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Science,1994

NUCLEOTIDE EXCISION REPAIR. 1874. XERODERMA PIGMENTOSUM. F. Hebra and M. Kaposi (1874), The New Sydenham Soc. London, 3, 252. Nature,1968. Science,1994. UV. 3H-TdR. IDENTIFICATION OF NER DEFECT Complementation analysis and UDS. DNA REPAIR - NUCLEOTIDE EXCISION REPAIR (NER).

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Science,1994

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  1. NUCLEOTIDE EXCISION REPAIR 1874 XERODERMA PIGMENTOSUM F. Hebra and M. Kaposi (1874), The New Sydenham Soc. London, 3, 252 Nature,1968 Science,1994

  2. UV 3H-TdR IDENTIFICATION OF NER DEFECTComplementation analysis and UDS

  3. DNA REPAIR - NUCLEOTIDE EXCISION REPAIR (NER) NER was the first DNA repair system that was associated with cancer; when James Cleaver reported that Xeroderma pigmentosum was caused by defective NER Type of damage repaired: Principally damage that causes helix distortion and damage to base (one of the criteria insufficient) a) cyclobutane pyrimidine dimers (CPD) b) (6-4) photoproducts (a different pyrimidine dimer) b) Benz(a)pyrene adducts, mostly to guanines c) some alkylation products (mostly longer than methyl) d) cisplatin and some other drugs NER requires approximately 30 different polypeptides; many in complex proteins or protein-protein interactions. NER differs from BER in that an invariant complex recognizes several different types of damage, as long as it causes helix distortion.

  4. Ratio CPD / 6-4PP 3 : 1 MAIN UV-INDUCED DNA LESIONS Cyclobutane ring UV Reactive 5 5 CPD double 6 6 bonds P P P P P P Thymine-thymine Adjacent thymines cyclobutane dimer (TT) Linked between carbon 6 of the 5` pyrimidine and carbon 4 of the 3`pyrimidine N H 2 U V Reactive 6-4 Photoproduct groups 4 6 P P P P P P and cytosine (right) Adjacent thymine (left) Trondheim 2006

  5. BULKY CHEMICAL ADDUCTS Polycyclic aromatic Monofunctional alkylating hydrocarbons: agents: DMBABNU B[a]P CH3 (CH2)3CH3 O = N N C NH2 CH3 O

  6. STEPS IN NER LESION RECOGNITION LESION DEMARCATION 30-35 bp DUAL INCISIONS (3', 5') REPAIR SYNTHESIS

  7. TRANSCRIPTION-COUPLED REPAIR (TCR) GLOBAL GENOME REPAIR (GGR) RNA pol. UV-DDB(XPE) XPC-hHR23B CSA CSB XPAB2 GLOBAL GENOME NER ‘INTENTIONAL’ RECOGNITION LESION-BINDING PROTEINS TRANSCRIPTION-COUPLED NER ‘ACCIDENTAL ’ RECOGNITIONRNA POLYMERASE II( STRAND-SPECIFIC) DNA Helix distortion Stalled RNA Polymerase NUCLEOTIDE EXCISION REPAIR Lesion recognition

  8. Nucleotide excision repair (NER) NER repairs: “bulky” lesions that cause helix distortions by an error-free mechanism Typical lesions: pyrimidine dimers and benz(a)pyrene adducts Requires: at least 30 different polypeptides Defective NER: causes cancer Types: Global genome repair (GGR) Takes place in all parts of the genome with equal rate Initiated by binding of XPC-hHR23B Transcription coupled repair (TCR): Takes place in protein encoding genes that are being transcribed. Transcribed strand more rapidly repaired. Requires CSA and CSB, but not XPC. Is defective in Cockayne syndr. Developmental defects, but not cancer.

