1&9.43=4+=CTD=4+=RNA=541=II='>=CAK=.8=-&25*7*)=1*&).3,=94=

.25&.72*39= .3= RNA= 8>39-*8.8= .38;.974= M&3:8(7.59= F.,:7*= /= C _= I3= &)).9.43= 94=

5-485-47>1&9.3,=RNA=541=II`=CAK=&184=5-485-47>1&9*8=9-*=3:(1*&7=7*(*5947=NR=&3)=

2:9&9.43= .3= C89*72.3&1= )42&.3= 4+= XPD= ).89:7'8= 9-:8= ).87:59= 9-*= 5-485-47>1&9.43=

7*8:19.3,=.3=9-*=97&38(7.59.43=)>87*,:1&9.43=4+=9-*=NR=7*85438.;*=,*3*8=R4(-*99*8E,1>`=

A)&2=*9=&1_=+331b=C425*`=D7&3*=*9=&1_=,**/_=F:79-*7247*`=C-IP=7*8:198=8-4<*)=9-&9=9-*=

5-485-47>1&9.43= 89&9:8= 4+= S*7/= 4+= CTD= 4+= RNA= 541= II= .8= <*&0*3*)= .3= 9-*8*= 9<4= XPD=

2:9&9.438=(425&7*)=94=9-*=XPD<9=(*118=M&3:8(7.59=F.,:7*=/=C=&3)=D_=T-.8=8-4<8=&=

(477*1&9.43=<.9-=&=2:(-=7*):(*)=RNA=*=57*88.43=4+= ! , =.3=9-*8*=XPD=2:9&39=(*118=

(425&7*)= 94= 9-*.7= 7*85*(9.;*= XPD<9= 4;*7*=57*88*)= (4397418= M&3:8(7.59= F.,:7*= ,_=

C43;*78*1>`= 4:7= :3)*789&3).3,= +47= 842*= 2:9&9.438= 1.0*= XPB2T++3P`= XPD2R++,H= &3)=

XPD2G0*,D=3**)*)=247*=.3;*89.,&9.43=&8=9-*=)*+*(9=.3=*3?>2&9.(=&(9.;.9>=841*1>=(&3349=

152 *=51&.3= 9-*= (4397&).(9.43= &'4:9= 9-*= 5*7+*(9= .38;.974= 97&38(7.59.43= &3)= 2:(-= 7*):(*)=

,*3*=*=57*88.43=.3=(*118=M&3:8(7.59=F.,:7*=,`=F.,:7*=/_==

A_,= TFIIH= )*514>8= 9-*= 7*(7:.92*39= 4+= NER= +&(9478= &3)= (-742&9.3= 7*24)*1.3,=*;*398=

A11=9-*=(*11:1&7=574(*88=.8=(&8(&)*=4+=*;*398=&3)=).89:7'&3(*=.3=&3>=4+=9-*=*;*398=2.,-9=

).89:7'= 9-*= 5&9-<&>= 14(&11>= &3)= 43= <-41*= ,*342*_= T-*= 8*6:*39.&1= &88*2'1>= 4+= NER=

+&(9478=.3=NER=&3)=.3=97&38(7.59.43=-&8='**3=.11:897&9*)='>=M4(6:*9=*9=&1_`=&3)=L*=M&>=

*9=&1`=7*85*(9.;*1>=M4(6:*9`=L&.3*=*9=&1_=,**2b=L*=M&>`=M49&8F*73&3)*8=*9=&1_=,*+*_=S:(-=

8*6:*39.&1= &88*2'1>= 4+= 5749*.38= (4:1)= '*= 7*6:.7*)= +47= 9-*= 5749*.385749*.3= .39*7&(9.43=

9-&9= 2*).&9*8= 9-*= 574,7*88.43= 4+= 9-*= NER= 5&9-<&>8= T&'1*= 0_= T-*= +:3(9.43&1= 1.30= 4+=

TFIIH=&3)=NER=+&(9478=.3=DNA=7*5&.7=-&8='**3=6:*89*)=4;*7=8*;*7&1=>*&78=&3)=.8=34<=

<*11= &((*59*)= ':9= 4:7= :3)*789&3).3,= 4+= 9-*.7= 7*1&9.438= 7*2&.38= +&7= +742= (4251*9*= .3=

97&38(7.59.43_=

T-*= C-IP= )&9&= &3&1>8.8= 7*;*&1*)= 9-&9= TFIIH`= .3= +&(9= .8= &(9.3,= 1.0*= &= 51&9+472= +47= 9-*=

7*(7:.92*39=4+=9-*=-./=+&(9478=):7.3,=9-*=97&38&(9.;&9.43=4+=/5/, `=&8=2:9&9.43=.3=XPB=

&3)=XPD=8:':3.98=4+=TFIIH=8.,3.+.(&391>=).89:7'8=9-*=7*(7:.92*39=4+=9-*=NER=+&(9478=&9=

9-*=574249*7_=N*;*79-*1*88`=2:9&9.43=.3=522TTD8A=)4*8=349=.25&.7=9-*=7*(7:.92*39=4+=

NER= +&(9478= <-.(-= (447).3&9*8= <.9-= 9-*= 57*;.4:8= 89:).*8= 8-4<.3,= = 522TTD8A= .8= &3=

*88*39.&1=(42543*39=+47=NER=':9=349=+47=97&38(7.59.43=C4.3`=D*=S&39.8=*9=&1_=,**0_=T-*=

57*8*3(*=4+=9-*=NER=+&(9478=&9=9-*=574249*7=.8=5*7-&58=7*6:.7*)=+47=9-*=*89&'1.8-2*39=4+=

9-*= &557457.&9*= (-742&9.3= 89&9*= &74:3)= 9-*= 574249*7= +47= &((:7&9*= &3)= 459.2&1= ,*3*=

*=57*88.43= B&77*94`= S(-&+*7= *9= &1_= ,**1b= L*= M&>`= M49&8F*73&3)*8= *9= &1_= ,*+*_= T-*=

.3;*89.,&9.43= 4+= 9-*= (-742&9.3= 89&9:8= &74:3)= 9-*= 574249*7= 7*;*&1*)= &= 8.,3&9:7*= 2&70=

<.9-=9-*=*37.(-2*39=4+=9-*=).82*9->1&9.43=4+=H-K.`=&(*9>1&9.43=4+=H-K3=&3)=)*(7*&8*=4+=

).82*9->1&9.43= 4+= H-K3`= <-.(-= (7*&9*8= 45*3= 47= &(9.;*= (-742&9.3= 2.1.*:= &74:3)= 9-*=

574249*7= +47= 9-*= 97&38(7.59.43= M&3:8(7.59= F.,:7*= -_= S.1*3(.3,= 4+= XPC`= XPA= &3)=

153

ERCC+=&3)=2:9&9.43=.3=XPC`=XPA`=ERCC+`=XPG=&3)=XPF=8-4<*)=9-*=).89:7'&3(*=.3=

9-*=5&99*73=4+=*37.(-2*39=4+=9-*8*=-.8943*=24).+.(&9.438L*=M&>`=M49&8F*73&3)*8=*9=&1_=

,*+*_=B*8.)*8=51&>.3,=&=741*=.3=*89&'1.8-.3,=-.8943*=PTM8`=.9=-&8='*(42*=.3(7*&8.3,1>=

&55&7*39=9-&9=9-*=7*(7:.92*39=4+=9-*=NER=+&(9478=43=9-*=&(9.;*=574249*78=4+=RNA=541=I=

&3)=541=II=,*3*8=(&3=.3):(*=DNA=)*2*9->1&9.43=&74:3)=9-*=574249*7_=I9=<&8=8-4<3=

9-&9=034(0)4<3=4+=G&))./&`=XPA`=XPG`=XPF`=47=TAF+,=47=97*&92*39=<.9-=)7:,8=9-&9=

.3-.'.9= NER= (&:8*8= ->5*72*9->1&9.43= 4+= 7DNA`= *89&'1.8-*8= -*9*74(-742&9.(= -.8943*=

2&708`=&3)=.25&.78=97&38(7.59.43=S(-2.9?`=S(-2.99=*9=&1_=,**3_==

I3=+&(9`=NER=+&(9478=8:(-=&8==*3)43:(1*&8*8=XPG`=XPF=&3)=TFIIH=-&;*='**3=574548*)=

8*;*7&1=>*&7='*+47*=94='*=.3;41;*)=.3=DNA=)*2*9->1&9.43=9-74:,-=NER=5&9-<&>=C-:=

&3)=M&>3*=+330_=S.3(*=NER=5&9-<&>=.8=57*+*7*39.&11>=.3;41;*)=.3=9-*=7*5&.7=4+=':10>=

1*8.438= &3)= &= 2*9->1= ,74:5= .8= 349= &9= &11= ':10>`= .9= .8= -&7)= 94= (43(*.;*= 9-&9= 9-*= 2*9->1=

(>948.3*= .8= 7*(4,3.?*)= &8= 8:(-= &3)= 9-&9= 9-*= NER= +&(9478= +:3(9.438= .3= 97&38(7.59.43&1=

7*,:1&9.43= .77*85*(9.;*= 4+= DNA= )*2*9->1&9.43_ = S:55479.3,= 9-*= 8&2*= (43(*59`= 4:7=

U3M*DIP=)&9&=8-4<8=9-&9=TFIIH=.8=5488.'1>=*3,&,*)=.3=DNA=)*2*9->1&9.43_=M:9&9.43=

.3= XPB= &3)= XPD= 8:':3.98= (1*&71>= ).87:598= 9-*= DNA= )*2*9->1&9.43= 1*&;.3,= 9-*=

574249*7= .3= ->5*72*9->1&9*)= 89&9*`= <-.1*= 522TTD8A= 2:9&9.43= )4*8= 349= &++*(9= 9-*=

574(*88_= A= +&.1:7*= 94= )*2*9->1&9*= 9-*= DNA= &3)= &(9.;&9*= 9-*= ,*3*= (4:1)= '7.3,=

97&38(7.59.43&1=)>87*,:1&9.43=5*7=8*=.3=9-*=(*11_=T-*=DNA=)*2*9->1&9.43=+4114<.3,=,*3*=

&(9.;&9.43=.8=3472&11>=4'8*7;*)=.3=9-*=(148*=574=.2.9>=94=9-*=7*,.438=<-*7*=DNA='7*&08=

4((:78_=T-*=DNA='7*&08=&7*=+472*)='>=9-*=*3)43:(1*&8*=XPG=&9=9-*=574249*7=&3)=XPF=

&9= 574249*7= &8= 4'8*7;*)= .3= XPB<9`= XPD<9= &3)= 522TTD8A<9= M&3:8(7.59`= F.,:7*= ,_=

T-*=+472&9.43=4+=DNA='7*&08=<&8=349=(438.89*39=<.9-=9-*=57*8*3(*=4+=XPG=&3)=XPF=&9=

574249*7=&3)=9*72.3&947=7*85*(9.;*1>`=<-.(-=<&8=14,.(&1=.3=&11=4+=9-*=2:9&39=(*11=1.3*_=

E3.,2&9.(&11>`=)*85.9*=4+=9-*=57*8*3(*=4+=&11=9-*=GTF8`=TFIIH=8:':3.98=&3)=NER=+&(9478`=

DNA=)*2*9->1&9.43`=9-*7*=<&8=34=DNA='7*&0=.3=522TTD8A=(*118_=T-*=8.,3.+.(&3(*=4+=

154

9-*= DNA= '7*&0= +472&9.43= .8= 89.11= *1:8.;*b= 5488.'1>= .9= (4:1)= '*= 7*6:.7*)= +47= 9-*=

3:(1*4842*= 7*&77&3,*2*39= &3)= (-742&9.3= 7*1&=&9.43`= 7*6:.7*)= +47= 9-*= +472&9.43= 4+=

(-742&9.3=1445.3,_=A184`=.9=.8=034<3=9-&9=TFIIH=.39*7&(98=5->8.(&11>=<.9-=XPG=):7.3,=

97&38(7.59.43=&8=<*11=&8=.3=NER=):7.3,=9-*=+472&9.43=4+=57*8.3(.8.43=(4251*=_=I3=+&(9`=

XPG=<&8=&184=(438.)*7*)=94='*=9-*=*1*;*39-=8:':3.9=4+=TFIIH=I94`=K:7&40&=*9=&1_=,**1b=

S(-C7*7=,**2_=M:9&9.43=.3=TFIIH=2.,-9=).89:7'=9-*=897:(9:7&1=&884(.&9.43=4+=TFIIH=&3)=

XPG=<-.(-=(4:1)=5488.'1>=&19*7=9-*=*3)43:(1*&8*=&(9.;.9>=4+=XPG`=9-:8=-.3)*7.3,=9-*=

+472&9.43=4+=DNA='7*&08_=I9=(4:1)=&184='*=5488.'1*=9-&9=XPG=+:3(9.43=&8=&=7*,:1&947=+47=

TFIIH`=89&'.1.?.3,=9-*=&884(.&9.43='*9<**3=(47*8TFIIH`=CAD=&3)=XPD_==

C439741= 4+= ,*3*= *=57*88.43= '>= +472&9.43= 4+= (-742&9.3= 14458= '*9<**3= 2:19.51*=

7*,:1&947>= *1*2*398= 14(&9*)= 4;*7= 1&7,*= ,*342.(= ).89&3(*8= -&8= '**3= )4(:2*39*)= .3=

