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Xeroderma pigmentosum and nucleotide excision repair of DNA
REVIEWS
TII~S 19 - FEBRUARY1994
DNA DAMAGING AGENTS are constantly encountered by cells, and a remarkable variety of strategies are used to defend the genome against this assault. For example, epithelia and membranes provide an initial physical barrier, while transporters associated with membranes can actively exclude damaging agents. In the cytoplasm, detoxification systems exist that can render reactive compounds less harmful. If nuclear DNA does suffer damage, cells respond by inducing the expression of genes whose products can limit the h~xmfui effects. One such response is the activation of cell-cycle checkpoints that help prevent cells with toxic an,d mutagenic lesions in their genomes from undergoing mitosis or DNA replication. Multicellular organisms have the additional option of activating apoptosis to precipitate the selfdestruction of a seriously damaged cell. The most direct response to DNA damage, however, is to repair it. The key strategy used by mammalian cells to remove carcinogenic lesions caused by IN light and many other common mutagens is nucleotide excision repair. During this process, a multiprotein system locates a lesien in DNA and catalyses enzymatic cleavage of the altered strand. The damaged oligonucleotide and the incision proteins are then displaced; DNA synthesis proceeds to form a short patch using the unmodified strand as a template; and repair is completed by a DNA ligase. The outline of the process is deceptively simple (Fig. 1), and at first glance it Is surprising to find that genetic and biochemical studies implicate more than 20 gene products in the full reaction. One reason for the involvement of so many proteins might be because the system must be both versatile and finely tuned, since many ¢lifferent types of DNA modifications have to be recognized in the midst of a tremendous excess of native, undamaged DNA. The best known of the gene products that participate in these reactions are those that correspond to the seven genetic complementation groups (A to (3) of the disease xeroderma pigmentosum (XP). Mutant cells representing any of these XP groups exhibit, to K. Tanaka is at the Institute of Molecular and Cellular Biology, Osaka University, 1-3 Yamada-Oka,Suita, Osaka 565, Japap; and R. D. Wood is at the Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts, UK EN6 3LD. © 1994,ElsevierScienceLtd 0968-0004/94/$07.00
Xeroderma pigmentosum and nucleotide excision repair of DNA Kiyoji Tanaka and Richard D. Wood Nucleotide excision repair is a versatile strategy for removing DNA damage from the genome. Tremendous progress in understanding this process has been made in the last few years, and the field continues to develop rapidly. Exciting connections have emerged between nucleotide excision repair, transcription, and DNA replication, but many mysteries remain concerning the biochemical details of the mechanism, the connection with several human inherited syndromes, and the role of DNA repair in preventing cancer.
varying degrees, defects in the first steps of nucleotide excision repairL Individuals affected with XP are hypersensitive to sunlight; most have a predisposition to skin cancer and some patients show severe neurological abnormalities. A related rare repair disorder is Cockayne's syndrome (CS), in which patients display photosensitivity, severe mental and physical retardation, skeletal abnormalities and a wizened appearance. Several complementation groups of CS have been described. The repair defect in CS is more subtle than is usually found in XP, and seems to be limited to a defect in the repair of
lesions in the transcribed strand of active genes. in addition to cells from people with XP and CS, other mammalian cell mutants are providing insight into nucleotide excision repair. Of particular interest are a set of IN-sensitive variants, isolated from rodent cells, which have been assigned to 11 different complementation groups. Human genes that can correct the repair defects of rodent mutants in these complementation groups are denoted ERCC (excision repair cross-complementing) genes and are referred to by number, ERCCI to ERCCII. As outlined below, some of
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Recognition
/ IIIIIIII
Incision
i i i i i ~ I I I I I I i i'13'rrrrl
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Excision
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Repair synthesis
Rgure 1 Nucleotide excision repair. During this process, a multiprotein system locates damage in DNA (represented here by the square box and local bulge in the duplex) and two enzymatic incisions are introduced on the affected st:and, one on each side of the lesion. The damaged oligonucleotide is then displaced; DNA synthesis creates a patch using the unmodifled strand as a template; and repair is completed by a DNA ligase. 83
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TIBS 19
Table I. Cloned human XP and ERCC genes involved in nucleotide excision repair Gene
Chromosomal location
Yeasthomologue ($. cerevisiae)
Proteinfunction
XPA
9q34.1
RAD14
Binding to damaged DNA
XPB(ERCC3)
2q21
SSL2/RAD25
DNA helicase
XPC
?
RAD4
?
XPD(ERCC2)
19q13.2
RAD3
DNA helicase
XPG(ERCC5)
13q33
RAD2
DNA nucleasea,b
CSB(ERCC6)
10q11-21
?
DNA helicase a
ERCC1
19q13.2
RADIO
Part of DNA nucleaseU;forms complex with XPF, ERCC4 and ERCCll
FEBRUARY .,~nA
from some cell lines derived from XP-E patients, which suggests that defects in the protein are responsible for this form of XP. The second damaged-DNAbinding activity is the one deficient in XP-A cells ~5. Despite a lower relative preference of the XPA protein for damaged DNA, loss of XPA function causes a more severe defect in nucleotide excision repair than the XP-E bi.r,dingfactor defect. Perhaps the XPA protein has important DNA repair function(s) other than DNA-damage recognition, such as in making key contacts with other proteins to coordinate the nucleotide excision repair process.
'Inferred from the predicted protein sequence. ~Predicted, based on experiments in yeast.
