Cancer in xeroderma pigmentosum and related disorders of DNA repair

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1 60. Bajenaru, M. L. et al. Optic nerve glioma in mice requires astrocyte Nf1 gene inactivation and Nf1 brain heterozygosity. Cancer Res. 63, (2003). 61. Bajenaru, M. L., Garbow, J. R., Perry, A., Hernandez, M. R. & Gutmann, D. H. Natural history of neurofibromatosis 1- associated optic nerve glioma in mice. Ann. Neurol. 57, (2005). 62. Reilly, K. M., Loisel, D. A., Bronson, R. T., McLaughlin, M. E. & Jacks, T. Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects. Nature Genet. 26, (2000). 63. Ridley, A. J., Paterson, H. F., Noble, M. & Land, H. Rasmediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation. EMBO J. 7, (1988). 64. Dasgupta, B., Li, W., Perry, A., Gutmann, D. H. Glioma formation in neurofibromatosis 1 reflects preferential activation of K-RAS in astrocytes. Cancer Res. 65, (2005). 65. King, D., Yang, G., Thompson, M. A. & Hiebert, S. W. 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Dasgupta, B., Yi, Y., Chen, D. Y., Weber, J. D. & Gutmann, D. H. Proteomic analysis reveals hyperactivation of the mtor pathway in NF1-associated human and mouse brain tumors. Cancer Res. 65, (2005). 73. Oliver T.G. et al. Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma. Development 132, (2005) Acknowledgements The authors thank our Neurofibromatosis Center Group at Washington University, especially A. Perry, J. Weber, M. Watson and J. Garbow, as well as R. Wechsler-Reya for critical reading of the manuscript. D.H.G. is supported by grants from the Department of Defense, National Institute of Neurological Disorders and Strokes, and James S. McDonnell Foundation. J.R. is a scholar of the Child Health Research Center of Excellence in Developmental Biology at Washington University School of Medicine and receives additional support from the National Institute of Child Health and Human Development, The American Cancer Society and Hope Street Kids. We also acknowledge the generous support from Schnuck Markets, Inc. Competing interests statement The authors declare no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Entrez Gene: CDNK2A NF1 National Cancer Institute: National Cancer Institute: leukaemia pheochromocytoma OMIM: neurofibromatosis type 1 FURTHER INFORMATION Gutmann lab: Rubin lab: Access to this interactive links box is free online. OPINION Cancer in xeroderma pigmentosum and related disorders of DNA repair James E. Cleaver Abstract Nucleotide-excision repair diseases exhibit cancer, complex developmental disorders and neurodegeneration. Cancer is the hallmark of xeroderma pigmentosum (XP), and neurodegeneration and developmental disorders are the hallmarks of Cockayne syndrome and trichothiodystrophy. A distinguishing feature is that the DNArepair or DNA-replication deficiencies of XP involve most of the genome, whereas the defects in CS are confined to actively transcribed genes. Many of the proteins involved in repair are also components of dynamic multiprotein complexes, transcription factors, ubiquitylation cofactors and signal-transduction networks. Complex clinical phenotypes might therefore result from unanticipated effects on other genes and proteins. Xeroderma pigmentosum (XP) is the archetype of an expanding family of nucleotideexcision repair (NER) diseases that includes XP itself, the XP variant (XP-V), Cockayne syndrome (CS), cerebro oculofacial skeletal syndrome (COFS), a mild ultraviolet (UV)-light-sensitive syndrome, trichothiodystrophy (TTD), and some diseases with combined symptoms of XP/CS and XP/ TTD 1. These diseases have complex overlapping symptoms associated with cancer, developmental delay, immunological defects, neurodegeneration, retinal degeneration and ageing. They represent a continuum with skin cancer alone at one extreme and neurodegeneration and developmental disorders at the other (BOX 1). The total number of genes directly involved in NER is estimated to be around 40 (REF. 2), but only about a dozen of these genes have been found to be deregulated in NER-related human diseases. The remainder represent either essential genes that would be lethal if mutated or that might have such mild clinical disease that they blend into the population of sun-sensitive individuals. The molecular defects in repair and the clinical symptoms of XP as compared with CS and TTD enable us to understand how certain pathways for processing DNA damage lead to cancer, whereas others lead to more complex disorders. Before the NER genes were cloned and identified, patients were assigned to complementation groups, such as XP-A or XP-D, by cell-fusion experiments 1. Once the mutated genes were identified and sequenced, they were named after the complementation group with which they associate. Many reviews have described the molecular mechanisms of NER and the amazing varieties of damage recognition and DNA manipulation that are required for the recognition and removal of DNA damage represented by the UV-light-induced dipyrimidine photoproducts, the 5 5, 6 6 cyclobutane pyrimidine dimers (CPDs) and the pyrimidine pyrimidone dimers, and other large carcinogen or oxidative products in DNA 3 6 (FIG. 1). I want to summarize these mechanisms briefly in terms of the function of the XP genes and address broad general questions about the comparison between XP and CS. These include two contrasting questions, the considerations of which are particularly informative. Why do patients with mutations in genes specific for global genomic repair (GGR) or bypass replication predominantly develop cancer but rarely neurodegeneration? And why do patients with mutations in genes specific for repair of damage in transcribed genes not get cancer but suffer from developmental and neurodegenerative disorders? Nucleotide-excision repair The question of how DNA damage is recognized can be viewed from two different perspectives: one question asks which proteins interact directly with damaged bases in DNA, and the other asks which protein acts first in this damage-response pathway (FIG. 1). Many of the NER proteins interact directly with damaged DNA, including XPE, XPC HR23B, XPA, replication protein A (RPA) and the transcription factor TFIIH 3 (TABLE 1). Which proteins act first depends on the transcriptional activity of the DNA. 564 JULY 2005 VOLUME 5