8*;*7&1= 89:).*8_= S:(-= 5->8.(&1= .39*7&(9.438= &243,= 7*,:1&947>= *1*2*398= &7*= &.)*)= '>=

(-742&9.3= 47,&3.?*78= 8:(-= &8= (4-*8.43= &3)= CTCF_= S.3(*= 4:7= C-IP= )&9&= 8-4<8= 9-*=

57*8*3(*= 4+=&11=9-*=GTF8`=TFIIH=&3)=NER= +&(9478=85*(.+.(&11>=&9=9<4=7*,.43b= 574249*7=

&3)= 9*72.3&947= M&3:8(7.59= F.,:7*= ,`= S:551*2*39&7>= F.,:7*= +`= <*= &3&1>?*)= 9-*=

(-742&9.3= 1445= +472&9.43= '*9<**3= 574249*7= &3)= 9*72.3&947_= W*= 4'8*7;*)= 9-7**=

(43).9.438b=+472&9.43=4+=(-742&9.3=1445.3,=&8=.3=XPB<9`=XPD<9=&3)=522TTD8A<9`=34=

(-742&9.3=1445.3,=&8=.3=XPB2F33S`=XPD2R++,H`=XPD2R02-W=&3)=XPD2R1,,W=&3)=&=

5*78.89*39= (-742&9.3= 1445.3,=&8=.3= XPB2T++3P`=XPD2G0*,D=&3)=522TTD8A_=A'8*3(*=

&3)= 57*8*3(*= 4+= (-742&9.3= 1445.3,= .8= ;*7>= 2:(-= :3)*789&3)&'1*= ':9= 9-*= 57*8*3(*= 4+=

(438.89*39= 1445= .8= &= 2&99*7= 4+= +:79-*7= .3;*89.,&9.43_= P7*8*3(*= 4+= 5*78.89*39= (-742&9.3=

1445.3,=2.,-9=&184=8*7;*=94=.3-.'.9=,*3*=*=57*88.43_==

A194,*9-*7`= 9-.8= 89:)>= -&8= :3)*78(47*)= 9-*= +:3(9.438= 4+= TFIIH= )**5*7`= <-.(-= 8:,,*89=

9-&9= *&(-= 2:9&9.43= .3= TFIIH= .8= 85*(.+.(_= E&(-= 4+= 9-*= 2:9&9.438= 89:).*)= 7*57*8*398=

97&38(7.59.43&1=)>87*,:1&9.43=<-*9-*7=.9=.8=XPB=&3)=XPD=2:9&398=<-*7*=.9=.8=&++*(9.3,=

7.,-9= &9= 9-*= 1*;*1= 4+= 7*(7:.92*39= 4+= NER= &3)= 9-*3= +:79-*7= )4<3897*&2= 47= 522TTD8A=

155 M% : 1QJs PBLF S PBLT P PDLRH PDLG D PDLR PDLR ] LLP R S ]

TFIIH CQJsV`0VR 1J CQJsV`0VR 1J LQ1V` TFIIH LQ1V` TFIIH LQ1V` TFIIH LQ1V`TFIIH LQ1V` TFIIH HQI]Qs1 1QJ HVCCs ` HVCCs ` :JR 1J HQJR HQJR HQJHVJ `: 1QJ HQJHVJ `: 1QJ CQJR :JR V:@ 1JR 01 `Q HVJ `: 1QJ V5 ` HVJ `: 1QJ ` :JR R1s %`Gs :JR R1s %`Gs HVJ `: 1QJ V5 ` 1J V`:H 1QJ 1J V`:H 1QJ Q` G1JR1J$ IJ V`:H 1QJ Q` IJ V`:H 1QJ Q` ^``QI PB 11 . ] :ss:7 G5 PD 11 . PD 11 . `Vs]VH 10V G7 1JR01 `Q ] G5 ` ] G5 ` HVCC C1JV_ G1JR1J$ :ss:7 G HVC1H:sV NQ R1s %`GVR G NQ CQI]CV VC7 M%H. CQI]CV VC7 CQI]CV VC7 NA ^1JR01 `Q_ R1s %`GVR G R1s`%] s `VR%HVR R1s`%] s R1s`%] s .VC1H:sV .VC1H:sV .VC1H:sV .VC1H:sV :H 101 7 ` :H 101 7 ` :H 101 7 ` :H 101 7 `

ATP:sV RVR%HVR ATP:sV RVR%HVR NQ RVR%HVR RVR%HVR NQ NA ^1JR01 `Q_ :H 101 7 ` ATP:sV R1s %`GVR ` ATP:sV ATP:sV R1s %`GVR ` :H 101 7 ` :H 101 7 ` :H 101 7 `

P.QsR D1s %`GVR ` NQ NQ NQ H7]QR D1s %`GVR ` NQ R1s %`GVR ` ].Q`7C: 1QJ R1s %`GVR ` R1s %`GVR ` R1s %`GVR ` ].Qs].Q`7C: 1 QJ Q` NR R NQ R1s %`GVR `

RNA RVR%HVR NQ NQ NQ RVR%HVR RVR%HVR NQ R1s %GVR : s7J .Vs1s RNA s7J .Vs1s ` R1s %`GVR ` R1s %`GVR ` R1s %`GVR ` RNA RNA s7J .Vs1s ` ^1JR01 `Q_ s7J .Vs1s `

P`QIQ V` D1s %`GVR G NQ NA NA II]VRV II]VRV NA Q]VJ1J$ R1s %`GVR G P`QIQ V` P`QIQ V` ^1JR01 `Q_ Q]VJ1J$ G Q]VJ1J$ G DNA `V]:1` CPD `V]:1` NQ CQI]CV V CQI]CV V LQ1 CV0VC Q` LQ1 CV0VC Q` LQ1 CV0VC Q` ^1JR01 `Q_ RV`1H1VJH7 G R1s %`GVR G NER NER NER :H 101 7 H5 ` NER :H 101 7 H5 ` NER :H 101 7 : RV`1H1VJH7 H5 ` RV`1H1VJH7 H5 `

T *# [T F3,!2'-,1 -$ TFIIH

NR8=N:(1*&7=7*(*5947`=NER8=N:(1*49.)*=*=(.8.43=7*5&.7`=NA8=N49=&;&.1&'1*==

&8C4.3`=D*=S&39.8=*9=&1_=,**0`='8C4.3`=O08*3>(-=*9=&1_=,**1`=(8=D:'&*1*`=P74.*99.=D*=

S&39.8=*9=&1_=,**-`=)8=K*7.*1`=S9&7>=*9=&1_=,**,`=*8B499&`=N&7)4=*9=&1_=,**,`=+8&3&1>?*)=

.3=57*8*39=89:)>=

156 2:9&39=<-*7*=.9=.8=).89:7'.3,=247*=&9=9-*=1*;*1=4+=(-742&9.3=7*24)*1.3,_=T-*8*=8:'91*=

)*+*(98=&9=97&38(7.59.43&1=1*;*1=7*897.(9=:8=+742=,.;.3,=&=(4-*7*39=*=51&3&9.43=+47=8:(-=

(4251*== 241*(:1&7= 2&3.+*89&9.43= 4+= 9-*8*= 8>3)742*8_= H4<*;*7`= &9= 9-*= 8&2*= 9.2*=

8:,,*89=9-&9=9-*=;&7>.3,=7&3,*=4+=(1.3.(&1=+*&9:7*8=.3=9-*=XP`=XP2CS=&3)=TTD=5&9.*398=

(4:1)=&7.8*=+742=9-*8*=8:'91*=)*+*(98=.3=97&38(7.59.43_=T&'1*=3`=7*57*8*39=9-*=(43(1:8.43=

4+=9-*=&3&1>8.8=4'9&.3*)=.3=9-*=57*8*39=89:)>=&8=<*11=&8=+742=57*;.4:8=7*1&9*)=89:).*8_==

BT F3230# "'0#!2'-, $-0 TFIIH ," NER $ !2-01

T7&38(7.59.43&1= 7*,:1&9.43= (&3= 4((:7= &9= 9<4= .39*7(433*(9*)= 1*;*18a= 9-*= +.789= .3;41;*8=

97&38(7.59.43= +&(9478= &3)= 9-*= 97&38(7.59.43= &55&7&9:8`= &3)= 9-*= 8*(43)= .3;41;*8=

(-742&9.3=897:(9:7*=&3)=.98=7*,:1&9478_=I3=+472*7=,*3*=7*,:1&9.43=9>5.(&11>=4((:78=<-*3=

97&38(7.59.43= +&(9478= '.3)8= 94= (4+&(9478= 8:(-= &8= (4&(9.;&9478= &3)= (47*57*88478= ):7.3,=

.3.9.&9.43=47=*143,&9.43`=<-.1*=.3=1&9*7=7*,:1&9.43=4((:78='>=(-742&9.3=7*24)*1*78=&3)=

5749*.38=9-&9='.3)=&3)=24).+>=9-*=-.8943*8=D*8(7.'*)=.3=C-&59*7=I_D_=T-*=97&38(7.59.43=

+&(947`= (4+&(9478= &3)= (-742&9.3= 7*,:1&9478= &7*= 7*6:.7*2*39= +47= &11= 9-*= (*11= 9>5*8= &3)=

2.87*,:1&9.43=4+=9-*8*=+&(9478=(&3=(&:8*=&='74&)=7&3,*=4+=).8*&8*_==

T-*=57*8*39=89:)>=,.;*8=&3=4;*7;.*<=9-&9=-4<=&=8:'91*=)*+*(9=.3=&3>=97&38(7.59.43=+&(947=

(&3=1*&)=94=9-*=97&38(7.59.43=)>87*,:1&9.43`=+:79-*7=,.;.3,=7.8*=94=).8*&8*8=1.0*=XP`=TTD`=

CS=&3)=XP2CS_=U8.3,=8:(-=).8*&8*=24)*1`=9-.8=89:)>=-&8=574;.)*)=.3+472&9.43=<-.(-=

)*+.3.9*1>=(4397.':9*)=94=4:7=034<1*),*=4+=,*3*=*=57*88.43_=O:7=4'8*7;&9.43=8:,,*898=

&3=.251.(&9.43=4+=TFIIH=.3=9-*=&(9.;*=DNA=)*2*9->1&9.43=&884(.&9*)=94=-.8943*=PTM8=

&7,:*8= +:79-*7= .3;*89.,&9.43_= T-*= 6:*89.43= &7.8*8= .8= 9-&9b= D4*8= TFIIH= &++*(98= &= 8*9= 4+=

,*3*8= 431>= 47= .9= .3+1:*3(*8= 9-*= &(9.;*= DNA= )*2*9->1&9.43= ,14'&11>= 9-74:,-4:9= 9-*=

,*342*`=.8=89.11=&=2&99*7=4+=.3;*89.,&9.43_=TFIIH=&184=.39*7&(98=<.9-=XPG`=XPF=&3)=XPC=

<-.(-= &7*= )*'&9*)= 94= '*= .3;41;*)= .3= DNA= )*2*9->1&9.43= 9-74:,-= BER= &3)= NER_=

U3)*789&3).3,= 9-*= (447).3&9.43= 4+= 9-*8*= 97&38(7.59.43= &3)= NER= +&(9478`= 5&79.(:1&71>=

157

9-*.7=1.30=94=9-*=DNA=2*9->1&9.43=&3)=)*2*9->1&9.43=2&(-.3*7.*8=<.11=8-4<=9-&9=.+=&3)=

-4<= 9-*= (-742&9.3= ).7*(98= DNA= 2*9->197&38+*7&8*= 47= )*2*9->1&8*_= T-.8= (4:1)= 8-*)=

842*=1.,-9=43=9-*=(&:8*=4+=8*;*7&1=).8*&8*8=.3(1:).3,=9-48*=).8(:88*)=.3=57*8*39=89:)>_=

M47*4;*7=&(9.;*=DNA=)*2*9->1&9.43=.8=&884(.&9*)=<.9-=9-*=DNA=(43+472&9.43b=R81445`=

+472*)=&9=9-*=8.9*=4+=97&38(7.59.43=':''1*_=O3*=0*>=+&(947=<-.(-=,.;*8=7.8*=94=9-*=R81445=

.8=9-*=GC=80*<`=<-.(-=.8=2*&8:7*=4+=9-*=).897.':9.43=4+=,:&3.3*=G=&3)=(>948.3*=C=

7*8.):*8=&(7488=9-*=9<4=DNA=897&3)8_=D:7.3,=97&38(7.59.43`=.9=.8=9-4:,-9=9-&9=9-*=3*<1>=

8>39-*8.?*)= RNA= 897&3)`= :543= 1*&;.3,= 9-*= RNA= *=.9= (-&33*1= 4+= 9-*= 2.,7&9.3,= RNA=

541>2*7&8*`= (&3= (425*9*= <.9-= 9-*= 34389*251&9*= DNA= 897&3)= +47= 7*8&33*&1.3,= 94= 9-*=

9*251&9*=DNA=897&3)_=G.334`=L499=*9=&1_=,*+,_=I9=(4:1)='*=5488.'1*=9-&9=R814458=2&>=

&184=8.,3&1=9-*=7*(7:.92*39=4+=DNA=)*2*9->1&9.3,=(4251*=*8=94=CG=574249*78=&3)=9-*=

:3<.3).3,=4+=9-*=DNA=7*1&9*)=94=9-*=TFIIH=-*1.(&8*8=.8=7*6:.7*8=+47=9-*=+472&9.43=4+=R8

1445=&3)=(438*6:*391>=9-*=DNA=)*2*9->1&9.43_=

F472&9.43= 4+= RNAaDNA= ->'7.)8= .8= &184= )*5*3)*39= 43= 9-*= 89&'.1.9>= 4+= 9-*= 7*8:19.3,=

RNAaDNA=->'7.)=&3)=9-*=*=548*)=897*9(-=4+=88DNA_=C1:89*78=4+=G=97&(98=43=9-*=3438

9*251&9*= 897&3)= (&3= +41)= .394= &= 89&'1*= G86:&)7:51*== 897:(9:7*`= <-.(-= 2&>= -*15= 94=

89&'.1.?*=9-*=*=548*)=88DNA=7*,.43=4+=9-*=R81445_=I9=.8=.25479&39=94=349*=9-&9=XPB=&3)=

XPD=-*1.(&8*=&7*=8-4<3=94='*=7*(7:.9*)=&9=9-*=8.9*=4+=G86:&)7:51*=*8=897:(9:7*=G7&>`=

V&11:7=*9=&1_=,*+._=T-*8*=89:).*8=&,&.3=8:,,*89=9-&9=TFIIH=-41)8=842*=247*=+:3(9.438=

&3)=94=)*1;*=)**5=(*79&.31>=247*=.3;*89.,&9.438=&7*=7*6:.7*)_=

A349-*7= .25479&39= &85*(9= <-.(-= (4397.':9*8= 94= 9-*= ,*3*= 7*,:1&9.43= .8= 9-*= 897:(9:7&1=

47,&3.?&9.43= 4+= 9-*= ,*342*_= I9= <.11= 9-*7*+47*= 349= '*= 8:757.8.3,= .+= 9-*= )*+*(98= .3= 9-*=

-.,-*7= 47)*7= (-742&9.3= 897:(9:7*= &3)= (-7424842*= 897:(9:7*= (&3= (&:8*= ).8*&8*8_=