A remaining puzzle: XP variants Roughly 20% of XP patients do not fall into the seven complementation L~,') these genes prove to be equivalent to :,isi~e RAD,., and a highly conserved groups described above, and instead particular XP-complementing genes"," core was identified that includes are designated as 'XP variants' (XP-V). while others are distinct. The process residues found in the acti,,'e sites of The nature of the XP variant is not well of nucleotide excision repair is evol- DNA nucleases s. RAD2 does indeed understood. XP-V patients have an utionarily conserved in eukaryotes, and have nuclease activity, and a corre- inherited predisposition to sunlightfunctional homologues of many of the sponding acti~,ity is expected for XPG"q. induced skin cancer, and develop cliniERCC and XP genes have been identified A cell-free in vitro repair system has cal characteristics that are generally
in other organisms. The identification and study of RAD genes and proteins in yeast has been especially informative. Identification of the classical XP groups is near completion The first reports of the isolation of cDNAs that could complement XP cells appeared only three years ago. Now, the gene or protein for each of the groups
has been identifiuti. The XPA ~ P group A complementing) and XPC ~ P group C complementing) genes were directly cloned by complementation of ~ sensitivity after transfecting XP cells with mouse genomic DNA or a human cDNA library, respectively'',s. The transfection cloning strategy was also used successfully to obtain a series of ERCC genes that correct the nucleotide excision repair defect in mutants of rodent cells. Some of these genes have proved to be XP-correcting genes. Specifically, the ERCC2, 3 and 6 genes are respectively equivalent to the XPD, XPB and CSB (Cockayne's syndrome group B complementing) genes 4: (TabJ,e i). Mutations were found in the appropriate genes of XP or CS patients, confirming that these are the causative genes for the corresponding form of each disorder. The XPG cDNA was fortuitously isolated when the protein product of its ~.gtll cDNA clone reacted with serum from a patient with the autoimmune disease systemic lupuc erythematosus ~. XPG resembles Saccharomyces c e r e -
84
shown that human XP complementation group G correspond:; to rodent repair complementation group 5 (Ref. 10), so the XPG gene is equivalent to the ERCC5 gene. in vitro studies have also recently revealed the existence of a repairenzyme complex involving XP group F complementing activity and activities that correct excision repair in ERCCI, ERCC4 and ERCCli cell extracts ~,v'. It is likely that ERCC4 or ERCCII is equivalent to XPF, which simplifies the situation. Further interpretation of this result is greatly aided by studies of S. cerevisiae, in which the RAD1 and RADIO proteins form a tight complex with DNA nuclease activity Is. P,AD10 shows sequence similarity to ERCCI, and it is predicted that when ERCC4, XPF and ERCCll cDNAs are isolated, one or more of them will show similarity to S. cerevisiae RADI. Two XP proteins have been found to preferentially bind damaged DNA, suggesting that they are involved in the damage-recognition step of nucleotide excision repair. One protein was found by several research groups, using a gel retardation assay to purify from crude mammalian cell extracts the major activity that can bind specifically to a UV-irradiated oligonucleotide. The activity includes a polypeptide of -125 kDa with a large discrimination for LN photoproducts over undamaged nucleotides 14. Significantly, this binding activity is completely absent in extracts
indistinguishable from the common symptoms of the classical XP groups described above. However, fibroblasts from XP-V individuals are only slightly more sensitive to the cytotoxic effects of IN than cells from normal donors, and XP-V cells excise ~ photoproducts from DNA at a normal, or near-normal rate. Thus, they do not appear to be defective in nucleotide excision repair! Some time ago, it was discovered that, after exposure to UV irradiation, XP-V cells have much more difficulty in carrying out normal DNA replication than do normal cells. This is manifested by a slower rat~ of joining together of newly synthesized DNA fragments, sometimes referred to as a 'postreplication repair' defect ~6. It has now become clear that mammalian cells can carry out semiconservative replication of DNA containing a large amount of damage, and have a mechanism for tolerating lesions, particularly pyrimidine dimers, by bypassing them during replication 17.The defect in replication of UVirradiated DNA in the XP variant has been suggested to be a defect in this ability to bypass pyrimidine dimers. A further well-established characteristic of skin fibroblasts from XP-V patients is that they are hypersensitive to the mutagenic action of UV. This hypermutability may explain why patients with XP-V are cancer-prone, but how does it arise? An important clue has recently been provided by the
REVIEWS
TIBS 1 9 - FEBRUARY1994
finding that UV irradiation produces different types of mutations in XP-V cells compared with normal cells 's. The DNA polymerase of normal cells usually seems to insert a purine, most often adenine, during bypass of a pyrimidine dimer in the template DNA. In XP-V cells, purines are not the most commonly inserted bases opposite a damaged area, suggesting that the mechanism of bypass by the DNA replication complex in XP-V cells is altered in some way. This is consistent with the earlier observations of a post-IN replication defect in XP-V. It seems possible that XP-V cells have alterations in a subunit of a DNA polymerase holoenzyme or in an accessory factor that helps the replication complex in normal cells bypass lesions in DNA.
