oxidants), DNA repair is probably the most important mechanism a cell has to .... syndrome had a genetically determined deficiency in the recovery of transcrip.
Nucleotide Excision Repair: Variations Associated with Cancer Development and Speciation J E CLEAVER1 • .I R SPEAKMAN2. J P G VOLPE1 1 Laboratory
of Radiobiology and Environmental Health, University of California, San Francisco, California 94143-0750; 2Depart17lent ofZnology, University ofAberdeen, Aberdeen AB92TN
Introduction Mechanism of nucleotide excision repair The coupling of transcription with repair Diseases of nucleotide excision repair Xeroderma pigmentosum Cockayne syndrome Trichothiodystrophy Interindividual variability of nucleotide excision DNA excision repair and speciation Implications for cancer and other health effects Summary
I~TRODUCTIO~
The genome of cells presents massive problems of fidelity and maintenance of accurate information (Lindahl, 1993). A diploid human cell has 1.3 X 10 10 bases that must be faithfully maintained, replicated and passed on to the daughter cells. Given the large number of endogenous and exogenous genotoxic agents that cells encounter during the lifetime of their host, it is not surprising that such hosts have evolved elaborate means of safeguarding their genomes. Although preventive mechanisms are by no means trivial (eg anti oxidants), DNA repair is probably the most important mechanism a cell has to maintain its genome faithfully. Major roles for repair systems have been recog nized in mending endogenous oxidative damage in nuclear and mitochondrial DNA, in correcting DNA mismatches associated with non-polyposis colon can cer, in immunodeficiencies, in skin carcinogenesis in xeroderma pigmentosum (XP) and in developmental and neurological abnormalities in XP, Cockayne syndrome (CS) and trichothiodystrophy (TTD) (Cleaver and Kraemer, 1989). Less obvious, but possibly equally important, are variations in repair between individuals and between species. Cancer Surveys Volume 25: Genetics and Cancer: A Second Look © 1995 Imperial Cancer Research Fund. 0-87969-469-6/95. $5.00 + .00
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Repair systems range from those with high specificity, such as photolvases and uracil-DNA glycosylase, to those with great versatility, such as nucleotide excision repair (Sancar and Sancar, 1988). Photoreactivation directlv reverts cyclobutane pyrimidine dimers formed by ultraviolet (DV) light to normal nucleotides by phototransduction. This has a wide distribution among species, but for unexplained reasons is missing in a variety of microorganisms such as Bacillus subtilis and Schizosaccharomyces pombe and eutherian mammals in cluding humans (Yasui et al, 1994). Dracil-DNA-glycosylase is widespread and corrects uracil produced by deamination and misincorporation, by a base exci sion mechanism (Sancar and Sancar, 1988; Cleaver and Layher, 1995). Mis match repair removes single mismatched bases or small loops created by replication slippage and is responsible for much tumour associated DNA se quence variability (Modrich, 1994). Double strand breaks in DNA are rejoined by non-homologous recombination in mammalian cells involving proteins that bind specifically to DNA ends. Defects in some of these proteins result in defective immunoglobulin rearrangement, and some circulating autoimmune antigens are directed against these proteins (eg the Ku antigen) (Taccioli et al, 1994; Finnie et al, 1995). Nucleotide excision repair (NER) removes photoproducts, bulky adducts and many other damaged regions of DNA (Sancar, 1994). This seems to be the most versatile way a cell has to repair its DNA, and this pathway is active when many of the other pathways are overloaded with damage. A great deal of prog ress has been made in recent years in the mechanism of nucleotide excision repair, its coupling with transcription, the complex diseases exhibited by muta tions in NER genes and variations between individuals and species (Hanawalt, 1994; Sancar, 1994). Consideration of this process not only highlights an ex quisite molecular mechanism, but also illustrates a broad range of intriguing questions in cancer and development.