2 Actively transcribed genes are repaired more rapidly than non-transcribed regions through transcription-coupled repair (TCR). Damage appears to be recognized by the arrest of RNA polymerase II (Pol II) that is relieved through an assembled protein complex that includes two proteins, CSA and CSB 7. Damage to non-transcribed regions of the genome is recognized by binding of XPE and XPC protein complexes through GGR 3 5,8. These recognition events in TCR and GGR initiate a common pathway through which the DNA is unwound (the XPB and XPD helicase components of TFIIH), XPA binds and presents docking sites for the structure-specific nucleases (XPG and XPF ERCC1) and the damage is excised and replaced (FIG. 1). Global damage recognition Both XPE and XPC exist as heterodimeric proteins: XPE as a damage-specific DNA binding protein 2 (DDB2) DDB1 (p48 p127) dimer 9 ; XPC as an XPC HR23B dimer 10. Patients are found only with mutations in DDB2 or XPC, not in their partner genes 11. These two main components of the GGR pathway, XPC and XPE, activate ubiquitylation and proteolytic pathways, and interact with the proteasome system (BOX 2). Box 1 Cockayne syndrome and endogenous DNA damage Cockayne syndrome (CS) is an autosomal-recessive disease characterized by cachectic dwarfism, retinopathy, microcephaly, ganglial calcification, deafness, neural defects, retardation of growth and development after birth, and sun sensitivity. It is not characterized by cancer 1,135. The clinical presentation ranges from the very mild ultraviolet (UV)-lightsensitive syndrome (UV s ) syndrome to a range classified as CS types I, II and III, and the severe neonatal lethal cerebro oculofacial skeletal syndrome 135. Neurological defects in xeroderma pigmentosum (XP) patients include diminished deep-tendon reflexes, sensorineural deafness, peripheral neuropathy, walking difficulties and progressive mental deterioration. These XP symptoms are sufficiently different from those in CS such that patients who have XP with neurological disease (typically XPA) can be discriminated clinically from those with XP plus CS. XP patients have cerebral atrophy and primary neuronal degeneration (in the grey matter), whereas CS patients have dysmyelination (in the white matter), retinal and Purkinje cell loss, and growth retardation, but without loss of personality. Mutations in the two CS genes, CSA and CSB, at present show no obvious clustering within the sequences that can be correlated with the disease. A null mutation in CSB has been identified in one mild UV s patient, whereas point mutations in CSB can be more severe 136, and sometimes the same mutation can give quite different symptoms 137. CSA and CSB are required for enhanced repair of actively transcribed genes and for ubiquitylation of RNA polymerase II after UV-light-induced damage (TABLE 1). Trichothiodystrophy (TTD) is another nucleotide-excision repair (NER)-defective disease characterized by sulphur-deficient brittle hair with characteristic tiger tail banding when observed in polarized light, and by skin photosensitivity. It is not characterized by increased pigmentation or cancer 47. TTD is a complex neuroectodermal disorder with a common deficiency in synthesis of high-sulphur matrix proteins that results in growth retardation, neurological abnormalities and sulphur-deficient hair and nails. TTD is caused by a subset of mutations in XPB, XPD or TFB5, all components of the TFIIH transcription factor that reduce the overall transcription capacity of differentiated cells 47,50. The DNA-binding protein XPE. XP-E patients are mildly affected their cells carry out near-normal levels of NER 12,13. Because of difficulties of quantifying small differences in DNA repair (unscheduled DNA synthesis), there was initial confusion about the assignment of cells to this group. This has been clarified and it is now generally agreed that XP-E represents a group of patients with mutations in the DDB2 component of the heterodimer 12. XPE binding to a damaged site facilitates the binding of XPC, and seems to suppress the mutagenicity rather than the toxicity of damage 14,15. The DDB1 component is present in excess over its partner, and is predominantly cytoplasmic but translocates to the nucleus after UV-light irradiation 16. Mutations in DDB2 also prevent the accumulation of DDB1 in the nucleus 17. DDB2 expression is induced by UV light through p53-dependent transcription in human cells (but not mouse or hamster cells) 9,18, which provides a partial explanation for early observations that excision repair (GGR) is low in mouse cell lines 19. The global damage recognition protein XPC. The XPC HR23B complex 10,20 is the main early damage detector and, with XPE, serves to stabilize the low-affinity XPA and RPA proteins that subsequently bind at damaged sites 21,22. The XPE subunit DDB2 seems to facilitate XPC translocation within the nucleus after irradiation 23. In the absence of XPC, the residual NER is biased towards transcribed regions, but the total amount of residual repair appears to involve large stretches of DNA, greater than the amount represented by the active genes alone 24,25 (TABLE 1). Highly distorting lesions provide sufficient opening of the DNA strands so, XPC can be dispensed with in cell-fee systems of NER 26,27. The XPC HR23B complex is also associated with CEN2, a protein that stabilizes XPC and that is involved in centrosome duplication, indicating a direct link between GGR and cell division 28. The constitutive level of expression of XPC is controlled by p53, and