Nuclectide excision repair occurs in cell-fTee systems Nucleotide excision repair can be carried out by extracts of mammalian cells. The repair synthesis that takes place towards the end of the reaction can be monitored by incubating damaged circular plasmid DNA with extracts in a reaction mixture that includes radiolabelled deoxynucleoside triphosphates. Quantification of incorporated radioactive nucleotides gives a measure of DNA repair. Proteins predicted by cell genetic studies to be involved in nucleotide excision repair have indeed proved to be required for the in vitro reaction. Extracts from most XP cell lines or UV-sensitive ERCCrodent cell lines are repair deficient. However, repair can be restored by mixing cell extracts from different repair complementation groups with one another, which permits an in vitro biochemical complementation assay. The in vitro system has been used to purify the XPA protein as an active polypeptide with an affinity for damaged DNA~s, and also led to the discovery of the correspondence of some XP and ERCC groupsl°-~2 in vitro studies also identified the human replication proteins RPA and PCNA as participants in nucleotide excision repair 19,2°. Analysis of repair in a cell-free system has also been a good way to learn about the relative affinity of nucleotide excision repair for different lesions in DNA, and is giving clues on how the repair system searches for damaged sites. The system has recently been extended to monitor the repair of SV40 minichromosomes, where the DNA is assembled into nucleosomes2L
A most important discovery, made using a cell-free system, has been the identification of the sites at which the nucleotide excision repair complex incises DNA22. A DNA duplex containing several specifically located UV-induced pyrimidine dimers was used in reaction mixtures with normal human cell extracts. Pyrimidine dimers were released in fragments of 27-29 nucleotides in length, with dual incisions flanking the damage, about 22 nucleotides 5' and six nucleotides 3' to the damaged site (see Fig. 1). Excision of a fragment of this size explains why nucleotide excision repair patches are found to be roughly 30 nucleotides long, in vivo and in vitro. The same pattern of dual incisions has been found in Xenopus oocytes, so this mode of dual incision by nucleotide excision repair may be a general one in eukaryotes.
What is the connection between nucleatide excision repair and transcdption? Some recent exciting findings have uncovered two clear (and conceptually separate) connections between DNA repair and the transcriptional process. One link is that the XPB (ERCC3) DNA repair protein is a component of TFIIH, a part of the RNA polymerase II transcription initiation complex 23. Functional XPB is needed for repair of nontranscribed, as well as transcribed, DNA. The protein is a DNA helicase and the best interpretation of the results, at present, is that the protein has a dual function, serving in a DNA unwinding step during nucleotide excision repair, as well as during transcription initiation. We have already noted examples in which one protein can participate in two distinct pathways of DNA metabolism, as exemplified by the replication/repair proteins RPA and PCNA. The XPD (ERCC2) protein is also a DNA helicase 24, and shows interesting parallels with the XPB protein, suggesting it may also be included in such a complex. The other link is the observation that pyrimidine dimers in transcribed genes are usually removed several-fold faster than from nontranscribed genes ~-s. There could be at least three reasons for this. Most directly, dimers in the transcribed strand of active genes are preferentially repaired, because transcription and nucleotide excision repair are directly coupled. The mechanism of such coupling has recently been elucidated in Escherichia coil, where the Mfd DNA helicase can bind to an RNA
polymerase complex stalled at a lesion on the transcribed DNA strand, and then recruit the E. coli !NrA protein to initiate repair at that ~,te 26. In mammalian cells less is known, but the ERCC6 DNA helicase appears to be involved 7. This direct linkage is only between transcription and nucleotide excision repair of some lesions (such as pyrimidine dimers), and the purpose might be to enhance the recognition of lesions that are normally difficult to repair. Furthermore, active genes might have a more open chromatin structure than inactive genes, allowing easier access for DNA repair proteins. Finally, the presence of one or more repair proteins, such as ERCC3, in the transcriptional initiation complex might mean that the local concentration of components of the repair apparatus is greater in transcribed regions, thus increasing the rate of repair in these regions by increasing the rate of assembly of a functional repair complex. These findings might at first lead to the impression that DNA nucleotide excision repair is dependent upon transcription of the repaired gene, but this is not the case. The bulk of the mammalian genome undergoes repair even though it is not transcribed, and the initial rate of repair in the bulk of the genome is not affected by incubating cells with the RNA polymerase Ii inhibitor a-amanitin. Some types of damage, such as IN.induced (6--4) photoproducts, are already good substrates for repair and are equally well removed from both nontranscribed and transcribed regions of DNA. Moreover, repair takes place in vitro in cell extracts in the absence of transcription.
Repair defects and neurological abnormalities Why do individuals with XP so frequently have neurological abnormalities in addition to a nucleotide excision repair defect? There appear to be at least two reasons: abnormal gene expression, and accelerated deterioration caused by the accumulation of unrepaired DNA damage. We will consider these in turn. Some patients with XP exhibit the characteristic neurological defects of CS, including retinal degeneration, normal-pressure hydrocephalug with increased deep-tendon reflexes, demyelination, and calcification of the basal ganglia. The three known patients with XP-B have these symptoras, as do at least one XP-D and three XP-G 85
REVIEWS patients. Fu:,~hermore, a third syndrome is sometimes associated with XP. Some patients with XP-D are additionally afflicted with trichothiodystrophy (TI"D), which is characterized by sulphur-deficient brittle hair, short stature, mental retardation and ichthyosis (coarse, dry, scaly skin). The XP-B/CS, YJ~-D/CS (or XP-D/TI"D) and CS-B patients have only subtle mutations in the XPB, XPD and CSB genes, but such mutations appear to be responsible for all of the XP, CS or TTD symptoms in a particular individual. The reason why some patients develop symptoms of more than one syndrome, while others do not, could be due to the specific nature of the mutation in the XPB or XPD genes..as mentioned above, the XPB protein is involved in transcription initiation as well as in nucleotide excision repair 23. A defect in a domain of XPB that is important for transcription might cause suboptimal expression of critical genes, which could result in the defective development of neural and ectodermal tissues found in CS or TTD. Mutations in XPD, CSB and XPG could have similar consequences. The above explanation does not seem to apply, however, to the more frequent XP-A form of XP. Here, severe repair defects occur without associated developmental dysfunctioa, suggesting that XPA is exclusively a repair protein, without a dual role in the transcription process. Instead, neurological abnormalities arise in a progressive fashion throughout the lifetime of the patient (typically 10-30 years). The'neurologi. cal abnormalities in XP-A are so different from those of C3 and TTD that they are sometimes designated as a separate disorder, 'DeSanctis-Cacchione syndrome'. It has been suggested that the accumulation of unrepaired DNA damage over the course of many years in the neural tissues of XP patients could lead to such symptoms 27. One likely source of such damage might be that produced by endogenous free radicals generated during metabolism. A candidate for an important causative agent of such damage has recently been described: oxygen-free radicals induce at least one frequent DNA lesion that is repaired in vitro by normal cell extracts, but not by XP-Acell extracts 28.