Mechanism of Nucleotide Excision Repair Early views on NER developed from the study of the capacity of cells to mend damage produced in DNA by DV light, either DVC (240-290 nm) or DVB (290-320 nm) (Cleaver and Mitchell, 1993). The most prevalent photo products induced are the cyclobutane pyrimidine dimer and, at about 25% the frequency, the pyrimidine-pyrimidone [6-4] photoproduct (Mitchell and Nairn, 1989; Mitchell and Cleaver, 1990). The [6-4] photoproduct can further undergo a DVB dependent conversion to its valence photoisomer, the Dewar pyrimidone (Taylor and Cohrs, 1987). The distribution of these photoproducts in human chromatin depends on base sequence, secondary DNA structure and DNA-protein interactions. Pyrimidines flanking a dipyrimidine sequence in crease photoproduct yield; purines suppress the yield. Photoproducts mapped to nucleosome structure indicate dimers are formed preferentially on the out er face of the DNA helix, but [6-4] photoproducts are much more common in linker than core DNA (Gold and Smerdon, 1990; Mitchell et al, 1990). The
Nucleotide Excision Repair
ratio of cytosine-containing cyclobutane dimers to thymine dimers increases significantly within the UVB region :\fitchell and Cleaver, 1990). Dimers con taining cytosine and [6-4J photoproducts, which are preferentially induced at TC sequences, may have a major role in UVB (solar) mutagenesis, producing C to T and CC to TT transition mutations (Brash and Haseltine, 1982; Wood et al, 1984; Brash et al, 1987; Dumaz et al, 1993). Damage by UV light has been mapped at nucleotide resolution in vivo using the ligation mediated polymerase chain reaction and has shown that the binding of transcription factors to promoter regions can both enhance and suppress photoproduct formation at specific sites (Tomaletti et al, 1993, 1994; Tornaletti and Pfeifer, 1994). The propensity of photoproducts to be formed in transcription factor binding sites would serve to enhance mutations affecting gene regulation and focus these mutations on specific tissues. Photoproducts produced in DNA by UVC or UVB radiation are repaired by a complex multistep process involving many interacting gene products (Fig. 1). These interactions can give rise to complex overlapping symptoms in patients with mutations in these genes. The repair process involves removal of a 27-29 nucleotide oligonucleotide that contains the photoproduct by endonucleolytic cleavage 5 nucleotides on the 3' side of the photoproduct and 24 nucleotides on the 5' side (Sancar, 1994). The single strand region is pro tected by Single strand binding protein (HSSB) and the gap is filled in by DNA polymerase () or E, proliferating cell nuclear antigen (PCNA) and ligase. These processes can be considered as involving sequential steps of photoproduct recognition, assembly of the excision complex, displacement of the excised fragment and polymerization of the replacement patch, but many puzzles remain. Photoproduct recognition is achieved by specific binding of the XPA gene product, a 273 aminoacid (30 kDa) protein. This protein appears to be rate limiting for repair in human cells but the precise mechanism of recognition is unknown. Its greater binding coefficient for [6-4J photoproducts over dimers influences the overall rates of repair such that [6-4J photoproducts are removed much faster than dimers (Jones and Wood, 1993; Asahina et al, 1994). The XPA protein bound to a photoproduct acts as a site for binding two nucleases: the XPG protein that cuts 3' to the dimer and a heterodimer of XPF and ERCC1 that cuts 5' to the dimer. Contact sites have been demonstrated between XPA and ERCC1 and ERCC1 and XPF, but XPG may act as an independent nuclease. Interestingly, another photoproduct binding protein, XPE, appears to playa minor part in quantitative levels of repair, but when it binds a photoproduct, it creates a DNase I hypersensitive site precise ly where the XPG nuclease cleaves DNA (Reardon et al, 1993). Even so, the role of the XPE gene product in repair remains a mystery: it is present in ex cess over XPA, but is not required for repair in vitro. The damaged region is unwound by the action of transcription factor TFIIH, which contains both 3' 5' (XPB) and 5' -3' (XPD) helicases. At least two components of TFIIH,
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..
24nt
5nt to . . . .
5' ~.I..J...LI..LJ....L.l..J..l...l...LJ....LL./"""".,,c...--~LL..LJ.....LL 5'
3'
TFIIH
B,D, ...
@HHR23B)
Fig. 1. Nucleotide excision repair: variation associated with cancer, development and specia tion. HHR23B = RAD23B
XPB and XPD, interact with TP53 and initiate a signal cascade in damaged cells that leads to cell cycle arrest and/or apoptosis. This functional process re quires about 100 nucleotides of DNA along which to operate (Huang and San car, 1994). This nuclease complex appears to have ready access to transcrip tionally active regions of the cell, but access to non-transcribed regions, or the non-transcribed strand, requires an additional set of gene products: the XPC/RAD23B heterodimer (Masutani et al, 1994).
The Coupling of Transcription with Repair The first evidence that excision repair might have a special relationship with transcription was the discovery that cell lines from patients with Cockayne syndrome had a genetically determined deficiency in the recovery of transcrip tion after UV irradiation (Lehman et al, 1979). Although these cells also fail to recover DNA replication, this second aspect of their phenotype has received little attention. The next discovery was that the transcriptionally active genes in normal cells were repaired faster than the rest of the genome and that the transcribed strand was better repaired than the non-transcribed strand (Mel lon et al, 1986). In expressed genes, a strand bias of mutations due to the per sistence of damage on the non-transcribed strand has been observed (Dumaz et al, 1993). An interesting question, therefore, is whether the lack of repair in non-transcribed regions of the genome means that these regions accumulate genetic variations that can emerge precipitously when cells enter altered states of gene expression through development or through translocation of promoter and enhancer regions. The relationship between repair and transcription involves two main sets of gene products. One set of gene products, those of the Cockayne syndrome genes (CKN] and CKN2), are human analogues of the mutation frequency decline locus in Escherichia coli and couple RNA polymerase II transcription to the excision complex (Venema et al, 1990a; Hanawalt, 1994). The precise
l'Jucleotide Excision Repair
TABLE 1. Nucleotide excision repair: genes identified in human disorders and UV sensitive Chinese hamster ovary ((HO) cells Human group
Rodent groupa
Chromosome location
Xeroderma pigmentosum A 9q34.1 Bb ERCC3 2q21 3p25.1 C Db,c ERCC2 19q13.2 E 11 Fd ERCC4 16p13 ERCC5 13q32-33 G Variant Cockayne syndrome A ERCC6 B
Relative repair (%)
Function