H4<*;*7`=.9=.8=4+9*3=349=;*7>=(1*&7=9-&9=9-*=5-*349>5*=4+=).8*&8*=4'8*7;*)=.8='*(&:8*=4+=

9-*= (-&3,*8= '74:,-9= :5= '>= 14(&1= -.8943*= 24).+.(&9.438= 7*8:19.3,= .3= &19*7*)= ,*3*8

158

*=57*88.43= 47= '*(&:8*= 4+= 897:(9:7&1= (-742&9.3= )*+*(98_= T-*7*= &7*= 8*;*7&1= &7(-.9*(9:7&1=

5749*.38`=1.0*=-*9*74(-742&9.3=5749*.3=+=HP+=)*(47&9.3,=9-*=(-742&9.3=+.'*78`=47=CTCF=

&3)= (4-*8.3= <-.(-= &7*= .3;41;*)= .3= 9-*= +472&9.43= 4+= (-742&9.3= 14458`= -&;*= '**3=

.251.(&9*)=.3=).8*&8*_=CTCF=&184=51&>8=&=741*=.3=89&'.1.?.3,=9-*=-.,-*7=47)*7=(-742&9.3=

897:(9:7*=&3)=-&8='**3=8:,,*89*)=94=51&>=&=741*=.3=-:2&3=)*;*1452*39&1=).847)*78=1.0*=

S.1;*78R:88*11= &3)= B*(0<.9-8W.*)*2&3= S>3)742*_= C4-*8.3= &143,= <.9-= 2*).&947= .8=

&349-*7=5749*.3=034<3=+47=.98=741*=.3=(-742&9.3=1445.3,_=I9=<4:1)=349='*=&=8:757.8*`=.+=

9-*8*= &7(-.9*(9:7&1= 5749*.38= <470= 94,*9-*7= .3= &= (42'.3&947.&1= 2&33*7= .3= 9-*= ,*3*=

7*,:1&9.43_==

T-*=TFIIH=<&8=&184=+4:3)=94='*=.39*7&(9.3,=<.9-=2*).&947=(4251*==&3)=9-*=2*).&947=

(4251*==(433*(98=9-*=DNA='.3).3,=5749*.38=94=9-*='&8&1=97&38(7.59.43=2&(-.3*7>=P488`=

E'2*.*7=*9=&1_=,*+-_=A184=2:9&9.43=.3=2*).&947=,.;*8=7.8*=94=8>3)742*8=*=-.'.9.3,=;*7>=

2:(-= 8.2.1&7= (1.3.(&1= 5-*349>5*8_= S9:).*8= '&8*)= 43= 9-*= +:3(9.43= &3)= 897:(9:7&1=

(-&7&(9*7.?&9.43= 4+= TFIIH= &3)= 2*).&947= ):7.3,= ).++*7*39= 97&38(7.59.43= *;*39= (4:1)=

+:79-*7= 574;.)*= 034<1*),*= 4+= ,*3*= *=57*88.43_= U3)*789&3).3,= 9-*= (*11:1&7= 241*(:1&7=

2*(-&3.82= '*-.3)= 97&38(7.59.43&1= 7*,:1&9.43= .3= XP`= TTD`= XP2CS= 5&9.*398= (4:1)=

)*+.3.9*1>=-*15=:8=94=)*;*145='*99*7=).&,3489.(=94418=&3)=9-*7&5*:9.(=897&9*,.*8=+47=9-*=

(4251*==).8*&8*8=.3=+:9:7*_=

159 C43(1:8.43=

T-*=57*8*39=89:)>=:3)*78(47*8=9-*=+:3(9.438=4+=TFIIH=.3=97&38(7.59.43_=U3)*789&3).3,=

9-*=7*1&9.438-.5=4+=TFIIH=94=NER=+&(9478=.3=97&38(7.59.43=(4:1)=,.;*=&3=.38.,-9=43=9-*=

<.)*=7&3,*=4+=9-*=(1.3.(&1=+*&9:7*8=8-4<3='>=9-*=XP`=TTD`=XP2CS=5&9.*398_==

M:9&9.438= .3= XPB= &3)= XPD= 8:':3.98= 1*&)= 94= 9-*= ).89:7'&3(*= .3= 9-*= *3?>2&9.(

&(9.;.9.*8= 4+= TFIIH_= T-*8*= &(9.;.9.*8= &7*= 7*6:.7*)= ):7.3,= 9-*= 97&38(7.59.43= &3)

):7.3,=9-*=NER_

P7*8*3(*= 4+= +:3(9.43&1= TFIIH= .8= *88*39.&1= (43).9.43= +47= 9-*= 7*(7:.92*39= 4+= NER

+&(9478_=TFIIH=&(98=&8=9-*=51&9+472=+47=9-*=&88*2'1>=4+=9-*=NER=+&(9478_=M:9&9.438

.3=XPB=&3)=XPD=-&25*7*)=9-*=5745*7=&884(.&9.43=4+=9-*=TFIIH=(42543*398=&9

9-*=574249*7`=<-.(-=9-<&79=9-*=97&38(7.59.43&1=3*9<470=)4<3897*&2_

T-*=(-742&9.3=7*24)*1.3,=&884(.&9*)=<.9-=NER=+&(9478=8**28=94='*=9-<&79*)='>

9-*= )&2&,*)= TFIIH_= T-*= 5&99*738= 4+= 9-*= -.8943*= 24).+.(&9.43= &3)= DNA

)*2*9->1&9.43=<*7*=7&9-*7=&19*7*)=.3=&11=9-*=XP`=TTD=&3)=XP2CS=(*118_

T-*=TFIIH=8-4<=.98=.25479&3(*=.3=DNA='7*&08=&884(.&9*)=<.9-=XPG=&3)=XPF=&9

574249*7=&3)=9*72.3&947=&3)=+:79-*7=&.)=9-*=CTCF=2*).&9*)=(-742&9.3=1445.3,

'*9<**3=9-*8*=9<4=7*,.438_

522TTD8A=(*118`=-4<*;*7=8-4<8=34=).89:7'&3(*=.3=NER=+&(9478=7*(7:.92*39=&3)

431>= &++*(9= DNA= '7*&0= &3)= (-742&9.3= 1445.3,_= T-.8= 8:,,*898= &= +:3(9.43

&884(.&9.43= 4+= 522TTD8A= <.9-= XPG= 47= XPF= ).7*(91>= 47= ;.&= 842*= 49-*7= 8:':3.9

<.9-.3=TFIIH_

160 C-,$#0#,!#S

R41*= 4+= TFIIH= .3= 7*(7:.92*39= 4+= NER= +&(9478= ):7.3,= 97&38(7.59.43= 57*8*39*)= &9

)4(947&1=8(-441=&9=-T2*=/4:73S*=)*8=)4(947&398=):=C&25:8=)*=2S)*(.3*=)*=S97&8'4:7,`=

N4;*2'*7=,*+,_=

R41*= 4+= TFIIH= 1.30*)= 94= NER= +&(9478= .3= 9-*= 7*,:1&9.43= 4+= ,*3*= *=57*88.43

57*8*39*)= &9= 9-*= G*72&38F7*3(-= DNA= R*5&.7= M**9.3,a= E5.,*3*9.(8= &3)= G*342*=

I39*,7.9>=I110.7(-`=F7&3(*`=O(94'*7=,*+-_=

C-301#1S

A11=74&)8=1*&)=94=RNAa=3*<8=&3)=5*785*(9.;*8`=J&3:&7>=,*++

C43,*3.9&1=).847)*78=&3)=9-*.7=*2'7>43.(=47.,.38`=J&3:&7>=,*+,

I39*,7&9*)=897:(9:7&1='.414,>`=J&3:&7>8F*'7:&7>=,*+,

I38.,-9=+742=.38;.;4=89:).*8`=M&7(-=,*+,

H4<= 94= 2&0*= 9-*= 7.,-9= .257*88.43= &3)= )*1.;*7= (1*&7= 2*88&,*8= &143,= 5:'1.(

85*&0.3,=8=M*7(0=M.11.547*`=M&7(-=,*+,

E++*(9.;*=8(.*39.+.(=<7.9.3,`=A57.1=,*+,

C&7**7=)*;*1452*39=<4708-45`=J:3*=,*+,

S(.*39.+.(=C422:3.(&9.43=]=P:'1.8-.3,`=F*'7:&7>=,*+-

I3):897.&1=;.8.9=94=F8H4++2&338=L&=R4(-*`=B&8*1`=S<.9?*71&3)`=J&3:&7>=,*+.

161 B.'1.4,7&5->=

AGR%C`:.I:J5 85 I8 IC 1s5 V :C8 ^_8 ARCH RQI:1J Q` PD5 :J :JH.Q`1J$ ]C: `QÌ `Q` CAK .: HQJR1 1QJs TFIIH DNA `V]:1` :JR `:JsH`1] 1QJ :H 101 1Vs8 P`QHVVR1J$s Q` .V N: 1QJ:C AH:RVI7 Q` SH1VJHVs Q` .V J1 VR S : Vs Q` AIV`1H: ^ _7 E R 8 A$%1C:`RF%VJ Vs5 J85 M8 F`V$QsQ5 V :C8 ^ _8 ] LTTDA O0V`V6]`Vss1QJ EJ.:JHVs RI``:R1: 1QJ RVs1s :JHV :JR S%]]`VssVs TFIIH M% : 1QJs 1J : \1 :C1H]D`QsQ].1C:\L1 :C1H] T`1H.Q .1QR7s `Q].7 MQRVC8 PLQS $VJV 1Hs ^_7 V 8 A@Q%C1 H.V05 S85 S8 C.%1@Q05 V :C8 ^_8 TFIIH 1s JV$: 10VC7 `V$%C: VR G7 HR@ RHQJ :1J1J$ IVR1: Q` HQI]CV6Vs8 N: %`V ^ _7 R 8 A@Q%C1 H.V05 S85 T8 P8 M:@VC:5 V :C8 ^ _8 RV_%1`VIVJ `Q` TFIIH @1J:sV :H 101 7 1J `:JsH`1] 1QJ G7 RNA ]QC7IV`:sV II8 N: %`V ^ _7 R 8 AC T:JQ%`75 85 A8 P1s@%JQ05 V :C8 ^_8 1 :I1J A :JR `V 1JQ1R s1$J:C1J$7 $VJQI1H :JR JQJ$VJQI1H V``VH s8 JQ%`J:C Q` C1]1R `VsV:`H. ^ _7 R 8 ACC``V75 8 G8 :JR A8 E8 M1`s@7 ^ _8 S `%H %`:C MQR1`1H: 1QJs Q` H1s QJVs :JR .V1` PQss1GCV RQCV 1J .V RV$%C: 1QJ Q` RNA S7J .Vs1s8 SH1VJHV ^ _7 8 AJR`:%5 J8RC85 L8 0:J RV P:sH.5 V :C8 ^ _8 GVJQIVR1RV LQH: 1QJ Q` .V CQ:H 10: Q` MVR1: Q`7 B1JR1J$ 11 .Q% AH 10: 1QJ :JR T`:Js1VJ CR@ IJ V`:H 1QJ QJ DNA8 MQCVH%C:` CVCC ^_7 R 8 A`H.:IG:%C 5 J85 G8 P:J5 V :C8 ^ _8 FCP5 .V RAP R1J V`:H 1J$ s%G%J1 Q` : .%I:J ]`Q V1J ].Qs].: :sV .: RV].Qs].Q`7C: Vs .V H:`GQ67CR VÌ1J:C RQI:1J Q` RNA ]QC7IV`:sV IIO8 T.V AÌ:H.V5 K8RJ85 H8 KV VJGV`$V`5 V :C8 ^_8 A`H.1 VH %`V Q` 1J1 1: 1QJRHQI]V VJ Rs%G%J1 RNA ]QC7IV`:sV II8 P`QHVVR1J$s Q` .V N: 1QJ:C AH:RVI7 Q` SH1VJHVs ^_7 R 8 B:JJ1s V`5 A8 J8 :JR T8 KQ%<:`1RVs ^_8 RV$%C: 1QJ Q` H.`QI: 1J G7 .1s QJV IQR1`1H: 1QJs8 CVCC `VsV:`H. ^_7 R 8 B:``V Q5 G85 A8 SH.:`V`5 V :C8 ^ _8 G:RR : ]`QIQ Vs V]1$VJV 1H $VJV :H 10: 1QJ G7 `V]:1`RIVR1: VR DNA RVIV .7C: 1QJ8 N: %`V ^ _7 R 8 BVJs:%RV5 O85 F8 BQJJV 5 V :C8 ^ _8 RV$%C: VR ].Qs].Q`7C: 1QJ Q` .V RNA ]QC7IV`:sV II CR VÌ1J:C RQI:1J ^CTD_8 B1QH.VI CVCC B1QC ^_7 R 8 B1s1:s5 T85 J8 M8 PV`Q5 V :C8 ^ _8 DNARDV]VJRVJ ATP:sV AH 101 7 Q` B:H V`1:C PB HVC1H:sVs8 B1QH.VI1s `7 ^_7 R 8 BC:H@5 JQs.%: C85 C8 :J RVH.VI5 V :C8 ^_8 H1s QJV L7s1JV MV .7C: 1QJ D7J:I1Hs7 Es :GC1s.IVJ 5 RV$%C: 1QJ5 :JR B1QCQ$1H:C II]:H 8 MQCVH%C:` CVCC ^_7 R 8 BQ :5 E85 T8 N:`RQ5 V :C8 ^_8 RVR%HVR CV0VC Q` .V `V]:1`L `:JsH`1] 1QJ `:H Q` TFIIH 1J `1H.Q .1QR7s `Q].78 H%I MQC GVJV ^_7 R 8 BQ%CQJ5 S85 B8 P`:RV RB:C:RV5 V :C8 ^_8 HSP :JR I s RTPLP`V`QCR1JRC1@V CQH.:]V`QJV A`V IJ0QC0VR 1J .V C7 Q]C:sI1H AssVIGC7 Q` RNA PQC7IV`:sV II8 MQCVH%C:` CVCC ^ _7 R 8 BQ%`$%V 5 85 8 AJR`75 V :C8 ^_8 HV V`QR1IV`1H HQI]CV6 Q` RAR :JR RR J%HCV:` `VHV] Q` C1$:JRR G1JR1J$ RQI:1Js7 ]%`1`1H: 1QJ5 H`7s :CC1<: 1QJ5 :JR ]`VC1I1J:`7 R`:7 R1```:H 1QJ :J:C7s1s8 P`Q V1J V6]`Vss1QJ :JR ]%`1`1H: 1QJ ^_7 R 8