Animalmodelsfor XP Animal models with specific mutations in nucleotide excision repair genes should be very useful in understanding the consequences of repair 86
TIBS 19 - FEBRUARY1994 2 Tanaka, K. et al. (1990) Nature 348, 73-76 defects and the etiology of neurological 3 Legerski, R. and Peterson, C. (1992) Nature abnormalities. The first animal model 359, 70-73 to be described was a mutant of the 4 Weber, C. A., Salazar, E. P., Stewart, S. A. and fruit fly. When the haywire gene of Thompson, L. H. (1990) EMBOJ. 9, 1437-1447 5 Flejter, W. L. et al. (1992) Proc. NatlAcad. ScL Drosophila was isolated, it was found to USA 89, 261-265 encode a protein with 66?/0 identity to 6 Weeda, G. et al. (1990) Cell 62, 777-791 the human XPB protein. Strong amino 7 Troelstra, C. et al. (1992) Cell 71, 939-953 8 Scherly, D. et al. (1993) Nature 363, 182-185 acid conservation throughout the 9 Habraken, Y., Sung, P., Prakash, L. and human and Drosophila proteins indiPrakash, $. (1993) Nature 366, 365-368 cates that they are homologues. The 10 O'Donovan, A. and Wood, R. D. (1993) Nature original haywire mutation is a recessive 363,185-188 lethal, but flies with weak mutations in 11 van Vuuren, A. J. et aL (1993) EMBO J. 12, 3693-3701 haywire express marginal levels of the I 2 8iggerstaff, M., Szymkowski, D. E. and Wood, gene product and sur~ve. These disR. D. (1993) EMBO J. 12, 3685-3692 play a variety of symptoms such as 13 Tomkinson, A. E. et aL (1993) Nature 362, 860-862 motor defects, reduced life span, sexual Hwang, B. J. and Chu, G. (1993) Biochemistry impairment, central nervous system I 4 32, 1657-1666 defects and ~ hypersensitivity~-9. 15 Robins, P. et al. (1991) EMBOJ. 10, 3913-3921 These results support the view that the neurological complications in XP-B/CS 16 Lehmann, A. et al. (1975) Proc. Natl Acad. Sci. USA 72, 219-223 are due to abnormal development of 17 Spivak, G. and Hanawalt, P. C. (1992) neural tissues. Biochemistry 31, 6794-6800 The ability to perform gene targetting 18 Wang, Y-C., Maher, V. M., Mitchell, D. L. and McCormick, J. J. (1993) Mol. Cell. Biol. 13, in mouse embryonic stem cells has 4276-4283 made it possible to construct mouse 19 Shivji, M. K. K., Kenny, M. K. and Wood, R. D. (1992) Cell 69, 367-374 models for XP, CS, TTD, and ERCC defects. These are now being studied [n 20 Nichols, A. E and Sancar, A. (1992) Nucleic Acids Res. 20, 3559-3564 several laboratories, and a first report 21 Masutani, C. et al. (1993) J. Biol. Chem. 268, on ERCCl-deficient mice has appeared 3°. 9105-9109 Such models will, no doubt, prove to be 22 Huang, J. C., Svoboda, D. L., Reardon, J. T. and Sancar, A. (1992) Proc. Natl Acad. ScL USA 89, extremely valuable for the study of DNA 3664-3668 repair, carcinogenesis and neurodegen- 23 8chaeffer, L. et al. (1993) Science 260, 58-63 eration in a very intriguing group of 24 Sung, P. et al. (1993) Nature 365, 852-855 25 Hanawalt, P, C. (1991) Murat, Res. 247, genetic disorders. 203-211
26 Selby, C. P. and Sancar, A. (1993) Science 260,
References Journal policy limits the number of references that can be given here to 30, and we apologize to all those who we were unable to cite explicitly. I Cleaver, J, E. and Kraemer, K. H. (1989)in The ' Metabolic Basis of Inherited Disease (6th edn) (Scriver, C. R., Beaudet, A. L., Sly, W. S. and Vatle, D., eds), pp. 2949-2971, McGraw-Hill
53-58 27 Robblns, J. H. (1988) J. Am. Med. Asscc 260, 384-388 28 Satoh, M. S., Jones, C. J., Wood, R. D. and Lindahl, T. (1993) Proc. Natl Acad. Sci. USA 90, 6335-6339 29 Mounkes, L. C. et aL (1992) Cell 71, 925-937 30 McWhir, J. et al. (1993) Nature Genet. 5, 217-224
TIBS reference lists Authors of TIBS articles are asked to limit the number of references cited to provide non-specialist readers with a concise list for further reading. It is hoped that the citation of other, more extensive review articles rather than a comprehensive list of original articles enables interested readers to delve more immediately into the topic.