can be induced by UV-light irradiation, enhancing GGR 29. Transcriptional recognition The initial damage-recognition mechanism for TCR is the stalled RNA Pol II itself 30,31. Arrested RNA Pol II is phosphorylated on its carboxy-terminal domain and, subsequently, is polyubiquitylated by a mechanism that involves CSA and CSB (BOX 2). Two interpretations of the role of ubiquitylation have been proposed. One indicates that ubiquitylation marks the RNA Pol II molecules for degradation, leaving the active genes accessible for repair and the resumption of transcription 7,34. The other indicates that a blocked RNA Pol II does not need to be degraded to facilitate repair 35, that the ubiquitylation is not essential for TCR 36, and the ubiquitylation linkage through Lys63 on RNA Pol II is a signal for activation of cellular processes including repair 37. The arrested RNA Pol II caused by TCR deficiency is a strong trigger for apoptosis in fibroblasts, but not necessarily for other cell types 38,39. Association of CSB with the transcription elongation complex enhances elongation of nascent RNA 40 and might also stabilize a blocked RNA Pol II (REF. 41), therefore increasing the efficiency with which RNA Pol II negotiates refractory secondary structures or T-rich regions in template DNA 42 (BOX 1). CSB can block retrograde movement of a blocked RNA Pol II 33, possibly by its ability to wrap the DNA 43. CSB is a member of the ATP-dependent SWI2 SNF2 chromatin-remodelling family and binds to DNA as a dimer. In the presence of ATP, CSB actively wraps the DNA around itself, and following ATP hydrolysis releases the NATURE REVIEWS CANCER VOLUME 5 JULY

3 Global genomic repair and replication cancer but no CNS disorder Pol-η-dependent replication of damaged DNA cancer but no disorder XPE XPC HR23B XPA RPA TFIIH (XPB XPD helicases) DNA 43. Irreversibly blocked RNA Pol II is degraded by the transcription release factor IIS 42. CSB deficiency might therefore affect the transcription efficiency of many other unrelated genes through its action on RNA Pol II. CSA is a cofactor for an SCF-type ubiquitin ligase (a SKP1 Cullin F-box multisubunit protein, with up to 60 different F-box proteins in human cells; BOX 2) 44 and translocates to the nuclear matrix after UVlight irradiation and associates with RNA Pol II by a process that requires CSB 45. CSA deficiency might therefore affect the functions and half-lives of those proteins that are substrates for SCF-mediated ubiquitylation, but might be functionally unrelated to DNA repair. XPA is common to GGR and TCR The XPA protein freely diffuses in the cell nucleus and binds to a damaged site that has been previously marked by XPE and XPC 8 and unwound by the action of TFIIH 3. The single-strand-binding protein RPA also binds at this site, and to XPA, with a DNA footprint that corresponds to the XPF ERCC1 5' nuclease Transcription-coupled repair CNS disorder but no cancer CSA CSB RNA Pol II XPG 3' nuclease PCNA Pol δ Common pathway both CNS disorder and cancer Figure 1 Nucleotide-excision repair showing the two main branches of transcription-coupled repair and global genomic repair, their convergence on a common pathway, and the main genes involved with various steps along the pathways. The initial damage response in transcriptioncoupled repair is mediated by the coupling factors CSA and CSB associated with the RNA polymerase II transcription elongation complex. The initial damage response in global genomic repair is mediated by the damage-recognition factors XPE (xeroderma pigmentosum, complementation group E) and XPC. Following damage recognition, the damaged site is remodelled by the helicase activities of XPB and XPD and binding of XPA and replication protein A (RPA). The nucleases XPG and XPF ERCC1 then cut the damaged oligonucleotide region either side of the damage and this is excised. The repair patch is synthesized by proliferating-cell nuclear antigen (PCNA) and replicative polymerase (Pol) δ. Damage that is not excised must be replicated by the action of Pol η. The precise order of some of these factors is still the subject of active research, and might not necessarily be invariant under all conditions. CNS, central nervous system. repair-patch size. XPA anchors the nuclease XPF ERCC1 and activates the nuclease activity of XPG 8, but is then released following binding of the excision nucleases 4. The core DNA-binding region of XPA, amino acids Met98 to Phe219 encoded by exons 2 through 5, contains a zinc finger and a positively charged cleft at its carboxy region, into which single-stranded damaged DNA can bind. Mutations in this core region generally give rise to severe symptoms, but mutations in the C-terminal exon 6 are generally milder 1,11. Some mutations, especially those near splice sites, can be leaky and permit sufficient normal protein to be made to minimize clinical symptoms. Mutation of an arginine at the C-terminal end of the core region (Arg207) to an uncharged glycine or lysine created an XPA revertant that no longer repaired CPDs in non-transcribed regions, but retained repair of more accessible damage such as [6-4] photoproducts and CPDs in transcribed regions 46. This XPA revertant might have a reduced binding to DNA owing to the loss of the positive charge on the arginine. Remodelling the TFIIH components The transcription factor TFIIH is a 10-member protein complex involved in transcription initiation and transcript elongation. The protein components are involved in the regulation of gene expression and the cell cycle, as well as in DNA repair. Two components, XPB and XPD, are helicases that serve to unwind the DNA, and TFIIH might in the process serve to distinguish damaged strands from undamaged strands 3 (TABLE 1). XP-B patients are exceedingly rare, being found only in three families to date, whereas XP-D patients are more common. The XPD gene is particularly complex, because mutations are associated with multiple disease