162 B`:R`Q`R5 P8 T85 A8 M8 GQCRs V1J5 V :C8 ^_8 C:JHV` :JR JV%`QCQ$1H RV$VJV`: 1QJ 1J 6V`QRVÌ: ]1$IVJ Qs%I7 CQJ$ VÌ `QCCQ1R%] H.:`:H V`1sVs .V `QCV Q` DNA `V]:1`8 JQ%`J:C Q` IVR1H:C $VJV 1Hs 7 R 8 B`:Rs.V`5 J85 F8 CQ1J5 V :C8 ^_8 D1s 1JH `QCVs `Q` .V .VC1H:sVs Q` TFIIH 1J `:JsH`1] 1J1 1: 1QJ :JR ]`QIQ V` VsH:]V8 J B1QC C.VI ^_7 R 8 B`:JR5 M85 C8 LV%`VJ 5 V :C8 ^ _8 T.`VVRR1IVJs1QJ:C s `%H %`Vs Q` .V TAFIIRHQJ :1J1J$ HQI]CV6Vs TFIID :JR TFTC8 SH1VJHV ^ _7 R 8 B`Q%$. QJ5 B8 C85 H8 S V1J$`1IsRQ 1`5 V :C8 ^ _8 M% : 1QJs 1J .V 6V`QRVÌ: ]1$IVJ Qs%I $`Q%] D DNA `V]:1`L `:JsH`1] 1QJ $VJV 1J ]: 1VJ s 11 . `1H.Q .1QR7s `Q].78 N: %`V $JV 1Hs 7 R 8 B%R.%5 A8 S8 :JR N8 NQ7 ^_8 D1`VH H.:JJVC1J$ Q` `V 1JQ1H :H1R GV 1VVJ HVCC%C:` `V 1JQ1H :H1RRG1JR1J$ ]`Q V1J II :JR `V 1JQ1H :H1R `VHV] Q` sVJs1 1

163 CQ1J5 F85 J8 A%`1QC5 V :C8 ^_8 P.Qs].Q`7C: 1QJ Q` PB .VC1H:sV `V$%C: Vs TFIIH J%HCVQ 1RV V6H1s1QJ `V]:1` :H 101 78 EIGQ J ^_7 R 8 CQ1J5 F85 E8 BV`$I:JJ5 V :C8 ^ _8 M% : 1QJs 1J PB :JR PD .VC1H:sVs `Q%JR 1J 6V`QRVÌ: ]1$IVJ Qs%I ]: 1VJ s 1I]:1` .V `:JsH`1] 1QJ `%JH 1QJ Q` TFIIH8 EIGQ J ^ _7 R 8 CQ1J5 F85 L8 P8 DV S:J 1s5 V :C8 ^ _8 ] LTTDRA :s : RV]:1`RS]VH1`1H TFIIH S%G%J1 8 MQC CVCC ^_7 R 8 CQ1J5 F85 8 [email protected] V :C8 ^ _8 D1s 1JH RQCVs `Q` .V PBL] :JR PDL] S%GHQI]CV6Vs Q` TFIIH 1J D:I:$VR DNA O]VJ1J$ R%`1J$ N%HCVQ 1RV E6H1s1QJ RV]:1`8 MQC CVCC ^_7 R 8 CQ1J5 F85 8 [email protected] V :C8 ^ _8 N%HCVQ 1RV V6H1s1QJ `V]:1` R`10VJ G7 .V R1ssQH1: 1QJ Q` CAK ``QI TFIIH8 MQC CVCC ^_7 R8 CQI]V5 E85 P8 D`:JV5 V :C8 ^ _8 D7s`V$%C: 1QJ Q` .V PV`Q61sQIV P`QC1`V`: Q`RAH 10: VR RVHV] Q` T:`$V GVJVs G7 PD M% : 1QJs8 MQC CVCC B1QC ^_7 R 8 CQI]V5 E8 :JR J8 M8 E$C7 ^_8 TFIIH7 1.VJ `:JsH`1] 1QJ IV DNA `V]:1`8 N: RV0 MQC CVCC B1QC ^ _7 R 8 CQI]V5 E85 M8 M:CV`G:5 V :C8 ^ _8 NV%`QCQ$1H:C RV`VH s 1J `1H.Q .1QR7s `Q].7 `V0V:C : HQ:H 10: Q` `%JH 1QJ Q` TFIIH8 N: %`V JV%`QsH1VJHV ^_7 R8 CQJ:1:75 J8 8 :JR R8 C8 CQJ:1:7 ^ _8 A I%C 1s%G%J1 `:JsH`1] 1QJ `:H Q` VssVJ 1:C `Q` :HH%`: V 1J1 1: 1QJ G7 RNA ]QC7IV`:sV II8 J8 B1QC8 C.VI8 ^_7 R 8 CQJ:1:75 J8 85 J8 P8 H:JCV75 V :C8 ^ _8 T`:JsH`1] 1QJ 1J1 1: VR G7 RNA ]QC7IV`:sV II :JR `:JsH`1] 1QJ `:H Q`s ``QI C10V`8 S `%H %`V :JR :H 1QJ Q` `:JsH`1] 1QJ `:H Q`s V]s1CQJ :JR :%8 J8 B1QC8 C.VI8 7 R 8 CQJ:1:75 R8 C8 :JR J8 8 CQJ:1:7 ^ _8 AJ RNA ]QC7IV`:sV II `:JsH`1] 1QJ `:H Q` .:s :J :ssQH1: VR DNARRV]VJRVJ ATP:sV ^RATP:sV_ :H 101 7 s `QJ$C7 s 1I%C: VR G7 TATA `V$1QJ Q` ]`QIQ V`s8 P`QH8 N: C AH:R8 SH18 8S8A8 7 R 8 CQ`RVJ5 J85 D8 L8 C:RVJ:5 V :C8 ^ _8 A %J1_%V s `%H %`V : .V H:`GQ67C VÌ1J%s Q` .V C:`$Vs s%G%J1 Q` .V V%@:`7Q 1H RNA ]QC7IV`:sV II8 P`QH8 N: C8 AH:R8 SH18 SA 7 R 8 C`:IV`5 P85 K8 J8 AÌ:H.V5 V :C8 ^ _8 S `%H %`V Q` E%@:`7Q 1H RNA PQC7IV`:sVs8 AJJ%:C RV01V1 Q` B1Q].7s1Hs ^_7 R 8 DACVss1Q5 A8 C85 I8 C8 G8 V:0V`5 V :C8 ^ _8 AHV 7C: 1QJRIJR%HVR T`:JsH`1] 1QJ Is RV_%1`VR `Q` AH 10V DNA DVIV .7C: 1QJ 1J MV .7C: 1QJRS1CVJHVR GVJVs8 MQCVH%C:` :JR CVCC%C:` B1QCQ$7 ^_7 R 8 RV BQV`5 J8 :JR J8 H8 HQV1=I:@V`s ^_8 N%HCVQ 1RV V6H1s1QJ `V]:1` :JR .%I:J s7JR`QIVs8 C:`H1JQ$VJVs1s ^_7 R 8 DV VV`RRK:s VCV1J5 E8 A85 8 KV1=

164 DVJ$5 85 B8 M:CVHQ0<5 V :C8 ^ _8 TFIIB RVHQ$J1 1QJ ECVIVJ s CQJ `QC .V TFIIARNC A61s 1J T`:JsH`1] 1QJ:C RV$%C: 1QJ8 MQCVH%C:` :JR CVCC%C:` B1QCQ$7 ^ _7 R8 D.:CC%1J5 C85 J8 E8 C:`CsQJ5 V :C8 ^ _8 S `%H %`V :JR C1$:JR Q` : .1s QJV :HV 7C `:Js`V`:sV G`QIQRQI:1J8 N: %`V ^ _7 R 8 D1G1Q0:JJ:5 J8 J8 :JR K8 H8 K`:VIV` ^_8 S.1J1J$ : C1$. QJ 6V`QRVÌ: ]1$IVJ Qs%I8 T.V JQ%`J:C Q` 1J0Vs 1$: 10V RVÌ: QCQ$7 ^ P _7 R 8 D1C1Q` .5 F8 J8 :JR P8 C.:IGQJ ^_8 N%HCV:` `VHV] Q`s HQQ`R1J: V .V :H 101 1Vs Q` H.`QI: 1J `VIQRVC1J$ HQI]CV6Vs :JR HQ:H 10: Q`s Q `:H1C1 : V 1J1 1: 1QJ Q` `:JsH`1] 1QJ8 OJHQ$VJV ^_7 R 8 D`:]@1J5 R85 J8 T8 RV:`RQJ5 V :C8 ^ _8 D%:C `QCV Q` TFIIH 1J DNA V6H1s1QJ `V]:1` :JR 1J `:JsH`1] 1QJ G7 RNA ]QC7IV`:sV II8 N: %`V 7 R 8 D%G:VCV5 S85 L8 P`Q1V 1 DV S:J 1s5 V :C8 ^_8 B:s:C `:JsH`1] 1QJ RV`VH R1sH`1I1J: Vs GV 1VVJ 6V`QRVÌ: ]1$IVJ Qs%I :JR `1H.Q .1QR7s `Q].7 1J PD ]: 1VJ s8 MQC CVCC ^ _7 R 8 D7sQJ5 H8 J8 :JR P8 E8 `1$. ^ _8 IJ `1Js1H:CC7 %Js `%H %`VR ]`Q V1Js :JR .V1` `%JH 1QJs8 N: %`V `V01V1s8 MQCVH%C:` HVCC G1QCQ$7 ^_7 R 8 E$CQ``5 S85 M8 D1VJs G1V`5 V :C8 ^_8 ]R: 1J$ .V RNA ]QC7IV`:sV CTD HQRV7 :RR1J$ $VJVRs]VH1`1H C:7V`s8 T`VJRs 1J $VJV 1Hs 7 TIG ^ _7 R8 EC K:RV`15 B85 S8 MVRCV`5 V :C8 ^ _8 GVJV CQQ]1J$ 1s HQJ`V``VR G7 :H 10: Q`RRV]VJRVJ 1J V`:H 1QJ Q` `:JsH`1] 1QJ 1J1 1: 1QJ :JR VÌ1J: 1QJ I:H.1JV`1Vs8 T.V JQ%`J:C Q` G1QCQ$1H:C H.VI1s `7 ^ _7 R 8 E0:Js5 E85 J8 G8 MQ$$s5 V :C8 ^ _8 MVH.:J1sI Q` Q]VJ HQI]CV6 :JR R%:C 1JH1s1QJ `QÌ: 1QJ G7 .%I:J J%HCVQ 1RV V6H1s1QJ `V]:1` `:H Q`s8 EIGQ J ^_7 R 8 F:J5 L85 A8 S8 A`0:15 V :C8 ^ _8 CQJsV`0VR PB HQ`V s `%H %`V :JR IQ 1`s `Q` DNA %J11JR1J$7 1I]C1H: 1QJs `Q` ]: .1:7 sVCVH 1QJ Q` `:JsH`1] 1QJ Q` V6H1s1QJ `V]:1`8 MQC CVCC ^_7 R 8 FV:0V`5 8 J85 O8 G1CV:R15 V :C8 ^ _8 P%`1`1H: 1QJ :JR H.:`:H V`1<: 1QJ Q` 7V:s RNA ]QC7IV`:sV II `:JsH`1] 1QJ `:H Q` G8 J8 B1QC8 C.VI8 ^ _7 R 8 FV:0V`5 8 J85 N8 L8 HVJ`75 V :C8 ^ _8 GVJVs `Q` T`G5 T`G5 :JR T`G s%G%J1 s Q` 7V:s `:JsH`1] 1QJL`V]:1` `:H Q` IIH8 HQIQCQ$7 Q .%I:J H7HC1JRRV]VJRVJ @1J:sV :H 10: 1J$ @1J:sV :JR IIH s%G%J1 s8 J B1QC C.VI ^_7 R 8 FV:0V`5 8 J85 J8 Q8 S0V=s `%]5 V :C8 ^ _8 D%:C `QCVs Q` : I%C 1]`Q V1J HQI]CV6 ``QI S8 HV`V01s1:V 1J `:JsH`1] 1QJ :JR DNA `V]:1`8 CVCC ^ _7 R 8 FCQ`Vs5 O85 I8 H:5 V :C8 ^ _8 F:H Q`s 1J0QC0VR 1J s]VH1`1H `:JsH`1] 1QJ G7 I:II:C1:J RNA ]QC7IV`:sV II8 P%`1`1H: 1QJ :JR s%G%J1 HQI]Qs1 1QJ Q` `:JsH`1] 1QJ `:H Q` IIF8 J8 B1QC8 C.VI8 ^_7 R 8 FCQ`Vs5 O85 H8 L%5 V :C8 ^ _8 T.V sI:CC s%G%J1 Q` `:JsH`1] 1QJ `:H Q` IIF `VH`%1 s RNA ]QC7IV`:sV II 1J Q .V ]`V1J1 1: 1QJ HQI]CV68 P`QH8 N: C8 AH:R8 N: C8 SA 7 R8 FCQ`Vs5 O85 H8 L%5 V :C8 ^ _8 F:H Q`s 1J0QC0VR 1J s]VH1`1H `:JsH`1] 1QJ G7 I:II:C1:J RNA ]QC7IV`:sV II8 IRVJ 1`1H: 1QJ :JR H.:`:H V`1<: 1QJ Q` `:H Q` IIH8 J B1QC C.VI ^_7 R 8 FQ%s V`15 M85 8 VÌV%CVJ5 V :C8 ^ _8 CQH@:7JV s7JR`QIV A :JR B ]`Q V1Js R1``V`VJ 1:CC7 `V$%C: V `VH`%1 IVJ Q` H.`QI: 1J `VIQRVC1J$ :JR `V]:1` `:H Q`s Q s :CCVR RNA ]QC7IV`:sV II 1J 010Q8 MQC CVCC ^_7 R 8 F`V$QsQ5 M85 J8 P8 L:1JV5 V :C8 ^ _8 DNA RV]:1` :JR T`:JsH`1] 1QJ:C DV`1H1VJH1Vs C:%sVR G7 M% : 1QJs 1J .V D`QsQ].1C: ] S%G%J1 Q` TFIIH GVJV`: V DV0VCQ]IVJ :C DV`VH s :JR C.`QIQsQIV F`:$1C1 78 MQC CVCC B1QC ^_7 R 8 F%@s5 F85 P8 J8 H%`R5 V :C8 ^_8 T.V IV .7CRC]GRG1JR1J$ ]`Q V1J MVCP C1J@s DNA IV .7C: 1QJ Q .1s QJV IV .7C: 1QJ8 T.V JQ%`J:C Q` G1QCQ$1H:C H.VI1s `7 ^ _7 R8