TII~S 19 - FEBRUARY1994
DNA DAMAGING AGENTS are constantly encountered by cells, and a remarkable variety of strategies are used to defend the genome against this assault. For example, epithelia and membranes provide an initial physical barrier, while transporters associated with membranes can actively exclude damaging agents. In the cytoplasm, detoxification systems exist that can render reactive compounds less harmful. If nuclear DNA does suffer damage, cells respond by inducing the expression of genes whose products can limit the h~xmfui effects. One such response is the activation of cell-cycle checkpoints that help prevent cells with toxic an,d mutagenic lesions in their genomes from undergoing mitosis or DNA replication. Multicellular organisms have the additional option of activating apoptosis to precipitate the selfdestruction of a seriously damaged cell. The most direct response to DNA damage, however, is to repair it. The key strategy used by mammalian cells to remove carcinogenic lesions caused by IN light and many other common mutagens is nucleotide excision repair. During this process, a multiprotein system locates a lesien in DNA and catalyses enzymatic cleavage of the altered strand. The damaged oligonucleotide and the incision proteins are then displaced; DNA synthesis proceeds to form a short patch using the unmodified strand as a template; and repair is completed by a DNA ligase. The outline of the process is deceptively simple (Fig. 1), and at first glance it Is surprising to find that genetic and biochemical studies implicate more than 20 gene products in the full reaction. One reason for the involvement of so many proteins might be because the system must be both versatile and finely tuned, since many ¢lifferent types of DNA modifications have to be recognized in the midst of a tremendous excess of native, undamaged DNA. The best known of the gene products that participate in these reactions are those that correspond to the seven genetic complementation groups (A to (3) of the disease xeroderma pigmentosum (XP). Mutant cells representing any of these XP groups exhibit, to K. Tanaka is at the Institute of Molecular and Cellular Biology, Osaka University, 1-3 Yamada-Oka,Suita, Osaka 565, Japap; and R. D. Wood is at the Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts, UK EN6 3LD. © 1994,ElsevierScienceLtd 0968-0004/94/$07.00
Xeroderma pigmentosum and nucleotide excision repair of DNA Kiyoji Tanaka and Richard D. Wood Nucleotide excision repair is a versatile strategy for removing DNA damage from the genome. Tremendous progress in understanding this process has been made in the last few years, and the field continues to develop rapidly. Exciting connections have emerged between nucleotide excision repair, transcription, and DNA replication, but many mysteries remain concerning the biochemical details of the mechanism, the connection with several human inherited syndromes, and the role of DNA repair in preventing cancer.
varying degrees, defects in the first steps of nucleotide excision repairL Individuals affected with XP are hypersensitive to sunlight; most have a predisposition to skin cancer and some patients show severe neurological abnormalities. A related rare repair disorder is Cockayne's syndrome (CS), in which patients display photosensitivity, severe mental and physical retardation, skeletal abnormalities and a wizened appearance. Several complementation groups of CS have been described. The repair defect in CS is more subtle than is usually found in XP, and seems to be limited to a defect in the repair of
lesions in the transcribed strand of active genes. in addition to cells from people with XP and CS, other mammalian cell mutants are providing insight into nucleotide excision repair. Of particular interest are a set of IN-sensitive variants, isolated from rodent cells, which have been assigned to 11 different complementation groups. Human genes that can correct the repair defects of rodent mutants in these complementation groups are denoted ERCC (excision repair cross-complementing) genes and are referred to by number, ERCCI to ERCCII. As outlined below, some of
lllllllllllllllllllllllllllllllllll~l[llllllllllll
\ IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~]T['] i i i i i ill
Recognition
/ IIIIIIII
Incision
i i i i i ~ I I I I I I i i'13'rrrrl
IIIIIIlllilllllll,,~ia,~,~,~J~,~il~llllllilll/'
Excision
Iltlllllllllllllllllllllllllllllllllllllllilllllllllllll
Repair synthesis
Rgure 1 Nucleotide excision repair. During this process, a multiprotein system locates damage in DNA (represented here by the square box and local bulge in the duplex) and two enzymatic incisions are introduced on the affected st:and, one on each side of the lesion. The damaged oligonucleotide is then displaced; DNA synthesis creates a patch using the unmodifled strand as a template; and repair is completed by a DNA ligase. 83
REVIEWS
TIBS 19
Table I. Cloned human XP and ERCC genes involved in nucleotide excision repair Gene
Chromosomal location
Yeasthomologue ($. cerevisiae)
Proteinfunction
XPA
9q34.1
RAD14
Binding to damaged DNA
XPB(ERCC3)
2q21
SSL2/RAD25
DNA helicase
XPC
?
RAD4
?
XPD(ERCC2)
19q13.2
RAD3
DNA helicase
XPG(ERCC5)
13q33
RAD2
DNA nucleasea,b
CSB(ERCC6)
10q11-21
?
DNA helicase a
ERCC1
19q13.2
RADIO
Part of DNA nucleaseU;forms complex with XPF, ERCC4 and ERCCll
FEBRUARY .,~nA
from some cell lines derived from XP-E patients, which suggests that defects in the protein are responsible for this form of XP. The second damaged-DNAbinding activity is the one deficient in XP-A cells ~5. Despite a lower relative preference of the XPA protein for damaged DNA, loss of XPA function causes a more severe defect in nucleotide excision repair than the XP-E bi.r,dingfactor defect. Perhaps the XPA protein has important DNA repair function(s) other than DNA-damage recognition, such as in making key contacts with other proteins to coordinate the nucleotide excision repair process.
'Inferred from the predicted protein sequence. ~Predicted, based on experiments in yeast.