  9. GENETICS OF XERODERMA PIGMENTOSUM Group % of XP Locus Function (defect) Other XPA 63% 9q34.1 DNA binding protein Most severe form with affinity for damaged DNA XPB rare 2q21 3’-5’ DNA helicase, in TFIIH 2 families reported XPC 25% 3p25.1 DNA damage recognition and ‘classic’ form of XP XPD 15% 19q13.2 5’-3’ DNA helicase, in TFIIH XPE (DDB1 rare 11q12-13 Uncertain, not required in vitro Relatively mild form and 2) 11p11-12 XPF rare 16p13 Structure-specific endonuclease, Mostly in Japan catalyses 5’ incision XPG rare 13q32 Structure-specific endonuclease, catalyses 3’ incision XPV common p21.1-6p12 DNA pol  (eta), translesion synth. Not NER defect. Error prone DNA pol  (iota) substitutes

  10. FEATURES OF HUMAN NER SYNDROMES Associated syndrome UDS UV-sens. Cancer Compl. group Relative occurence <2 3-40 15-30 15-30 >50 15-30 2-25 100 100 XP-A XP-B XP-C XP-D XP-E (DDB1 and 2) XP-F XP-G CS-A CS-B XP/CS, TTD XP/CS, TTD XP/CS +++ ++ + ++ + + ++ + + + +/- + +/- +/- +/- +/- - - XP XP CM

  11. Xeroderma pigmentosum Characteristics:  1/250 000 (varies), less USA/Europe  Wide range of symptoms  Skin and eye cancers, dry pigmented skin  Very UV-sensitive  Blindness and deafness  Developmental disabilities  Dwarfism and hypergonadism  Mental retardation Genetics:  Seven genes (XPA, XPB, XPC, XPD, XPE, XPF and XPG) cause defect in NER  XPV caused by defect in DNA pol  (eta)

  12. Cockayne syndrome Characteristics:  Only 200 cases known world wide  Normal first year  Onset symptoms 2. Year  Dwarfism  Microcephaly - mental deficiency  Neurodevelopmental delay, deafness  Unsteady gait  Typical facial appearance  Premature ageing - shortened lifespan  Sunburn, but not cancer Genetics:  Two genes: CS-A and CS-B  Defective in TCR, but proficient in GGR  Transcription defect, not really repair defect Candice 5 years - died at 8

  13. SUMMARY • NER is mediated by sequential (diffusion controlled) assembly of repair proteins to the site of DNA damage (data not presented). • All XP factors are assigned to specific functions. • XPC-hHR23B is the principal damage recognition factor, but UV-DDB comes first. • UV-DDB (deficient in XPE) is part of ubiquitin ligase complex and is essential for repair of less helix distorting lesions. Involved in ubiquitination of histones H3 and H4. • XPA participates in a later step of NER allowing binding of ERCC1-XPF, but is not required for recruitment of XPG or RPA. • Repair synthesis is carried out by DNA polymerase delta and /or epsilon • and ligase 1. • Assembly of NER complex does not follow strict order.

  14. UV-DDB (XPE) NUCLEOTIDE EXCISION REPAIR Transcription-Coupled Repair (TCR) Global Genome Repair (GGR) XPC-hHR23B RNA pol. TFIIH XPA • CSA • CSB • XAB2 • HMGN1 CAK-subunit of TFIIH RPA XPF-ERCC1 XPG

  15. NER factors RNAPIIo CS Remodelling ?? UV damage RNAPIIo NER factors RNAPIIo Degradation of RNA polymerase II CS TRANSCRIPTION-COUPLED REPAIR - Stalling of RNA polymerase II is central as signal STRATEGY: In vivo crosslinking. Isolation of stalled RNAPIIo complexes by Chromatin immunoprecipitation (ChIP) from TCR proficient and deficient human cells.

  16. MEASUREMENT OF DNA LESIONS IN SPECIFIC DNA SEQUENCES Treatment of cells, isolation and restriction of DNA Enzymatic or chemical conversion of DNA lesions into DNA breaks or alkali labile sites by UVDE (an endonuclease cleaving at CPD) Alkaline agarose gel plus Southern blot Ratio of the intensity of the bands: Poisson distribution Time (h) 0 4 24 - - + + - + TS UVDE: Transcribed strand

  17. DIFFERENT LEVELS OF NUCLEOTIDE EXCISION REPAIR STRAND SPECIFIC REPAIR OF CPD IN AN ACTIVE AND AN INACTIVE ADA GENE Active ADA Inactive ADA 100 100 80 80 60 60 % CPD Removed 40 40 20 20 0 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 Time after UV (Hr) TS ADA BclI NTS ADA BclI Factor IX