165 F%ss5 J8 O8 :JR J8 A8 T:1JV` ^_8 PB :JR PD .VC1H:sVs 1J TFIIH Q`H.Vs `: V DNA R%]CV6 Q]VJ1J$ :JR R:I:$V 0V`1`1H: 1QJ Q HQQ`R1J: V `V]:1` 11 . `:JsH`1] 1QJ :JR HVCC H7HCV 01: CAK @1J:sV8 DNA `V]:1` ^ _7 R 8 G:J%<:5 M8 S8 L8 C8 C8 M8 G8 G8 S8 R8 B8 M8 A8 P8 D8 P8 J8 M8 S8 D8 B8 M8 ^_8 GVJV 1H 1J:H 10: 1QJ Q` CR@ CV:Rs Q HVCC H7HCV :``Vs :JR 1JR%HVs ]`VI: %`V :$1J$ R%V Q :R%C s VI HVCC V6.:%s 1QJ8 T.V EMBO =Q%`J:C ^_7 R 8 GV5 H8 :JR R8 G8 RQVRV` ^ _8 T.V .1$. IQG1C1 7 $`Q%] ]`Q V1J HMG H:J `V0V`s1GC7 1J.1G1 HC:ss II $VJV `:JsH`1] 1QJ G7 1J V`:H 1QJ 11 . TATARG1JR1J$ ]`Q V1J8 J8 B1QC8 C.VI8 7 R 8 GV`:`R5 M85 L8 F1sH.V`5 V :C8 ^ _8 P%`1`1H: 1QJ :JR 1J V`:H 1QJ ]`Q]V` 1Vs Q` .V .%I:J RNA ]QC7IV`:sV BÎI_ $VJV`:C `:JsH`1] 1QJ `:H Q` BTF8 T.V JQ%`J:C Q` G1QCQ$1H:C H.VI1s `7 ^_7 R 8 GV`GV`5 H8 P85 M8 H:$I:JJ5 V :C8 ^ _8 RNA ]QC7IV`:sV II CR VÌ1J:C RQI:1J `V_%1`VR `Q` VJ.:JHV`R R`10VJ `:JsH`1] 1QJ8 N: %`V 7 R 8 GVÌ:1J5 P85 B8 S :VCs5 V :C8 ^ _8 O0V`01V1 Q` JQIVJHC: %`V Q` J%HCV:` `VHV] Q`s8 P.:Ì:HQCQ$1H:C `V01V1s ^_7 R 8 GV`s.VJ

166 HV5 85 J8 F:J$5 V :C8 ^_8 S `%H %`:C 01s%:C1<: 1QJ Q` @V7 s V]s 1J .%I:J `:JsH`1] 1QJ 1J1 1: 1QJ8 N: %`V ^ _7 R 8 HVG`:5 F8 :JR M8 K:]Qs1 ^ _8 OJ R1sV:sVs Q` .V s@1J 1JHC%R1J$ V6:J .VI: :5 0QC%IV III8 NV1 S7RVJ.:I SQH 7 R 8 HVI:0: .75 K8 C8 :JR 8 N:$:`:=: ^ _8 DNA IV .7C: 1QJ 1J I7HQG:H V`1:7 AGsVJHV Q` IV .7C: 1QJ : GATC ^D:I_ :JR CCALTGG ^DHI_ sV_%VJHVs8 FEMS III%JQCQ$7 MVR1H:C M1H`QG1QCQ$7 ^_7 R 8 H1`QsV5 8 :JR J8 L8 M:JCV7 ^_8 RNA ]QC7IV`:sV II :JR .V 1J V$`: 1QJ Q` J%HCV:` V0VJ s8 GVJVs RV0VCQ]IVJ ^_7 R 8 HQV1=I:@V`s5 J8 H8 ^_8 GVJQIV I:1J VJ:JHV IVH.:J1sIs `Q` ]`V0VJ 1J$ H:JHV`8 N: %`V ^ _7 R 8 HQQ$s `: VJ5 D85 A8 L8 N1$$5 V :C8 ^_8 R:]1R s11 H.1J$ Q` TFIIH GV 1VVJ RNA ]QC7IV`:sV I :JR II `:JsH`1] 1QJ :JR DNA `V]:1` 1J 010Q8 MQC CVCC ^ _7 R 8 HQ`[email protected] M85 T8 :I:IQ Q5 V :C8 ^ _8 AJ:C7s1s Q` s `%H %`V `VC: 1QJs.1]s Q` 7V:s TATARGQ6 G1JR1J$ `:H Q` TFIID8 CVCC 7 R 8 Hs1J5 J8RP85 K8 1:J$5 V :C8 ^_8 F%JH 1QJ :JR CQJ `QC Q` RNA PQC7IV`:sV II CRTV`I1J:C DQI:1J P.Qs].Q`7C: 1QJ 1J V` VG`: V T`:JsH`1] 1QJ :JR RNA P`QHVss1J$8 MQCVH%C:` :JR CVCC%C:` B1QCQ$7 ^_7 R 8 I 1J5 P8 H85 A8 S:`:s1J5 V :C8 ^_8 T`1H.Q .1QR7s `Q].77 %]R: V QJ .V s%C`%`RRV`1H1VJ G`1 CV .:1` s7JR`QIVs8 J AI AH:R DV`I: QC ^ _7 R 6 _%1< R 8 I Q5 S85 I8 K%`:Q@:5 V :C8 ^ _8 PG S :G1C1

167 K:I1%H.15 S85 M8 S:1=Q5 V :C8 ^_8 T`:JsCQH: 1QJ Q` CQH@:7JV s7JR`QIV $`Q%] A ]`Q V1J Q .V J%HCV:` I: `167 ]Qss1GCV `VCV0:JHV Q `:JsH`1] 1QJRHQ%]CVR DNA `V]:1`8 P`QH N: C AH:R SH1 S A ^_7 R 8 K:J$5 J8 J85 D8 T8 A%GCV5 V :C8 ^ _8 AJ:C7s1s Q` .V 7V:s `:JsH`1] 1QJ `:H Q` TFIIA7 R1s 1JH `%JH 1QJ:C `V$1QJs :JR : ]QC7IV`:sV IIRs]VH1`1H `QCV 1J G:s:C :JR :H 10: VR `:JsH`1] 1QJ8 MQC8 CVCC8 B1QC8 7 R8 KVVJV75 S85 G8 J8 C.:J$5 V :C8 ^ _8 C.:`:H V`1<: 1QJ Q` : .%I:J DNA R:I:$V G1JR1J$ ]`Q V1J 1I]C1H: VR 1J 6V`QRVÌ: ]1$IVJ Qs%I E8 J8 B1QC8 C.VI8 7 R8 KVCCVJGV`$V`5 E85 C8 DQI1J$%V<5 V :C8 ^ _8 SQC% 1QJ s `%H %`V Q` .V CR VÌ1J:C RQI:1J Q` TFIIH P s%G%J1 `V0V:Cs : JQ0VC 7]V Q` CC `1J$ RQI:1J 1J0QC0VR 1J ]`Q V1JR]`Q V1J 1J V`:H 1QJs8 J B1QC C.VI8 KV`1VC5 A85 A8 S :`75 V :C8 ^_8 PD I% : 1QJs ]`V0VJ TFIIHRRV]VJRVJ `:Js:H 10: 1QJ G7 J%HCV:` `VHV] Q`s :JR ].Qs].Q`7C: 1QJ Q` RAR:C].:8 CVCC ^_7 R 8 KV`s.J:`5 E85 S8 8 %5 V :C8 ^ _8 III%JQ:``1J1 7 ]%`1`1H: 1QJ :JR `%JH 1QJ:C H.:`:H V`1<: 1QJ Q` .%I:J `:JsH`1] 1QJ `:H Q` IIH :JR RNA ]QC7IV`:sV II ``QI HCQJ:C HVCC C1JVs .: HQJR1 1QJ:CC7 V6]`Vss V]1 Q]VR :$$VR s%G%J1 s Q` .V I%C 1]`Q V1J HQI]CV6Vs8 J B1QC C.VI ^ _7 R 8 K1I5 J8 K85 D8 P: VC5 V :C8 ^ _8 CQJ `:s 1J$ s `%H %`:C 1I]:H s 1JR%HVR G7 H1sRs7J H7HCQG% :JV R1IV` :JR ^ R_ :RR%H 1J DNA R%]CV6 RVH:IV`s7 1I]C1H: 1QJ 1J I% :$VJVs1s :JR `V]:1` :H 101 78 P.Q QH.VI1s `7 :JR ].Q QG1QCQ$7 ^_7 R 8 K1I5 M85 H8 S%.5 V :C8 ^ _8 P.Qs].Q`7C: 1QJ Q` .V 7V:s R]G CR VÌ1J:C RQI:1J : sV`1JVs 5 5 :JR 8 J B1QC C.VI ^ _7 R 8 KJV