A remaining puzzle: XP variants Roughly 20% of XP patients do not fall into the seven complementation L~,') these genes prove to be equivalent to :,isi~e RAD,., and a highly conserved groups described above, and instead particular XP-complementing genes"," core was identified that includes are designated as 'XP variants' (XP-V). while others are distinct. The process residues found in the acti,,'e sites of The nature of the XP variant is not well of nucleotide excision repair is evol- DNA nucleases s. RAD2 does indeed understood. XP-V patients have an utionarily conserved in eukaryotes, and have nuclease activity, and a corre- inherited predisposition to sunlightfunctional homologues of many of the sponding acti~,ity is expected for XPG"q. induced skin cancer, and develop cliniERCC and XP genes have been identified A cell-free in vitro repair system has cal characteristics that are generally
in other organisms. The identification and study of RAD genes and proteins in yeast has been especially informative. Identification of the classical XP groups is near completion The first reports of the isolation of cDNAs that could complement XP cells appeared only three years ago. Now, the gene or protein for each of the groups
has been identifiuti. The XPA ~ P group A complementing) and XPC ~ P group C complementing) genes were directly cloned by complementation of ~ sensitivity after transfecting XP cells with mouse genomic DNA or a human cDNA library, respectively'',s. The transfection cloning strategy was also used successfully to obtain a series of ERCC genes that correct the nucleotide excision repair defect in mutants of rodent cells. Some of these genes have proved to be XP-correcting genes. Specifically, the ERCC2, 3 and 6 genes are respectively equivalent to the XPD, XPB and CSB (Cockayne's syndrome group B complementing) genes 4: (TabJ,e i). Mutations were found in the appropriate genes of XP or CS patients, confirming that these are the causative genes for the corresponding form of each disorder. The XPG cDNA was fortuitously isolated when the protein product of its ~.gtll cDNA clone reacted with serum from a patient with the autoimmune disease systemic lupuc erythematosus ~. XPG resembles Saccharomyces c e r e -
84
shown that human XP complementation group G correspond:; to rodent repair complementation group 5 (Ref. 10), so the XPG gene is equivalent to the ERCC5 gene. in vitro studies have also recently revealed the existence of a repairenzyme complex involving XP group F complementing activity and activities that correct excision repair in ERCCI, ERCC4 and ERCCli cell extracts ~,v'. It is likely that ERCC4 or ERCCII is equivalent to XPF, which simplifies the situation. Further interpretation of this result is greatly aided by studies of S. cerevisiae, in which the RAD1 and RADIO proteins form a tight complex with DNA nuclease activity Is. P,AD10 shows sequence similarity to ERCCI, and it is predicted that when ERCC4, XPF and ERCCll cDNAs are isolated, one or more of them will show similarity to S. cerevisiae RADI. Two XP proteins have been found to preferentially bind damaged DNA, suggesting that they are involved in the damage-recognition step of nucleotide excision repair. One protein was found by several research groups, using a gel retardation assay to purify from crude mammalian cell extracts the major activity that can bind specifically to a UV-irradiated oligonucleotide. The activity includes a polypeptide of -125 kDa with a large discrimination for LN photoproducts over undamaged nucleotides 14. Significantly, this binding activity is completely absent in extracts
indistinguishable from the common symptoms of the classical XP groups described above. However, fibroblasts from XP-V individuals are only slightly more sensitive to the cytotoxic effects of IN than cells from normal donors, and XP-V cells excise ~ photoproducts from DNA at a normal, or near-normal rate. Thus, they do not appear to be defective in nucleotide excision repair! Some time ago, it was discovered that, after exposure to UV irradiation, XP-V cells have much more difficulty in carrying out normal DNA replication than do normal cells. This is manifested by a slower rat~ of joining together of newly synthesized DNA fragments, sometimes referred to as a 'postreplication repair' defect ~6. It has now become clear that mammalian cells can carry out semiconservative replication of DNA containing a large amount of damage, and have a mechanism for tolerating lesions, particularly pyrimidine dimers, by bypassing them during replication 17.The defect in replication of UVirradiated DNA in the XP variant has been suggested to be a defect in this ability to bypass pyrimidine dimers. A further well-established characteristic of skin fibroblasts from XP-V patients is that they are hypersensitive to the mutagenic action of UV. This hypermutability may explain why patients with XP-V are cancer-prone, but how does it arise? An important clue has recently been provided by the
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finding that UV irradiation produces different types of mutations in XP-V cells compared with normal cells 's. The DNA polymerase of normal cells usually seems to insert a purine, most often adenine, during bypass of a pyrimidine dimer in the template DNA. In XP-V cells, purines are not the most commonly inserted bases opposite a damaged area, suggesting that the mechanism of bypass by the DNA replication complex in XP-V cells is altered in some way. This is consistent with the earlier observations of a post-IN replication defect in XP-V. It seems possible that XP-V cells have alterations in a subunit of a DNA polymerase holoenzyme or in an accessory factor that helps the replication complex in normal cells bypass lesions in DNA.