outcomes including XP, XP/CS and TTD Deletions of XPD are embryonic lethal, and only missense mutations cause clinical phenotypes 49. One mutation, R683W, is exclusively associated with XP-D, whereas others R112H, R658H and R722W are found in TTD patients. Certain mutations in XPB and XPD, or in a third component, p8, regulate quantitative levels of TFIIH and are associated with the short brittle hair disorder (due to a lack of sulphur and cystine because of impaired synthesis of high-sulphur matrix protein) that is a diagnostic hallmark of TTD 50 (BOX 1). Removal of damage The structure-specific endonucleases XPG and XPF ERCC1 are directed to the damaged site by XPA and make the 3 and 5 incisions either side of the damaged site 51,52. Previous helicase activity of TFIIH is required before loading of XPF ERCC1 (REF. 53). These nucleases are also involved in repair of DNA DNA crosslinks and homologous recombination where single-strand double-strand DNA junctions are found, similar to those in an opened excision site 51,54. XPF ERCC1 is also a component of the telomeric TRF2 complex and removes the 3 overhang from uncapped telomeres 55. Complex phenotypes involving multiple organ systems could therefore be caused by the involvement of these nucleases in multiple pathways of gene expression, recombination and chromosome stability. XPG makes the first incision, 3 to damage, and patients with mutations in XPG are generally the more severely affected, showing both XP and XP/CS symptoms 51. Most mutations are truncations, resulting in very low levels of repair. XPG also activates a glycosylase involved in repair of oxidative damage 51. One mild case of XP-G has been reported in which an alternative splicing event eliminated the TFIIH-interacting region of the XPG protein, but retained other functions JULY 2005 VOLUME 5

4 Table 1 Genes affected in xeroderma pigmentosum/cockayne syndrome Repair mechanism Global genome repair Transcriptioncoupled repair Common pathway Postreplication repair Gene Chromosome Size (aa) Residual levels of repair (%) XPE (DDB2) Table compiled from data in REF The symptoms of XP-F patients are mild, with considerable levels of residual repair and very rare cases of neurodegeneration 57 (TABLE 1), and mutations in ERCC1 have not yet been reported. This is probably because mutations that would inactivate either protein would be embryonic or neonatal lethal in humans, as they are in mice, or cause very severe symptoms such as those seen in COFS patients 58. Replication of damaged DNA Pol η DNA replication is a crucial process by which many proteins are coordinated to pass on genetic information in a stable, accurate fashion to succeeding progeny. The process is even more complex when damaged DNA is to be processed, mutations minimized and genomic stability maintained 59. An impressive array of checkpoints delays cell proliferation to permit time for repair to be completed. Loss of replication control by mutation or loss of one or more checkpoint or replication components is a major contributor to the genetic instability leading to chromosomal and gene copy number changes associated with cancer 60 and is the underlying cause of the XP-V disease. XP-V patients have mutations in a lowfidelity Y-family DNA polymerase, Pol η, that is required for bypass replication of CPDs and other lesions (TABLE 1; BOX 3). The enzyme has a catalytic region at the 5 end of the gene and signal sequences at the 3 end for translocation to arrested DNA replication Function 11p >50 Damage binding, E3 ligase XPC 3p Damage binding CSA 5q Ubiquitylation E3 ligase CSB 10q DNA-dependent ATPase XPA 9p Damage verification XPB 2q Helicase, 3-5 XPD 19q Helicase, 5-3 XPG 13q <2 3ʹ nuclease XPF 16p ʹ nuclease XPV (Pol η) 6p Bypass polymerase sites and binding to proliferating-cell nuclear antigen (PCNA) 64,65. Pol η is also involved in somatic hypermutation during B-cell immunoglobulin class-switch recombination One of the distinctive features of XP-V cells has been their increased sensitivity to toxic effects of high concentrations of caffeine after UV-light irradiation 70. One interpretation attributes the sensitization to the interference of caffeine with members of the phosphatidylinositol-3-kinase-like family of proteins, ATM, ATR and DNA-dependent protein kinase, which are involved in the DNA-damage response 71, but the mechanism is still obscure. The presence of p53 is required for sensitization, because p53-null XP-V cells show a greatly increased sensitivity to UV light with no enhancement by caffeine 72. XP-V cells accumulate in the S-phase after UV-light-induced damage, indicating that Pol η is required for UV-light-damaged cells to pass through the S-phase checkpoint 73. During this arrest, XP-V cells demonstrate increased DNA-fork breakage that might signify chromosome instability 74,75. The stability of these arrested replication forks is strongly dependent on the DNAbinding capacity of p53 for DNA junctions and termini 76,77, on the single-strand-binding protein RPA 78, and on p53-dependent repression of RAD51 recombination 79. Recovery of DNA replication after UV-lightinduced damage then triggers a range of Box 2 Ubiquitylation and the response to ultraviolet-light damage Many of the major pathways of the DNA-damage response involve protein modification and degradation by ubiquitylation 82,138. Ubiquitin is a 76-residue polypeptide that is conjugated to target protein substrates to modify their functions (mono-ubiquitylation) or mark them for degradation through the proteasomal system (poly-ubiquitylation) The ubiquitylation and de-ubiquitylation of a substrate involves four classes of enzyme: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin protein ligase (E3) and a de-ubiquitylation enzyme (E4). Many of these are themselves complex multicomponent protein systems 44. The E3 ligases are primarily responsible for conferring substrate specificity and there are many more E3 