168 L:``:JHVR:J:ssV5 J85 G8 A`sVJV:%C 5 V :C8 ^_8 S `%H %`:C :JR `%JH 1QJ:C V01RVJHV .: R:R HQI]V Vs 11 . R:R `Q` G1JR1J$ Q .V T`G s%G%J1 Q` TFIIH 1J NER8 N%HCV1H :H1Rs `VsV:`H. ^_7 R 8 L:$`:J$V5 T85 A8 N8 K:]:J1R1s5 V :C8 ^ _8 NV1 HQ`V ]`QIQ V` VCVIVJ 1J RNA ]QC7IV`:sVRII RV]VJRVJ `:JsH`1] 1QJ7 sV_%VJHVRs]VH1`1H DNA G1JR1J$ G7 `:JsH`1] 1QJ `:H Q` IIB8 GVJVs DV08 7 R8 L:1JV5 J8 P85 B8 N8 S1J$.5 V :C8 ^ _8 A ].7s1QCQ$1H:C `QCV `Q` $VJV CQQ]s 1J 7V:s 8 GVJVs RV0VCQ]IVJ ^_7 R 8 L:@V5 R8 J85 E8 L8 BQV V`%V`5 V :C8 ^_8 T.V SV_%VJHVRS]VH1`1H T`:JsH`1] 1QJ F:H Q` HRJ%J T:`$V s CQH@:7JV S7JR`QIV P`Q V1J B Q RV$%C: V T`:JsH`1] 1QJ :JR C.`QI: 1J S `%H %`V8 PLQS $VJV 1Hs ^_7 V 8 L:%$VC5 85 C8 D:CCQ<5 V :C8 ^_8 M% : 1QJ %]R: V `Q` .V CSBLERCC :JR CSALERCC $VJVs 1J0QC0VR 1J CQH@:7JV s7JR`QIV8 H%I:J I% : 1QJ ^_7 R 8 L:%`VJ F1sH.V`5 M8 G85 C.`1s 1:J C.:C% 5 0Vs L% <5S:JR`1JV H%IGV` 5 M:s:IQ Q K:JJQ5 P1V``V C.:IGQJ5JV:JRM:`H E$C7 ^ _8 \SH1VJHVR RF1sH.V`R R 8]R`]8 CV M:1`V5 A85 C8 TV7ss1V`5 V :C8 ^_8 A %J1_%V sVHQJR:`7Rs `%H %`V s11 H. HQJ `QCs HQJs 1 % 10V $VJV `V]`Vss1QJ G7 `V 1JQ1H :H1R `VHV] Q`8 N: %`V s `%H %`:C IQCVH%C:` G1QCQ$7 ^ _7 R 8 LV M:75 N85 D8 F`:R1J5 V :C8 ^_8 PG :JR PF EJRQJ%HCV:sVs T`1$$V` C.`QI: 1J LQQ]1J$ :JR DNA DVIV .7C: 1QJ `Q` AHH%`: V E6]`Vss1QJ Q` AH 10: VR GVJVs8 MQC CVCC ^_7 R 8 LV M:75 N85 D8 MQ :RFV`J:JRVs5 V :C8 ^_8 NER `:H Q`s :`V `VH`%1 VR Q :H 10V ]`QIQ V`s :JR `:H1C1 : V H.`QI: 1J IQR1`1H: 1QJ `Q` `:JsH`1] 1QJ 1J .V :GsVJHV Q` V6Q$VJQ%s $VJQ Q61H LVV5 J8 H85 C8 J8 P:`@5 V :C8 ^_8 NMR s `%H %`V Q` .V DNA RVH:IV` R%]CV6 HQJ :1J1J$ RQ%GCV T G I1sI: H.Vs Q` H1sRs7J H7HCQG% :JV ]7`1I1R1JV R1IV`7 1I]C1H: 1QJs `Q` DNA R:I:$V `VHQ$J1 1QJ G7 .V PCR.HRB HQI]CV68 N%HCV1H :H1Rs `VsV:`H. ^ _7 R 8 LV.I:JJ5 A8 R85 D8 MHG1GGQJ5 V :C8 ^_8 V`QRVÌ: ]1$IVJ Qs%I8 O`].:JV J R:`V D1s 7 8 LV15 L85 D8 RVJ5 V :C8 ^ _8 T.V RAP s%G%J1 Q` .%I:J `:JsH`1] 1QJ `:H Q` IIF .:s s1I1C:` `QCVs 1J 1J1 1: 1QJ :JR VCQJ$: 1QJ8 MQCVH%C:` CVCC%C:` B1QCQ$7 ^_7 R 8 LV11s5 B8 A85 T8 K8 K1I5 V :C8 ^_8 A RQ1Js `V:I VCVIVJ 1J .V .%I:J GV :R$CQG1J ]`QIQ V`7 V01RVJHV Q` V6 VJRVR sV_%VJHVRs]VH1`1H `:JsH`1] 1QJ `:H Q` IID HQJ :H s8 P`QHVVR1J$s Q` .V N: 1QJ:C AH:RVI7 Q` SH1VJHVs Q` .V J1 VR S : Vs Q` AIV`1H: ^_7 R 8 L15 E8 :JR 8 .:J$ ^_8 DNA MV .7C: 1QJ 1J M:II:Cs8 CQCR S]`1J$ H:`GQ` ]V`s]VH 10Vs 1J G1QCQ$7 L1JR:.C5 T8 ^ _8 AJ NR$C7HQs1R:sV ``QI EsH.V`1H.1: HQC1 .: `VCV:sVs ``VV %`:H1C ``QI DNA HQJ :1J1J$ ^ _8 RV:I1J: VR H7 Qs1JV `Vs1R%Vs8 P`QHVVR1J$s Q` .V N: 1QJ:C AH:RVI7 Q` SH1VJHVs Q` .V J1 VR S : Vs Q` AIV`1H: ^ _7 R 8 L1JRVJG:%I5 85 D8 D1H@sQJ5 V :C8 ^_8 V`QRVÌ: ]1$IVJ Qs%ILHQH@:7JV s7JR`QIV HQI]CV67 `1`s JV%`Q]: .QCQ$1H:C s %R7 :JR `V01V1 Q` V1$. Q .V` H:sVs8 E%`Q]V:J =Q%`J:C Q` ]:VR1: `1H JV%`QCQ$7 7 EJPN 7 Q``1H1:C =Q%`J:C Q` .V E%`Q]V:J P:VR1: `1H NV%`QCQ$7 SQH1V 7 ^ _7 R8 L1%5 H85 J8 R%RQC`5 V :C8 ^ _8 S `%H %`V Q` .V DNA `V]:1` .VC1H:sV PD8 CVCC ^ _7 R 8 L1%5 85 D8 A8 B%s.JVCC5 V :C8 ^_8 S `%H %`V Q` :J RNA ]QC7IV`:sV IIRTFIIB HQI]CV6 :JR .V `:JsH`1] 1QJ 1J1 1: 1QJ IVH.:J1sI8 SH1VJHV ^ _7 R 8 L%5 H85 L8 :1VC5 V :C8 ^ _8 H%I:J $VJV`:C `:JsH`1] 1QJ `:H Q` IIH ].Qs].Q`7C: Vs .V CR VÌ1J:C RQI:1J Q` RNA ]QC7IV`:sV II8 N: %`V ^ _7 R 8

169 L%sV5 D8 S85 L8 C8 S]:J$CV`5 V :C8 ^_8 E``1H1VJ :JR `:]1R J%HCVQsQIV `:0V`s:C G7 RNA ]QC7IV`:sV II RV]VJRs QJ : HQIG1J: 1QJ Q` `:JsH`1] VCQJ$: 1QJ `:H Q`s8 T.V JQ%`J:C Q` G1QCQ$1H:C H.VI1s `7 ^ _7 R 8 M:1 15 A8 :JR A8 C8 D`Q.: ^_8 T.7I1JV DNA $C7HQs7C:sV H:J `:]1RC7 V6H1sV R`Q`I7CH7 Qs1JV :JR R H:`GQ67CH7 Qs1JV7 PQ VJ 1:C 1I]C1H: 1QJs `Q` :H 10V RVIV .7C: 1QJ Q` C]G s1 Vs8 JQ%`J:C Q` B1QCQ$1H:C C.VI1s `78 M:CRQJ:RQ5 E85 R8 S.1V@.: :`5 V :C8 ^ _8 A .%I:J RNA ]QC7IV`:sV II HQI]CV6 :ssQH1: VR 11 . SRB :JR DNAR`V]:1` ]`Q V1Js8 N: %`V ^ _7 R 8 M:J$VCsRQ``5 D8 J8 :JR R8 M8 E0:Js ^ _8 T.V RR .V V`QR1IV`s :JR Q`].:J `VHV] Q`s8 CVCC ^ _7 R 8 M:J$VCsRQ``5 D8 J85 C8 T.%IIVC5 V :C8 ^ _8 T.V J%HCV:` `VHV] Q` s%]V``:I1C77 .V sVHQJR RVH:RV8 CVCC ^ _7 R 8 M:` V1=J5 J8 A85 H8 L:Js5 V :C8 ^_8 JRV`s :JR1J$ J%HCVQ 1RV V6H1s1QJ `V]:1` :JR 1 s `QCVs 1J H:JHV` :JR :$V1J$8 N: %`V `V01V1s8 MQCVH%C:` HVCC G1QCQ$7 ^ _7 R 8 M: s%15 T85 J8 SV$:CC5 V :C8 ^ _8 M%C 1]CV `:H Q`s `V_%1`VR `Q` :HH%`: V 1J1 1: 1QJ Q` `:JsH`1] 1QJ G7 ]%`1`1VR RNA ]QC7IV`:sV II8 J8 B1QC8 C.VI 7 R 8 M:6QJ5 M8 E85 J8 A8 GQQR`1H.5 V :C8 ^ _8 T`:JsH`1] 1QJ `:H Q` IIE G1JRs ]`V`V`VJ 1:CC7 Q RNA ]QC7IV`:sV II: :JR `VH`%1 s TFIIH7 : IQRVC `Q` ]`QIQ V` HCV:`:JHV8 GVJVs DV0 ^ _7 R 8 MVCCQJ5 I85 G8 S]10:@5 V :C8 ^ _8 SVCVH 10V `VIQ0:C Q` `:JsH`1] 1QJRGCQH@1J$ DNA R:I:$V ``QI .V `:JsH`1GVR s `:JR Q` .V I:II:C1:J DHFR $VJV8 CVCC 7 R 8 MV`1JQ5 A85 K8 R8 M:RRVJ5 V :C8 ^ _8 DNA Q]Q1sQIV`:sV I 1s 1J0QC0VR 1J GQ . `V]`Vss1QJ :JR :H 10: 1QJ Q` `:JsH`1] 1QJ8 N: %`V 7 R8 M1s VC15 T8 ^_8 CVCC G1QCQ$7 Q` `:JsH`1] 1QJ :JR ]`VRIRNA s]C1H1J$7 J%HCV:` :`H.1 VH %`V IVV s J%HCV:` `%JH 1QJ8 JQ%`J:C Q` HVCC sH1VJHV ^_7 R 8 MQH_%V 5 85 J8 P8 L:1JV5 V :C8 ^ _8 SV_%VJ 1:C `VH`%1 IVJ Q` .V `V]:1` `:H Q`s R%`1J$ NER7 .V `QCV Q` PG 1J 1J1 1: 1J$ .V `Vs7J .Vs1s s V]8 EIGQ J ^_7 R 8 MQJ :0QJ5 T8 :JR D8 D%GQ%CV ^_8 L:JRsH:]Vs :JR :`H.1]VC:$Qs7 s]: 1:C Q`$:J1<: 1QJ Q` $VJV `V$%C: 1QJ 1J 0V` VG`: Vs8 T`VJRs 1J CVCC B1QCQ$7 ^ _7 R 8 MQ`1HVRP1H:`R5 F85 M8 C:`1QRAJR`X5 V :C8 ^ _8 NV1 HC1J1HQR$VJV 1H HC:ss1`1H: 1QJ Q` `1H.Q .1QR7s `Q].78 AIV`1H:J JQ%`J:C Q` MVR1H:C GVJV 1Hs P:` A A ^ _7 R8 N:J5 85 H8 H8 N$5 V :C8 ^ _8 T`:JsH`1] 1QJ:C `V]`Vss1QJ G7 .V IV .7CRC]GRG1JR1J$ ]`Q V1J MVCP 1J0QC0Vs : .1s QJV RV:HV 7C:sV HQI]CV68 N: %`V ^ _7 R 8 NQJJV@VJs5 J85 S8 C:G:J Q%s5 V :C8 ^_8 IJ 010Q 1J V`:H 1QJs Q` TTDA I% :J ]`Q V1Js 11 .1J TFIIH8 JQ%`J:C Q` HVCC sH1VJHV ^ _7 R 8 ORV1CC75 D8 :JR D8 R8 G`V:0Vs ^ _8 CVCCR 7]VRs]VH1`1H V6]`Vss1QJ Q` .V .%I:J CD $VJV 1s :ssQH1: VR 11 . H.:J$Vs 1J PQC II ].Qs].Q`7C: 1QJ :JR s.Q` R`:J$V 1J `:H.`QIQsQI:C $VJV CQQ]1J$8 GVJQI1Hs ^_7 R 8 O.@%I:5 8 :JR R8 G8 RQVRV` ^ _8 RV$%C: 1QJ Q` TFIIH ATP:sV :JR @1J:sV :H 101 1Vs G7 TFIIE R%`1J$ :H 10V 1J1 1: 1QJ HQI]CV6 `Q`I: 1QJ8 N: %`V ^ _7 R 8 O@:JQ5 M85 D8 8 BVCC5 V :C8 ^ _8 DNA IV .7C `:Js`V`:sVs DJI : :JR DJI G :`V VssVJ 1:C `Q` RV JQ0Q IV .7C: 1QJ :JR I:II:C1:J RV0VCQ]IVJ 8 CVCC ^_7 R 8 [email protected] 85 B8 BV`J:`RVs RV JVs%s5 V :C8 ^ _8 MQCVH%C:` 1Js1$. s 1J Q .V `VH`%1 IVJ Q` TFIIH Q s1 Vs Q` DNA R:I:$V8 T.V EMBO =Q%`J:C ^ _7 R 8 O@%R:5 M85 A8 T:J:@:5 V :C8 ^_8 A JQ0VC <1JH `1J$V` s `%H %`V 1J .V C:`$V s%G%J1 Q` .%I:J $VJV`:C `:JsH`1] 1QJ `:H Q` TFIIE8 J B1QC C.VI ^ _7 R 8 O`].:J1RVs5 G85 T8 L:$`:J$V5 V :C8 ^ _8 T.V $VJV`:C `:JsH`1] 1QJ `:H Q`s Q` RNA ]QC7IV`:sV II8 GVJVs DV0 ^_7 R 8