Nuclectide excision repair occurs in cell-fTee systems Nucleotide excision repair can be carried out by extracts of mammalian cells. The repair synthesis that takes place towards the end of the reaction can be monitored by incubating damaged circular plasmid DNA with extracts in a reaction mixture that includes radiolabelled deoxynucleoside triphosphates. Quantification of incorporated radioactive nucleotides gives a measure of DNA repair. Proteins predicted by cell genetic studies to be involved in nucleotide excision repair have indeed proved to be required for the in vitro reaction. Extracts from most XP cell lines or UV-sensitive ERCCrodent cell lines are repair deficient. However, repair can be restored by mixing cell extracts from different repair complementation groups with one another, which permits an in vitro biochemical complementation assay. The in vitro system has been used to purify the XPA protein as an active polypeptide with an affinity for damaged DNA~s, and also led to the discovery of the correspondence of some XP and ERCC groupsl°-~2 in vitro studies also identified the human replication proteins RPA and PCNA as participants in nucleotide excision repair 19,2°. Analysis of repair in a cell-free system has also been a good way to learn about the relative affinity of nucleotide excision repair for different lesions in DNA, and is giving clues on how the repair system searches for damaged sites. The system has recently been extended to monitor the repair of SV40 minichromosomes, where the DNA is assembled into nucleosomes2L
A most important discovery, made using a cell-free system, has been the identification of the sites at which the nucleotide excision repair complex incises DNA22. A DNA duplex containing several specifically located UV-induced pyrimidine dimers was used in reaction mixtures with normal human cell extracts. Pyrimidine dimers were released in fragments of 27-29 nucleotides in length, with dual incisions flanking the damage, about 22 nucleotides 5' and six nucleotides 3' to the damaged site (see Fig. 1). Excision of a fragment of this size explains why nucleotide excision repair patches are found to be roughly 30 nucleotides long, in vivo and in vitro. The same pattern of dual incisions has been found in Xenopus oocytes, so this mode of dual incision by nucleotide excision repair may be a general one in eukaryotes.
What is the connection between nucleatide excision repair and transcdption? Some recent exciting findings have uncovered two clear (and conceptually separate) connections between DNA repair and the transcriptional process. One link is that the XPB (ERCC3) DNA repair protein is a component of TFIIH, a part of the RNA polymerase II transcription initiation complex 23. Functional XPB is needed for repair of nontranscribed, as well as transcribed, DNA. The protein is a DNA helicase and the best interpretation of the results, at present, is that the protein has a dual function, serving in a DNA unwinding step during nucleotide excision repair, as well as during transcription initiation. We have already noted examples in which one protein can participate in two distinct pathways of DNA metabolism, as exemplified by the replication/repair proteins RPA and PCNA. The XPD (ERCC2) protein is also a DNA helicase 24, and shows interesting parallels with the XPB protein, suggesting it may also be included in such a complex. The other link is the observation that pyrimidine dimers in transcribed genes are usually removed several-fold faster than from nontranscribed genes ~-s. There could be at least three reasons for this. Most directly, dimers in the transcribed strand of active genes are preferentially repaired, because transcription and nucleotide excision repair are directly coupled. The mechanism of such coupling has recently been elucidated in Escherichia coil, where the Mfd DNA helicase can bind to an RNA
polymerase complex stalled at a lesion on the transcribed DNA strand, and then recruit the E. coli !NrA protein to initiate repair at that ~,te 26. In mammalian cells less is known, but the ERCC6 DNA helicase appears to be involved 7. This direct linkage is only between transcription and nucleotide excision repair of some lesions (such as pyrimidine dimers), and the purpose might be to enhance the recognition of lesions that are normally difficult to repair. Furthermore, active genes might have a more open chromatin structure than inactive genes, allowing easier access for DNA repair proteins. Finally, the presence of one or more repair proteins, such as ERCC3, in the transcriptional initiation complex might mean that the local concentration of components of the repair apparatus is greater in transcribed regions, thus increasing the rate of repair in these regions by increasing the rate of assembly of a functional repair complex. These findings might at first lead to the impression that DNA nucleotide excision repair is dependent upon transcription of the repaired gene, but this is not the case. The bulk of the mammalian genome undergoes repair even though it is not transcribed, and the initial rate of repair in the bulk of the genome is not affected by incubating cells with the RNA polymerase Ii inhibitor a-amanitin. Some types of damage, such as IN.induced (6--4) photoproducts, are already good substrates for repair and are equally well removed from both nontranscribed and transcribed regions of DNA. Moreover, repair takes place in vitro in cell extracts in the absence of transcription.
Repair defects and neurological abnormalities Why do individuals with XP so frequently have neurological abnormalities in addition to a nucleotide excision repair defect? There appear to be at least two reasons: abnormal gene expression, and accelerated deterioration caused by the accumulation of unrepaired DNA damage. We will consider these in turn. Some patients with XP exhibit the characteristic neurological defects of CS, including retinal degeneration, normal-pressure hydrocephalug with increased deep-tendon reflexes, demyelination, and calcification of the basal ganglia. The three known patients with XP-B have these symptoras, as do at least one XP-D and three XP-G 85
REVIEWS patients. Fu:,~hermore, a third syndrome is sometimes associated with XP. Some patients with XP-D are additionally afflicted with trichothiodystrophy (TI"D), which is characterized by sulphur-deficient brittle hair, short stature, mental retardation and ichthyosis (coarse, dry, scaly skin). The XP-B/CS, YJ~-D/CS (or XP-D/TI"D) and CS-B patients have only subtle mutations in the XPB, XPD and CSB genes, but such mutations appear to be responsible for all of the XP, CS or TTD symptoms in a particular individual. The reason why some patients develop symptoms of more than one syndrome, while others do not, could be due to the specific nature of the mutation in the XPB or XPD genes..as mentioned above, the XPB protein is involved in transcription initiation as well as in nucleotide excision repair 23. A defect in a domain of XPB that is important for transcription might cause suboptimal expression of critical genes, which could result in the defective development of neural and ectodermal tissues found in CS or TTD. Mutations in XPD, CSB and XPG could have similar consequences. The above explanation does not seem to apply, however, to the more frequent XP-A form of XP. Here, severe repair defects occur without associated developmental dysfunctioa, suggesting that XPA is exclusively a repair protein, without a dual role in the transcription process. Instead, neurological abnormalities arise in a progressive fashion throughout the lifetime of the patient (typically 10-30 years). The'neurologi. cal abnormalities in XP-A are so different from those of C3 and TTD that they are sometimes designated as a separate disorder, 'DeSanctis-Cacchione syndrome'. It has been suggested that the accumulation of unrepaired DNA damage over the course of many years in the neural tissues of XP patients could lead to such symptoms 27. One likely source of such damage might be that produced by endogenous free radicals generated during metabolism. A candidate for an important causative agent of such damage has recently been described: oxygen-free radicals induce at least one frequent DNA lesion that is repaired in vitro by normal cell extracts, but not by XP-Acell extracts 28.