enzymes other than ubiquitin-associated proteins A deficiency in an enzyme on the ubiquitylation pathway, especially E3 but occasionally earlier enzymes, can present unpredictable phenotypes that depend on the particular substrate proteins targeted by the system 139,141. E1 activating enzyme and the 19S and 20S proteasome subunits regulate both global genomic repair (GGR) and transcription-coupled repair (TCR) 82. The XPE heterodimer has a role both as a component of an SCF-type ubiquitylation E3 ligase and as a target for ubiquitylation itself 44,142. Damage-specific DNA binding protein 1 (DDB1), part of the XPE heterodimer, also acts as a cofactor for E3 ubiquitylation ligases for both DDB2 and CSA 44. The HR23B partner of the XPC HR23B damage-recognition complex interacts with XPC by its carboxy-terminal end, but its amino-terminal region has ubiquitin-like regions 143,144. These regions interact with other ubiquitylated proteins and with proteasome subunits to inhibit protein degradation 138, Recruitment of XPC to damaged sites also requires the function of the 20S proteasome subunit. The replication of damaged DNA involves mono-ubiquitylation, and the initial enzymes on the post-replication repair pathway RAD6 and RAD18 are ubiquitylation enzymes 148 that might be responsible for modifying proliferating-cell nuclear antigen to recruit the bypass polymerases η and ι (REF. 65) (BOX 3). Ubiquitylation has consequently emerged as a new important factor in modulating the activity of repair enzymes along each of the main branches of the ultraviolet-light response, GGR, TCR and bypass replication, and might represent novel targets for modifying repair activities in therapies for repair-deficient diseases. NATURE REVIEWS CANCER VOLUME 5 JULY

5 Box 3 The Y-family polymerases DNA photoproducts are blocks to the replicative DNA polymerases (Pols) α, δ and ε, which cannot accommodate large distortions, such as DNA photoproducts or adducts, in their active sites 149. Replicative bypass of these photoproducts is achieved by damagespecific Y-family polymerases that have larger active sites for nucleotide binding and relaxed substrate specificity 150,151. The Y-family polymerases have unique properties of accommodating large adducts within their active sites for replicating past such damage, with a wide range of substrate specificity, albeit with a significant error rate that can be as high as 1 2% Three Y-family polymerases have been identified in the mammalian genome Pol η, ι and κ 150. Mutations in Pol η are associated with a subset of xeroderma pigmentosum (XP) patients classified as the XP variants (XP-V; TABLE 1) Pol η specifically replicates past ultraviolet (UV)-light photoproducts and other large adducts. Pol ι has a poorer capacity for replication of UV-light damage, and Pol κ seems unable to replicate UV-light damage but might replicate other damage. The base specificity for Pol η and Pol ι indicate that Pol η might exercise a preference for replicating T-containing photoproducts, and Pol ι a preference for C-containing sites. Other polymerases, especially Pol κ and Pol ζ, can extend a DNA chain from mismatched termini, leading to a two-step process of bypassing damage 155,156. The loss of Pol η function results in increased UV-light-induced mutagenesis because of the need to recruit other, less accurate replication processes 157. Y-family polymerases need to be excluded from replication forks except when needed, otherwise the results would be catastrophic to the cells owing to their low fidelity. Several mechanisms regulate access to the replication forks. Replication arrest might facilitate increased access to blocked forks. The polymerases only translocate to replication foci after irradiation 70,84, and proliferating-cell nuclear antigen is modified by mono-ubiquitylation to facilitate polymerase binding 65. Since Pol η acts distributively, extending the nascent DNA chain by only a few bases across from photoproducts, before the replicative polymerases resume chain extension, the absolute error frequency will be low compared with replication on extended regions of undamaged DNA. Several early studies on nucleotide-excision repair (NER) showed a small but consistently greater DNA strand break frequency associated with excision in XP-V cells than normal cells 158,159. This could now be re-interpreted to indicate that Pol η might have a small role in the re-synthesis step of NER, perhaps when the parental strand also contains a photoproduct or other refractory sequence. recombination processes, involving both homologous recombination that is dependent on RAD51, and fork breakage associated with the MRE11 RAD50 NBS1 and γh2ax 74,75,80 complex. Enhanced replication-fork instability could be an important contributor to genomic instability that presages cancer. XP-V patients are clinically very similar to XP-C patients, indicating that failure of either global repair or of DNA bypass replication contribute equivalently to genomic instability and cancer incidence 1,60,61 (BOX 3). Tissue-specific modifications of NER Many of the tissues involved in the clinical presentation of XP and related diseases are partially or terminally differentiated, which can affect their repair capacity. The precise mechanisms of DNA repair that operate in different terminally differentiated tissues are unclear. Some differentiated cells of blood and neural origin, for example, appear to strongly downregulate GGR, leaving residual repair in transcribed regions of the genome 81. Keratinocytes, in contrast, upregulate GGR relative to TCR and show an altered apoptotic and p53 response to UVB light that is independent of TCR 38. The residual repair in neural tissues is subtly different from TCR in proliferating cells. The transcribed and non-transcribed strands of active genes appear to be repaired initially at similar rates (named differentiation-associated repair), in contrast to the enhanced repair on