170 P: VC5 S8 A8 :JR M8 C8 S1IQJ ^_8 F%JH 1QJ:C AJ:C7s1s Q` .V CR@ \C7HC1J H\M: CQI]CV6 1J MQ%sV EIG`7QJ1H S VI CVCCs :JR EIG`7Qs8 JQ%`J:C Q` B1QCQ$1H:C C.VI1s `7 ^_7 R 8 PV`1ss15 85 K8 JV]sVJ5 V :C8 ^_8 DVHQJs `%H 1J$ `V]`Vss1QJ7 V0QC01J$ IQRVCs Q` HQR`V]`VssQ` :H 1QJ8 N: %`V `V01V1s8 GVJV 1Hs ^_7 R8 PV VÌ:JJ5 E85 M8 1V$CV`5 V :C8 ^_8 ATPRRV]VJRVJ sVCVH 1QJ GV 1VVJ s1J$CV J%HCVQ 1RV :JR CQJ$ ]: H. G:sV V6H1s1QJ `V]:1`8 DNA `V]:1` ^_7 R8 PQCC1 5 R8 J85 F8 A8 JVJJV`5 V :C8 ^ _8 S1Gs 11 . IVJ :C :JR ].7s1H:C `V :`R: 1QJ :JR `1H.Q``.V61s JQRQs: 11 . :GJQÌ:C :I1JQ :H1R HQI]Qs1 1QJ Q` .V .:1`8 A`H. D1s C.1CR ^ _7 R 8 PQ` :C5 M8 M8 ^_8 M1H`QRNAR : `V$%C: Vs G:s:C `:JsH`1] 1QJ G7 :`$V 1J$ .V ] s%G%J1 Q` $VJV`:C `:JsH`1] 1QJ `:H Q` IIH8 P`QHVVR1J$s Q` .V N: 1QJ:C AH:RVI7 Q` SH1VJHVs ^_7 R 8 PQss5 8 C85 C8 C8 EGIV1V`5 V :C8 ^_8 T.V MVR1: Q` HQI]CV6 :JR `:JsH`1] 1QJ `V$%C: 1QJ8 C`1 1H:C `V01V1s 1J G1QH.VI1s `7 :JR IQCVH%C:` G1QCQ$7 ^ _7 R 8 P`1HV5 8 H85 R8 B8 ORQI5 V :C8 ^ _8 T`1H.Q .1QR7s `Q].77 s%C`%`RRV`1H1VJ G`1 CV .:1` :s : I:`@V` `Q` : JV%`QVH QRVÌ:C s7I] QI HQI]CV68 A`H. DVÌ: QC ^_7 R 8 P`Q1V 1RDVRS:J 1s5 L85 P8 D`:JV5 V :C8 ^ _8 CQH@:7JV s7JR`QIV B ]`Q V1J `V$%C: Vs .V `:JsH`1] 1QJ:C ]`Q$`:I :` V` 1``:R1: 1QJ8 EIGQ J ^ _7 R 8 P`Q%R`QQ 5 N8 J85 A8 F%`$V`5 V :C8 ^_8 IJ V$`: 1J$ IRNA P`QHVss1J$ 11 . T`:JsH`1] 1QJ8 CVCC ^_7 R 8 Q15 8 P8 R8 A8 S8 M8 C8 8 R8 ^_8 SV_%VJHVRRV]VJRVJ G:sV ]:1` s V]]1J$ R7J:I1Hs 1J PD .VC1H:sV %J11JR1J$8 VL1`V 8 R:J1s.5 J8 A85 S8 H:.J5 V :C8 ^_8 IRVJ 1`1H: 1QJ Q` TFB 5 : JV1 HQI]QJVJ Q` $VJV`:C `:JsH`1] 1QJ :JR DNA `V]:1` `:H Q` IIH8 N: %`V $VJV 1Hs ^ _7 R 8 R:]1J5 I8 ^_8 C.:] V` R D1sQ`RV`s Q` J%HCVQ 1RV V6H1s1QJ `V]:1`8 H:JRGQQ@ Q` CC1J1H:C NV%`QCQ$78 M8 L8 OC101V` D%C:H :JR B8 S8 H:`0V75 ECsV01V`8 QC%IV 7 R 8 RV:5 S85 F8 E1sVJ.:GV`5 V :C8 ^_8 RV$%C: 1QJ Q` H.`QI: 1J s `%H %`V G7 s1 VRs]VH1`1H .1s QJV H IV .7C `:Js`V`:sVs8 N: %`V ^ _7 R 8 RV1JGV`$5 D8 :JR R8 G8 RQVRV` ^ _8 F:H Q`s 1J0QC0VR 1J s]VH1`1H `:JsH`1] 1QJ G7 I:II:C1:J RNA ]QC7IV`:sV II8 P%`1`1H: 1QJ :JR `%JH 1QJ:C :J:C7s1s Q` 1J1 1: 1QJ `:H Q`s IIB :JR IIE8 J B1QC C.VI ^ _7 R8 RVJ:%R5 J8 P8 :JR D8 MQ`:s ^_8 S `%H %`:C s %R1Vs QJ J%HCV:` `VHV] Q`s8 CVCC%C:` :JR IQCVH%C:` C1`V sH1VJHVs 7 CMLS ^_7 R 8 R1VRC5 T8 H8 F8 E8 J8 M8 ^_8 T.V HQI1J$s :JR $Q1J$s Q` J%HCVQ 1RV V6H1s1QJ `V]:1` `:H Q`s QJ R:I:$VR DNA8 T.V EMBO =Q%`J:C ^ _7 R 8 R1Q%5 L85 L8 VJ$5 V :C8 ^ _8 T.V `VC: 10V V6]`Vss1QJ Q` I% : VR PB $VJVs `Vs%C s 1J 6V`QRVÌ: ]1$IVJ Qs%ILCQH@:7JVs s7JR`QIV Q` `1H.Q .1QR7s `Q].7 HVCC%C:` ].VJQ 7]Vs8 H%I MQC GVJV ^ _7 R8 RQGG1Js5 J8 ^ _8 V`QRVÌ: ]1$IVJ Qs%I HQI]CVIVJ : 1QJ $`Q%] H 1s 11 .R`:1J :JR `V:ss1$JVR Q $`Q%] D8 H%I:J $VJV 1Hs ^_7 R8 RQH.V VRE$C75 C8 ^_8 RV 1JQ1H :H1R s1$J:C1J$ :JR IQ%sV VIG`7QJ1H s VI HVCC R1``V`VJ 1: 1QJ7 C`Qss :C@ GV 1VVJ $VJQI1H :JR JQJR$VJQI1H V``VH s Q` RA8 B1QH.1I1H: V G1Q].7s1H: :H :8 RQH.V VRE$C75 C85 S8 AR:I5 V :C8 ^ _8 S 1I%C: 1QJ Q` RAR :C].: :H 10: 1QJ `%JH 1QJ AFR .`Q%$. G1JR1J$ Q .V $VJV`:C `:JsH`1] 1QJ `:H Q` TFIIH :JR ].Qs].Q`7C: 1QJ G7 CDK 8 CVCC ^_7 R 8 RQVRV`5 R8 G8 ^ _8 N%HCV:` RNA ]QC7IV`:sVs7 `QCV Q` $VJV`:C 1J1 1: 1QJ `:H Q`s :JR HQ`:H Q`s 1J V%@:`7Q 1H `:JsH`1] 1QJ8 MV .QRs 1J VJ<7IQCQ$7 7 R 8

171 RQVRV`5 R8 G8 :JR 8 J8 R% V` ^ _8 S]VH1`1H J%HCVQC:` :JR J%HCVQ]C:sI1H RNA ]QC7IV`:sVs8 P`QHVVR1J$s Q` .V N: 1QJ:C AH:RVI7 Q` SH1VJHVs Q` .V J1 VR S : Vs Q` AIV`1H: ^_7 R 8 RQss1$JQC5 M85 I8 KQCGRC.V7JVC5 V :C8 ^ _8 S%Gs `: V s]VH1`1H1 7 Q` .V HR@R:H 10: 1J$ @1J:sV ^CAK_ 1s :C V`VR %]QJ :ssQH1: 1QJ 11 . TFIIH8 T.V EMBO =Q%`J:C ^ _7 R 8 RQ .G:` 5 S8 B8 :JR B8 D8 S `:.C ^_8 IJ V`]`V 1J$ .V C:J$%:$V Q` .1s QJV :JR DNA IQR1`1H: 1QJs8 B1QH.1I1H: V B1Q].7s1H: AH : ^BBA_ R GVJV RV$%C: Q`7 MVH.:J1sIs ^ _7 R 8 RQ75 R85 J8 P8 AR:IH

172 SH`1I:5 A85 R8 KQJ1H@Q0:5 V :C8 ^ _8 S `%H %`:C G:s1s Q` DNARR:I:$V `VHQ$J1 1QJ G7 .V DDBR DDB HQI]CV68 CVCC ^ _7 R8 SV`1<:1:5 H85 J8 8 CQJ:1:75 V :C8 ^ _8 P.Qs].Q`7C: 1QJ Q` CR VÌ1J:C RQI:1J Q` RNA ]QC7IV`:sV II 1s JQ `V_%1`VR 1J G:s:C `:JsH`1] 1QJ8 N: %`V ^ _7 R 8 SV`Q<5 T85 C8 PV`V<5 V :C8 ^_8 ]LSSL5 .V `V$%C: Q`7 s%G%J1 Q` .V PDLRAD .VC1H:sV5 ]C:7s : H`%H1:C `QCV 1J .V `:JsH`1] 1QJ:C :H 101 7 Q` TFIIH8 J B1QC C.VI ^_7 R 8 S.:JR1C7:5 J8 :JR S8 G8 RQGV` s ^_8 T.V `:JsH`1] 1QJ H7HCV 1J V%@:`7Q Vs7 ``QI ]`QR%H 10V 1J1 1: 1QJ Q RNA ]QC7IV`:sV II `VH7HC1J$8 B1QH.1I1H: V G1Q].7s1H: :H : ^ _7 R8 S.1C: 1`:`R5 A85 R8 C8 CQJ:1:75 V :C8 ^_8 T.V RNA ]QC7IV`:sV II VCQJ$: 1QJ HQI]CV68 AJJ%:C `V01V1 Q` G1QH.VI1s `7 7 R 8 S.7@1JR5 B8 M85 J8 K1I5 V :C8 ^ _8 AH 10: 1QJ Q` .V TFIIDRTFIIA HQI]CV6 11 . HMGR8 GVJVs RV0VCQ]IVJ ^_7 R 8 S1IQJ5 JV```V7 A8 :JR RQGV` E8 K1J$s QJ ^_8 OHH%]71J$ C.`QI: 1J7 PQC7HQIG MVH.:J1sIs `Q` GV 1J$ Q GVJQI1H T:`$V s5 S Q]]1J$ T`:JsH`1] 1QJ:C T`:``1H5 :JR S :71J$ P% 8 MQCVH%C:` CVCC ^ _7 R 8 SI:CV5 S8 T8 :JR D8 B:C 1IQ`V ^ _8 T.V 1J1 1: Q` :s `:JsH`1] 1QJ HQJ `QC VCVIVJ 8 CVCC 7 R8 SI:CV5 S8 T8 :JR J8 T8 K:RQJ:$: ^_8 T.V RNA ]QC7IV`:sV II HQ`V ]`QIQ V`8 AJJ%:C `V01V1 Q` G1QH.VI1s `7 7 R 8 SQCQ$%G5 M85 D8 L1 QJ1J5 V :C8 ^ _8 TFB Is : T`:Js1VJ CQI]QJVJ Q` .V C: :C7 1H S1 V Q` .V H%I:J M1 QH.QJR`1:C RNA PQC7IV`:sV8 CVCC ^ _7 R 8 S]1C1:J:@1s5 C8 G8 :JR R8 A8 FC:0VCC ^_8 LQJ$R`:J$V 1J `:H.`QIQsQI:C 1J V`:H 1QJs 1J .V T .VC]V` 7]V H7 Q@1JV CQH%s8 N: %`V 1II%JQCQ$7 ^_7 R 8 S]C1J V`5 E85 H8 HV: .5 V :C8 ^ _8 CTCF IVR1: Vs CQJ$R`:J$V H.`QI: 1J CQQ]1J$ :JR CQH:C .1s QJV IQR1`1H: 1QJ 1J .V R$CQG1J CQH%s8 GVJVs DV0VCQ]IVJ ^ _7 R 8 S V`:J1J15 M85 S8 G1C1:J15 V :C8 ^ _8 DNA `V]:1` 1J0Vs 1$: 1QJs 1J J1JV I :C1:J ]: 1VJ s :``VH VR G7 `1H.Q .1QR7s `Q].78 M% : RVs ^_7 R 8 S V`:J1J15 M85 P8 L:$QI:`s1J15 V :C8 ^ _8 V`QRVÌ: ]1$IVJ Qs%I ^HQI]CVIVJ : 1QJ $`Q%] D_ I% : 1QJ 1s ]`VsVJ 1J ]: 1VJ s :``VH VR 11 . `1H.Q .1QR7s `Q].7 11 . ].Q QsVJs1 101 78 H%I8 GVJV 8 7 R8 S V`:J1J15 M85 P8 L:$QI:`s1J15 V :C8 ^ _8 GVJV 1H .V V`Q$VJV1 7 Q` .V V6H1s1QJ `V]:1` RV`VH :ssQH1: VR 11 . `1H.Q .1QR7s `Q].78 C:`H1JQ$VJVs1s ^ _7 R 8 S V`:J1J15 M85 8 VÌV%CVJ5 V :C8 ^ _8 A JV1 J%HCVQ 1RVRV6H1s1QJ `V]:1` $VJV :ssQH1: VR 11 . .V R1sQ`RV` `1H.Q .1QR7s `Q].78 AI8 J8 H%I8 GVJV 8 ^_7 R 8 S%$:s:1:5 K85 8 O@%R:5 V :C8 ^ _8 R1JR%HVR %G1_%1 7C: 1QJ Q` PC ]`Q V1J IVR1: VR G7 RDDBR %G1_%1 1J C1$:sV HQI]CV68 CVCC ^_7 R8 T:.1C1:J15 M85 K8 P8 KQ.5 V :C8 ^ _8 CQJ0V`s1QJ Q` RIV .7CH7 Qs1JV Q R.7R`Q67IV .7CH7 Qs1JV 1J I:II:C1:J DNA G7 MLL ]:` JV` TET8 SH1VJHV ^ _7 R 8 T:@:7:I:5 K85 E8 P8 S:C:<:`5 V :C8 ^ _8 DV`VH s 1J .V DNA `V]:1` :JR `:JsH`1] 1QJ $VJV ERCC 1J .V H:JHV`R]`QJV R1sQ`RV` 6V`QRVÌ: ]1$IVJ Qs%I $`Q%] D8 C:JHV` RVs8 7 R 8 T:@VR:H.15 A85 M8 S:1=Q5 V :C8 ^_8 DDB HQI]CV6RIVR1: VR %G1_%1 7C: 1QJ :`Q%JR DNA R:I:$V 1s Q]]Qs1 VC7 `V$%C: VR G7 PC :JR K% :JR HQJ `1G% Vs Q .V `VH`%1 IVJ Q` PA8 MQCVH%C:` :JR CVCC%C:` B1QCQ$7 ^_7 R 8 T:JRQJ$5 S8 M85 J8 D8 F`VJH.5 V :C8 ^ _8 D7J:I1H 1J V`:H 1QJs GV 1VVJ .V ]`QIQ V` :JR VÌ1J: Q` `V$1QJs Q` .V I:II:C1:J BRCA $VJV8 P`QHVVR1J$s Q` .V N: 1QJ:C AH:RVI7 Q` SH1VJHVs ^_7 R 8 T:J:@:5 A85 T8 : :J:GV5 V :C8 ^ _8 CVJ `:C `Q`@.V:R RQI:1J Q` .%I:J TFIIE GV : ]C:7s : ]`1I:`7 `QCV 1J G1JR1J$ RQ%GCVRs `:JRVR DNA : `:JsH`1] 1QJ 1J1 1: 1QJ8 GVJVs Q HVCCs 7 RV0Q VR Q IQCVH%C:` HVCC%C:` IVH.:J1sIs ^_7 R 8