  18. TS 80 NORMAL HUMAN CPD NTS 40 0 0 8 16 24 TS 80 NTS 40 0 0 8 16 24 TCR AND DNA LESIONS TCR OF BASE DAMAGE? 8-oxoG? NAAAF

  19. UV DAMAGE, REPAIR AND TRANSCRIPTION RESPONSE NORMAL HUMAN CS 100 100 100 TS WT 80 80 80 60 60 60 NTS 40 40 40 CS 20 20 20 0 0 0 0 4 8 12 16 20 24 0 2 4 6 8 0 4 8 12 16 20 24 CPD REPAIR IN ACTIVE GENES TRANSCRIPTION RESPONSE

  20. Courtesy of Alan Lehmann COCKAYNE SYNDROME • Clinical phenotype • Complex: from photosensitivity up to dwarfism and premature aging • Cellular phenotype • Impaired transcription recovery after DNA damage • Defective transcription–coupled repair • CS genes • CSB: ATPase of the SW12/SNF2 family • Chromatin remodeling factor (Citterio et al,2000) • Stimulation transcription elongation (Selby et al,1997) • CSA: WD40 (Trp and Asp) repeat protein • interacts with DDB1 and COP9 signalosome • (Groisman et al,2003). Involved in ubiquitination.

  21. IN VIVO CROSSLINKINGAND CHROMATIN IMMUNOPRECIPITATION OF MOCK OR UV-IRRADIATED CELLS Cross-linking of cell monolayers Cell lysis in the presence of non-ionic detergents Sonication of crude nuclei 150-300 bp DNA fragments Chromatin- IP Chromatin-IP:-RNAPIIo or -CSB

  22. Cross-linking of cell monolayers Soluble Chromatin Cell lysis in the presence of non-ionic detergents RNAPIIo CSB - + - + UV (20J/m2) Recovery (hr) - 1 - 1 Sonication of crude nuclei δ=1.66g/cm3 δ=1.39g/cm3 Cpm 150-300 bp DNA fragments Gradient Fraction Chromatin- IP IN VIVO CROSSLINKINGAND CHROMATIN IMMUNOPRECIPITATION

  23. XPA and ERCC1/XPF INTERACT WITH RNAPIIo Chromatin-IP: -RNAPIIo XP-C Normal CS-B -RNAPIIo -ERCC1 -XPA UV (20J/m2) - + - + - + Recovery time (h) 1 1 1

  24. BIOLOGICAL CONSEQUENCES OF DEFICIENCIES IN GLOBAL GENOME REPAIR AND TRANSCRIPTION COUPLED REPAIR

  25. Transcription-Coupled Repair (TCR) Global Genome Repair (GGR) UV-DDB (XPE) XPC-hHR23B RNA pol. • CSA • CSB • XAB2 TFIIH XPA CAK-subunit of TFIIH RPA XPF-ERCC1 XPG NUCLEOTIDE EXCISION REPAIR

  26. RESPONSES TO DNA DAMAGE IMPACT OF TCR AND GGR • Cell cycle arrest • Apoptosis • Mutagenesis • Carcinogenesis TCR GGR + + WT - - XPA + - XPC + - CSB - + + XPE TCR is a survival pathway – this is the major function

  27. ERYTHEMA / EDEMA TCR GGR MED + + WT 500 J/m2 - - XPA 40 J/m2 - + CSB 40 J/m2 + - XPC 500 J/m2 + - + XPE 500 J/m2

  28. TCR IS SURVIVAL PATHWAY VIA ACTIVATION OF p53 dependent APOPTOSIS

  29. Mismatch repair (MMR) Mismatches result from: * Replication errors * Deletions/insertions * Meiotic recombination * Some chemicals? The mammalian proteins involved in MMR Homologues of E. coli MMR proteins MutS and MutL. There are several homologues of each, but apparently no homologue of MutH. It is not clear how the strand carrying the incorrect nucleotide(s) is specifically identified. Mismatches are recognized by different complexes; MutS (hMSH2/6) for single-base loops and mismatches; MutS (hMSH2/3) for larger loops (resulting from deletions or insertions). Several other proteins are required. From Hoeijmakers, Nature 2001

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