the transcribed strand usually associated with TCR in proliferating cells 81. A recent study has shown that a temperature-sensitive mutation in the E1 activation enzyme of the ubiquitylation system (BOX 2) can downregulate both GGR and TCR, which might be one mechanism for a global control of NER in differentiated tissues 82. The testis is also inherently repair deficient, lacking XPA, both excision nucleases, XRCC1 and Pol η, perhaps explaining the sensitivity of testicular cancer to treatment by chemotherapeutic agents that are DNA damaging (such as cisplatin) 83,84. Cancer and chromosomal instability Cancer is the characteristic disease of most XP patients, but not of TCR-defective CS patients or TTD patients, despite their sun sensitivity. Although the absence of repair in XP can be readily linked to cancer through increased UV-light-induced mutagenesis, the lack of cancer in CS remains an enigma. Studies with UV-light-damaged episomal plasmids show that CS cells specifically fail to repair CPDs, and that both XP and CS cells show increased mutagenesis 85,86, indicating that a difference in mutagenesis levels does not strongly discriminate between these two diseases. However, mutagenesis in episomal vectors might not involve the full spectrum of damage responses, such as apoptosis, cell-cycle checkpoints and chromosomal instability, that occur when whole cells are damaged. The increased mutagenesis seen in both XP and CS cells might therefore be necessary but not sufficient for carcinogenesis. It has been inferred that that XP-related cancers should not involve chromosome instability and aneuploidy because of their high gene mutation rates 87. Indeed, mutation rates are increased in TP53 (REF. 88) and patched (PTC) genes 89 in XP tumours, but experimental evidence for genome copy number changes or aneuploidy has yet to be obtained. But the absence of cancer in CS patients compared with XP patients, both of whom have similarly increased mutation rates in response to UV light, indicates that additional chromosome instability might actually be required for malignant transformation in XP. Chromosomal instability has been considered as crucial in carcinogenesis for other tissues 60. Chromosomal instability in XP, in contrast to CS, might therefore represent a global genome response when GGR or bypass replication is defective. The absence of such increased genomic instability from CS cells, which have a normal global damage response, would minimize the development of cancer in TCR-defective cells despite their increased point-mutation rates. Clinical symptoms The clinical spectrum of disorders presented by XP and related diseases clearly indicated early on that more than just DNA repair was impaired 1. Alternative targets for repair might be generated by endogenous oxidative damage in specific tissues like brain 90. However, once a link between repair and transcription was established, these diseases could be envisaged as also being transcription-defective diseases. More recent discoveries that certain repair proteins are associated with ubiquitylation 44,91 indicate that the secondary modification of proteins 568 JULY 2005 VOLUME 5

6 Cancer induction by direct defects in global responses Global damage DNA damage NER (XPC, XPE) Neuronal apoptosis DNA replication arrest (XP-V) (XPA, TFIIH, XPG, XPF) Genomically unstable cells CNS disorder to be unusually sensitive to oxygen levels, including the Purkinje and retinal cells Defects in protein processing would make CS a disease similar in principle to other neurodegenerative disorders such as Alzheimer s, Parkinson s, Lou-Gehrig s and prion diseases that involve ubiquitylation and defects in protein degradation Non-dividing cells Transcribed genes RNA Pol II arrest Genomically stable cells (CSA,CSB) Neurodegeneration by apoptosis and transcription arrest Figure 2 Mechanisms by which cancer or neurodegeneration could be caused by defects in global genomic repair and bypass replication or by defects in transcription-coupled repair. Defects in the common pathway can give rise to both sets of symptoms according to individual gene functions. Damage to the whole genome (predominantly non-transcribed regions) is processed by XPE (xeroderma pigmentosum, complementation group E) and XPC, other nucleotide-excision repair (NER) components and polymerase (Pol) η, and defects could result in genomically unstable cells that, under selection, could result in malignant transformation. Damage to transcriptionally active genes causes transcription arrest, and defects in transcription-coupled repair might not cause a high degree of genomic instability, but might enhance apoptosis that causes neuronal loss in the brain. CNS, central nervous system; XP-V, xeroderma pigmentosum variant. for signalling and degradation is also important. A significant number of the clinical symptoms of these diseases might therefore be distantly related to their repair defects, but depend on the targets for disruption of gene expression, protein stability and signal transduction. The protein products of XPB and XPD, for example, are involved in basal and hormonal-induced transcription through the AB domain of nuclear receptors such as retinoic acid and oestrogen receptors and in transcription of MYC 92,93. Neurodegeneration and developmental disorders are major features of the NER disorders (BOXES 1,2). Patients with mutations in genes unique to GGR (for example, XPC and XPE) and DNA replication (for example, Pol η) have fewest clinical symptoms outside of cancer. Patients with mutations in components of TCR (CSA and CSB) and common elements (XPA, XPB, XPD, XPF and XPG) (TABLE 1; FIG. 1) show very complex clinical symptoms involving the central nervous system and other organ systems 1. Three of these genes XPB, XPD and XPG give rise to combined symptoms of XP/TTD and XP/CS, according to the particular mutations carried by the patients 11. Mutations in XPD have relatively