Animalmodelsfor XP Animal models with specific mutations in nucleotide excision repair genes should be very useful in understanding the consequences of repair 86
TIBS 19 - FEBRUARY1994 2 Tanaka, K. et al. (1990) Nature 348, 73-76 defects and the etiology of neurological 3 Legerski, R. and Peterson, C. (1992) Nature abnormalities. The first animal model 359, 70-73 to be described was a mutant of the 4 Weber, C. A., Salazar, E. P., Stewart, S. A. and fruit fly. When the haywire gene of Thompson, L. H. (1990) EMBOJ. 9, 1437-1447 5 Flejter, W. L. et al. (1992) Proc. NatlAcad. ScL Drosophila was isolated, it was found to USA 89, 261-265 encode a protein with 66?/0 identity to 6 Weeda, G. et al. (1990) Cell 62, 777-791 the human XPB protein. Strong amino 7 Troelstra, C. et al. (1992) Cell 71, 939-953 8 Scherly, D. et al. (1993) Nature 363, 182-185 acid conservation throughout the 9 Habraken, Y., Sung, P., Prakash, L. and human and Drosophila proteins indiPrakash, $. (1993) Nature 366, 365-368 cates that they are homologues. The 10 O'Donovan, A. and Wood, R. D. (1993) Nature original haywire mutation is a recessive 363,185-188 lethal, but flies with weak mutations in 11 van Vuuren, A. J. et aL (1993) EMBO J. 12, 3693-3701 haywire express marginal levels of the I 2 8iggerstaff, M., Szymkowski, D. E. and Wood, gene product and sur~ve. These disR. D. (1993) EMBO J. 12, 3685-3692 play a variety of symptoms such as 13 Tomkinson, A. E. et aL (1993) Nature 362, 860-862 motor defects, reduced life span, sexual Hwang, B. J. and Chu, G. (1993) Biochemistry impairment, central nervous system I 4 32, 1657-1666 defects and ~ hypersensitivity~-9. 15 Robins, P. et al. (1991) EMBOJ. 10, 3913-3921 These results support the view that the neurological complications in XP-B/CS 16 Lehmann, A. et al. (1975) Proc. Natl Acad. Sci. USA 72, 219-223 are due to abnormal development of 17 Spivak, G. and Hanawalt, P. C. (1992) neural tissues. Biochemistry 31, 6794-6800 The ability to perform gene targetting 18 Wang, Y-C., Maher, V. M., Mitchell, D. L. and McCormick, J. J. (1993) Mol. Cell. Biol. 13, in mouse embryonic stem cells has 4276-4283 made it possible to construct mouse 19 Shivji, M. K. K., Kenny, M. K. and Wood, R. D. (1992) Cell 69, 367-374 models for XP, CS, TTD, and ERCC defects. These are now being studied [n 20 Nichols, A. E and Sancar, A. (1992) Nucleic Acids Res. 20, 3559-3564 several laboratories, and a first report 21 Masutani, C. et al. (1993) J. Biol. Chem. 268, on ERCCl-deficient mice has appeared 3°. 9105-9109 Such models will, no doubt, prove to be 22 Huang, J. C., Svoboda, D. L., Reardon, J. T. and Sancar, A. (1992) Proc. Natl Acad. ScL USA 89, extremely valuable for the study of DNA 3664-3668 repair, carcinogenesis and neurodegen- 23 8chaeffer, L. et al. (1993) Science 260, 58-63 eration in a very intriguing group of 24 Sung, P. et al. (1993) Nature 365, 852-855 25 Hanawalt, P, C. (1991) Murat, Res. 247, genetic disorders. 203-211
26 Selby, C. P. and Sancar, A. (1993) Science 260,
References Journal policy limits the number of references that can be given here to 30, and we apologize to all those who we were unable to cite explicitly. I Cleaver, J, E. and Kraemer, K. H. (1989)in The ' Metabolic Basis of Inherited Disease (6th edn) (Scriver, C. R., Beaudet, A. L., Sly, W. S. and Vatle, D., eds), pp. 2949-2971, McGraw-Hill
53-58 27 Robblns, J. H. (1988) J. Am. Med. Asscc 260, 384-388 28 Satoh, M. S., Jones, C. J., Wood, R. D. and Lindahl, T. (1993) Proc. Natl Acad. Sci. USA 90, 6335-6339 29 Mounkes, L. C. et aL (1992) Cell 71, 925-937 30 McWhir, J. et al. (1993) Nature Genet. 5, 217-224
TIBS reference lists Authors of TIBS articles are asked to limit the number of references cited to provide non-specialist readers with a concise list for further reading. It is hoped that the citation of other, more extensive review articles rather than a comprehensive list of original articles enables interested readers to delve more immediately into the topic.