173 T:7CQ`5 E85 B8 B`Q%$. QJ5 V :C8 ^ _8 V`QRV`I: ]1$IVJ Qs%I :JR `1H.Q .1QR7s `Q].7 :`V :ssQH1: VR 11 . R1``V`VJ I% : 1QJs 1J .V PD ^ERCC_ `V]:1`L `:JsH`1] 1QJ $VJV8 P`QH8 N: C8 AH:R8 SH18 SA ^ _7 R 8 T.V1C5 A8 F85 J8 NQJJV@VJs5 V :C8 ^_8 SCQ1C7 P`Q$`Vss1J$ N%HCVQ 1RV E6H1s1QJ RV]:1` 1J T`1H.Q .1QR7s `Q].7 G`Q%] A P: 1VJ F1G`QGC:s s8 MQCVH%C:` :JR CVCC%C:` B1QCQ$7 ^ _7 R 8 T.QI:s5 M8 C8 :JR C8 M8 C.1:J$ ^ _8 T.V $VJV`:C `:JsH`1] 1QJ I:H.1JV`7 :JR $VJV`:C HQ`:H Q`s8 C`1 RV0 B1QH.VI MQC B1QC ^_7 R 8 T.QI]sQJ5 L8 L85 B8 J8 G%]]75 V :C8 ^_8 RV$%C: 1QJ Q` H.`QI: 1J s `%H %`V 01: .1s QJV ]Qs R `:JsC: 1QJ:C IQR1`1H: 1QJ :JR .V C1J@ Q H:`H1JQ$VJVs1s8 C:JHV` IV :s :s1s `V01V1s ^R_7 R 8 T1`QRV5 F85 D8 B%ssQ5 V :C8 ^ _8 RVHQJs 1 % 1QJ Q` .V `:JsH`1] 1QJ `:H Q` TFIIH7 :ss1$JIVJ Q` `%JH 1QJs `Q` .V .`VV VJ<7I: 1H s%G%J1 s5 PB5 PD5 :JR HR@ 8 MQC CVCC ^_7 R 8 TQ@%s%I15 85 8 M:5 V :C8 ^ _8 T.V NV1 CQ`V P`QIQ V` ECVIVJ CPE ^ CQ`V P`QIQ V` ECVIVJ _ D1`VH s AH 10: Q`R5 MVR1: Q`R5 :JR TATARB1JR1J$ P`Q V1JRDV]VJRVJ G% TFIIDRIJRV]VJRVJ RNA PQC7IV`:sV II T`:JsH`1] 1QJ ``QI TATARLVss P`QIQ V`s8 MQCVH%C:` :JR CVCC%C:` B1QCQ$7 ^ _7 R 8 =0<`15 A85 M8 P:C5 V :C8 ^_8 T.V F%JH 1QJs Q` TFIIF R%`1J$ IJ1 1: 1QJ :JR T`:JsH`1] ECQJ$: 1QJ A`V D1``V`VJ 1:CC7 A``VH VR G7 P.Qs].Q`7C: 1QJ G7 C:sV1J K1J:sV 8 JQ%`J:C Q` B1QCQ$1H:C C.VI1s `7 ^ _7 R 8 :J D7@V5 M8 85 R8 G8 RQVRV`5 V :C8 ^ _8 P.7s1H:C :J:C7s1s Q` `:JsH`1] 1QJ ]`V1J1 1: 1QJ HQI]CV6 :ssVIGC7 QJ HC:ss II $VJV ]`QIQ V`8 SH1VJHV 7 R 8 XCV

174 QJ$5 KQQJ H85 8 J1J5 V :C8 ^_8 TFIIH P.Qs].Q`7C: 1QJ Q` .V PQC II CTD S 1I%C: Vs MVR1: Q` D1ssQH1: 1QJ ``QI .V P`V1J1 1: 1QJ CQI]CV6 :JR P`QIQ V` EsH:]V8 MQCVH%C:` CVCC ^_7 R 8 QQRHQH@5 C8 L8 :JR S8 D1I1 `Q0 ^_8 H1$.V`RQ`RV` s `%H %`V Q` H.`QI: 1J :JR H.`QIQsQIVs8 C%``VJ Q]1J1QJ 1J $VJV 1Hs RV0VCQ]IVJ ^_7 R 8 Qss1RCQ5 M8 A8 J8 S8 8 L8 K8 B8 M8 R8 R8 S8 H8 8 J8 ^_8 D7J:I1H C1J@ Q` DNA RVIV .7C: 1QJ5 DNA s `:JR G`V:@s :JR `V]:1` 1J IQ%sV <7$Q Vs8 T.V EMBO =Q%`J:C ^_7 R 8 `:J$V5 O85 S8 O@`V 5 V :C8 ^ _8 C.:`:H V`1<: 1QJ Q` .V ]%`1`1VR :H 10: VR $C%HQHQ` 1HQ1R `VHV] Q` ``QI `: C10V` H7 QsQC8 T.V JQ%`J:C Q` G1QCQ$1H:C H.VI1s `7 ^ _7 R 8 %5 85 H8 L1%5 V :C8 ^ _8 GVJV 1H 0:`1:J s 1J GTFH :JR `1s@ Q` C%J$ H:JHV`7 A H:sVRHQJ `QC :J:C7s1s 1J : C.1JVsV ]Q]%C: 1QJ8 L%J$ C:JHV` ^_7 R 8 7sQH@:5 J85 T8 S11$% 5 V :C8 ^ _8 A PHD `1J$V` Q` NRF HQ%]CVs .1s QJV H C7s1JV `1IV .7C: 1QJ 11 . H.`QI: 1J `VIQRVCC1J$8 N: %`V ^ _7 R 8 1:Q5 H85 A8 PV:`sQJ5 V :C8 ^ _8 B1JR1J$ Q` G:s:C `:JsH`1] 1QJ `:H Q` TFIIH Q .V :H1R1H :H 10: 1QJ RQI:1Js Q` P :JR ] 8 MQC CVCC B1QC ^_7 R 8 1V5 85 T8 KQ@%GQ5 V :C8 ^ _8 S `%H %`:C s1I1C:`1 7 GV 1VVJ TAFs :JR .V .V V`Q V `:IV`1H HQ`V Q` .V .1s QJV QH :IV`8 N: %`V ^ _7 R8 :I:R:5 T85 8 :I:$%H.15 V :C8 ^ _8 PRTEFGRIVR1: VR ].Qs].Q`7C: 1QJ Q` .S] CR VÌ1J:C `V]V: s 1s H`1 1H:C `Q` ]`QHVss10V `:JsH`1] 1QJ VCQJ$: 1QJ8 MQCVH%C:` CVCC ^_7 R 8 :I:IQ Q5 8 85 H8 IH.1R:5 V :C8 ^ _8 D1``V`VJ 1: 1QJ Q` HQ`V ]`QIQ V` :`H.1 VH %`V GV 1VVJ ]C:J s :JR I:II:Cs `V0V:CVR G7 LDSS :J:C7s1s8 N%HCV1H :H1Rs `VsV:`H. ^ _7 R 8 :J5 Q85 R8 J8 MQ`VC:JR5 V :C8 ^ _8 D%:C `QCVs `Q` `:JsH`1] 1QJ `:H Q` IIF 1J ]`QIQ V` VsH:]V G7 RNA ]QC7IV`:sV II8 J B1QC C.VI ^ _7 R 8 Q@Q15 M85 C8 M:s% :J15 V :C8 ^_8 T.V 6V`QRVÌ: ]1$IVJ Qs%I $`Q%] C ]`Q V1J HQI]CV6 PCRHRB ]C:7s :J 1I]Q` :J `QCV 1J .V `VH`%1 IVJ Q` `:JsH`1] 1QJ `:H Q` IIH Q R:I:$VR DNA ÌJ P`QHVss C1 : 1QJa8 J B1QC C.VI ^_7 R 8 QJ:.:5 M85 T8 Ts%H.17:5 V :C8 ^ _8 CVCCRH7HCVRRV]VJRVJ ].Qs].Q`7C: 1QJ Q` .V G:s:C `:JsH`1]1 QJ `:H Q` RAP 8 FEBS CV V`s 7 R 8 %5 85 8 G1J=:C:5 V :C8 ^_8 PQC7ÂDPR`1GQs7C_: 1QJ `V$%C: Vs CTCFRRV]VJRVJ H.`QI: 1J 1Js%C: 1QJ8 N: %`V $VJV 1Hs ^_7 R8 VH.VC5 C85 8 Q8 S.VJ5 V :C8 ^ _8 D1IV`1<: 1QJ 1J V``:HVs `QÌVR GV 1VVJ .V DNA G1JR1J$ RQI:1Js RV VÌ1JV .V HQQ]V`: 10V G1JR1J$ Q` RRLRAR :JR RRLTR .V V`QR1IV`s Q DR :JR DR VCVIVJ s8 T.V EMBO =Q%`J:C ^ _7 R8 VJ JV`5 G8 E8 :JR S8 HVJ1@Q`` ^_8 RV$%C: 1QJ Q` J%HCVQsQIV R7J:I1Hs G7 .1s QJV IQR1`1H: 1QJs8 N: %`V s `%H %`:C IQCVH%C:` G1QCQ$7 ^_7 R 8 %`1 :5 M8 :JR C8 MV`1JQ ^_8 T.V `:JsH`1] 1QJ:C HQI]CV61 7 Q` .V TFIIH HQI]CV68 T`VJRs 1J $VJV 1Hs 7 TIG ^_7 R 8

175 Amita SINGH Contribution de TFIIH dans le remodelage de la chromatine dépendant des facteurs NER lors de la transcription

Résumé en Français

!"#$!%&'$()#(*%"+(, ./",R1%"2 %/".*$&",/"&*%"!'#(3!#(*%"% '/&&(#/".R!&&/56.!2/",R1%"/%&/56./" de protéines autour du promoteur. Parmi ces protéines, le complexe TFIIH joue un rôle central et important au travers de ses sous-unités enzymatiques. Des mutations dans les sous-unités XPB, XPD et p8/TTD-A de TFIIH conduisent à trois maladies autosomiques récessives distinctes : xeroderma pigmentosum (XP), parfois associés avec le syndrome de Cockayne (XP/CS) et la trichothiodystrophie (TTD). En étudiant différentes mutations dans ces trois sous- unités de TFIIH, nous avons montré que chaque mutation analysée conduit à une dérégulation transcriptionnelle spécifique du gène RAR !2, gène cible des RAR. R(%# 2$(# "!$'8(#/'#1$!./"/#" enzymatique de TFIIH conditionne le bon recrutement du complexe TFIIH et également des facteurs de réparation par excision de nucléotides (NER). TFIIH muté perturbe leur recrutement et par conséquence compromet le remodelage de la chromatine médiée par les facteurs NER tels que les modifications post- #$!,1'#(*%%/../&"9:;<&=",/&"8(&#*%/&>".R(%,1'#(*%",/&"'!&&1$/&" ,/".R?@A>".!", 5 #8B.!#(*%",/".R?@A"/#"./ s boucles de chromatine. Par conséquence, en plus de ses activités enzymatiques, TFIIH forme une plate-forme afin de recruter les facteurs NER et orchestres les fonctions connexes de la transcription. Cette pénétrance variable parmi les mutants donne lieu à un gradient de phénotype observé chez les patients TTD, XP ou XP/CS. Mot-clés : TFIIH, NER, transcription, chromatine

Résumé en anglais

Fidelity in transcription of the gene requires assembly of set of proteins around the promoter, upon gene activation. The TFIIH complex is central among these proteins and plays a key role through its enzymatic subunits. Mutations in TFIIH subunits XPB, XPD and p8/TTD-A leads to three distinct autosomal recessive disorders: xeroderma pigmentosum (XP), sometimes !&&*'(!#/,"C(#8"D*'E!B%/R&"&B%,$*5/"9F:GDH="!%,"#$('8*#8(*,B&#$*)8B"9;;@=I"JB",B(%2"#8/" different mutation in these three subunits of TFIIH from mentioned genetic disease models, we have shown that each mutation analyzed led to a specific transcriptional dysregulation of the RAR-target gene RAR 2. The architectural and enzymatic integrity of TFIIH condition the appropriate recruitment of TFIIH complex and further the arrival of the Nucleotide Excision Repair (NER) factors. By disturbing their recruitment, mutated TFIIH consequently compromised the chromatin remodeling mediated by NER factors such as histones post- translational modifications (PTMs), DNA breaks induction, DNA demethylation and gene looping. Hence it can be concluded that in addition to its enzymatic activities, TFIIH provide a platform to recruit the NER factors and orchestrates the related functions in transcription. Such varying penetrance among mutants gives rise to a phenotype gradient as observed in TTD, XP or XP/CS patients. Keywords: TFIIH, NER, transcription, chromatin.

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