similar levels of repair deficiencies, but elicit a wide range of clinical disorders ranging across the complete spectrum of XP, CS and TTD that are influenced by transcription capacity 94,95. Reductions in levels of TFIIH due to certain mutations in several components XPB, XPD or TFB5 result in the TTD disorder 50,95, indicating this disease can be caused by limiting amounts of TFIIH in differentiating keratinocytes and hair-follicle cells, and other cell types, and can even be associated with β-thallasaemia 96, a genetic disorder of anaemia. Neurodegeneration might be ascribed to the greater relative importance of a TCR-like repair in differentiated brain cells transcription-blocking lesions that persist due to a failure of TCR could trigger an apoptotic response that would be pathological 30,31,39. The most obvious source for damage would be the high level of oxidative metabolism that occurs in the brain and produces lesions that cause transcription arrest 90,97. Active transcription can be estimated to involve only about 1 2% of total genomic DNA 24,25, indicating that the lethal (apoptotic) signal generated by a failure of TCR of active genes 30,39 must be about 50 to 100 times as potent per unit of repairable DNA, as is the signal for lethal events from a failure of GGR. This potent apoptotic signal might be a source for the pathological consequences in CS and cause cell loss from non-dividing tissues such as brain and retina (FIG. 2). Apoptosis that removed damaged cells from the skin would conversely prevent UV-light carcinogenesis, especially in CS cells 38. Oxidative damage has been reported in the brains of repair-deficient patients 98. Some tissues that degenerate in CS appear Mouse models There has been steady progress in making mouse knockout strains that reflect the human DNA-repair defects 105. In general, these display the cancer symptoms associated with repair defects, and some additional phenotypes of severe neonatal death that would be missed in humans. Thus, Xpa /, Xpc / and Xpe / mice all show increased skin cancer from UV light or chemical carcinogens, but so do Csa /, Csb / and Ttd / mice. Xpd / and Xpb / mice are embryonic lethal, consistent with the human, in which only point mutations are viable, and a mouse mimicking a human TTD mutation is viable. Xpg /, Xpf / and Ercc1 / strains all have severe developmental disorders. Human XP or CS heterozygotes appear to have no clearly demonstrable symptoms; however, some (but not all) Xpc +/ and Xpe +/ and other heterozygous knockouts show increased cancer incidence after UVB or carcinogen exposure 106,107. The variability points to additional roles in skin carcinogenesis from strain backgrounds and modifier genes. A comparison between Xpc / and Csb / mice on the same genetic background showed that although cancer incidence from UVB light was increased in both, Xpc / mice were more sensitive than Csb / mice 108. These outcomes indicate that failure of GGR is more strongly associated with cancer predisposition, and failure of TCR is associated with acute effects such as immunosuppression and erythema (UV-light-induced skin irritation). But there are other areas of major difference between human and mice: neurodegeneration, especially, is much milder and more difficult to develop in mice 105,108,109. The Xpa / knockout strain, despite corresponding to one of the more severe groups of human patients, shows no neurological disorder throughout its lifespan Several examples have shown that reducing overall repair capacity by crossing repair-deficient mouse strains with Xpa / mice that have no NER can increase the severity of neurodegenerative phenotypes. These include Csb / mice crossed with Xpa / mice 113 ; Ttd / mice crossed with Xpa / mice 114 ; and NATURE REVIEWS CANCER VOLUME 5 JULY

7 Box 4 Clinical and cellular characteristics unique to GGR or TCR Xeroderma pigmentosum groups C, E, V The clinical characteristics of these diseases include sun sensitivity, freckling, skin cancers and corneal damage. Individuals with XPE are very mildly affected. The clinical characteristics of XPC and XPV are indistinguishable, and the symptoms are moderate to severe. Most cases are neurologically normal, though there have been some very rare cases of neurological abnormalities. The cellular characteristics include ultraviolet (UV)-light sensitivity. Individuals with XPE and XPV show slight sensitivity, and individuals with XPC are more sensitive but highly variable. The damage induced by UV light causes reduced DNA repair or DNA replication. Cockayne syndrome The clinical characteristics of Cockayne syndrome include sun sensitivity, growth failure, absence of subcutaneous fat, sunken facial features, short life expectancy, progressive neurological impairment, deafness, retinal degeneration, dysmyelination, and brain and ganglial calcification. The cellular characteristics include ultraviolet (UV)-light sensitivity. The damage induced by UV light causes reduced transcription-coupled repair. Xpg / mice crossed with Xpa / mice 115. But crossing Xpc / mice with Xpa / mice does not enhance the cancer susceptibility or cause the development of neurological symptoms 105. The repair function of Xpc must therefore be channelled completely within the functions of Xpa, whereas the functions of the other genes involve downstream end points additional to those determined by Xpa. TFIIH is an essential transcription factor, so complete knockouts of these are lethal at the 2-cell embryo stage 105. Engineering a human TTD mutation in the mouse Xpd gene recapitulated most of the human disease except for an observed increase in cancer susceptibility in the mouse, and a potential role for XPD-mediated DNA repair in ageing 116,117. The knockout of Xpg reflects the severe disorders seen in human XP-G patients and has been used to define the last 360 amino acids as crucial for nuclease function and growth retardation 118. Similarly, knockouts of either Ercc1 (REF. 119) or Xpf 58 show severe neonatal-lethal phenotypes. Mouse strains with defective Y-family polymerases are all viable. Deletion of a large 5ʹ region of the gene was not viable. Targeting exons 4 or 8 in the catalytic region at the 5ʹ end of the gene 120 or exon 11 at the 3ʹ end of the gene that interacts with PCNA has produced viable fertile mice (R.R. Laposa and C.J.E., unpublished observations). An inactivating mutation in the Pol ι gene has occurred spontaneously in the mouse 129 strain, a strain often used as a source of embryonic stem cells for generation of knockout mice by homologous recombination 121. Generalized defects in oxidative repair generally do not cause the kind of neurological symptoms associated with CS. Neither mice defective in repair of 8-oxo-guanine 99,122 nor mice with defective Xrcc1 (which is crucial for most base-excision repair 123,124 ) show neurodegenerative symptoms. However, oxidative stress has been suggested as a mechanism for the loss of Purkinje cells in repair-deficient Xpg / mice 101 and in Atm-null mice 100, with stimulation of inappropriate activation of the cell cycle 125. The offending lesions that cause endogenous DNA damage relevant to neurodegeneration might therefore be a subset of chemical modifications caused by reactive oxidative species such as lipid peroxidation products 97 or cyclo-da 90 (a cyclic oxidative product of adenine formed in DNA) that act as transcription-blocking lesions subject to TCR, rather than single base lesions like 8-oxo-guanine that are repaired by base-excision repair. The cancer incidence in Csa /, Csb / and Ttd / mice could be an artefact, caused by several peculiarities of NER in the mouse genome. Generally, mouse cells require fewer steps for malignant transformation than human cells, and contain longer telomeres 126,127. Additionally, DDB2 in mouse cells is not upregulated by UV light, as in human cells, because the promoter contains a mutated inactive p53 response element 13, which might contribute inherent genomic instability above that in human cells. The mouse models of NER-defective diseases have therefore become an extremely informative set of strains with many features that recapitulate the human condition, but with some features that could be unique to the mouse. These strains will surely increase in value not only for basic understanding of these diseases but also as platforms to develop and test therapies that will aid the patients themselves 128. Diagnosis, treatment and cure? The NER diseases present major challenges in diagnosis and in patient care. UV-light sensitivity and DNA-repair deficiency can be used for patient and prenatal diagnosis 129. These procedures, though easy for a DNA-repair laboratory, are specialized techniques and a more general approach would be direct DNA sequencing available to most clinical genetic diagnostic laboratories. No strong hot spots have been identified in the general population, so each gene and mutation has to be identified directly. A problem then lies in what should be done with the information prognostically, especially in light of available treatment. Evidence for sun sensitivity and cancer predisposition in XP launches patients and their families into radical lifestyle changes of extreme solar protection. But the mutations in XP and CS genes are not sufficiently correlated directly with clinical symptoms to predict with certainty the future progress of disease, especially when the mutation is novel and the patient young. Unique cures are at present unavailable, and standard clinical care is the norm, though some approaches show promise. Bacterial repair enzymes in liposomes have been delivered to the skin and show reduction of UV-light-induced damage 130, and chemoprevention with retinoids showed promise, but with severe side effects 131. An XPC gene expressed from an adenovirus vector injected into the skin of an XPC mouse strain reduces skin carcinogenesis 128. Final comments XP was first identified as a DNA-repairdeficient human disease approximately 35 years ago with a somewhat overconfident statement: Patients with xeroderma pigmentosum develop fatal skin cancers when exposed to sunlight, and so the failure of DNA repair must be related to carcinogenesis 132. This has proven a true, but over-simplified statement. Here, I have attempted to provide a perspective on the relationships between molecular understanding of the NER diseases and the general nature of their clinical symptoms. Many case-by-case analyses still need to be done for individual patients according to their particular combination of mutations. Major frontiers remain. One is the elucidation of the structure of each of the NER proteins and polymerases 133,134, together with an understanding of how they interact and enter into molecular regulatory networks. A final frontier is to relate, in ways that will ultimately benefit patients, 570 JULY 2005 VOLUME 5

8 the clinical features of the NER diseases to the developing map of the purturbed molecular pathways. XP patients with mutations in the GGR recognition genes, XPC and XPE, or in Pol η are at high risk for solar-induced skin cancer, but rarely, if ever, have significant neurodegenerative disorders (BOX 4), whereas the situation for CS is the converse 1. This suggests to me that an element of cancer induction in these diseases involves a global destabilization of the genome leading to increased copy number changes and aneuploidy, whereas neurodegeneration and other symptoms are more specifically related to targets of gene transcription and protein modification (FIG. 2). James E. Cleaver is at the Auerback Melanoma Laboratory, Room N431, UCSF Cancer Center, Box 0808, Room N431, UCSF Cancer Center, University of California, San Francisco, California, , USA. jcleaver@cc.ucsf.edu doi: /nrc Bootsma, D., Kraemer, K. H., Cleaver, J. E. & Hoeijmakers, J. H. 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