- Email: [email protected]
Repair and Transcription: Collision or collusion?
P.C. HANAWALT, B.A. DONAHUE AND K.S. SWEDER
REPAIR AND TRANSCRIPTION
Collision or collusion? While some proteins have distinct responsibilities in both transcription and DNA repair, additional proteins are needed to couple these essential DNA transactions in expressed genes. Unrepaired damage in genomic DNA can interfere with the essential transactions of replication and transcription. The formidable task faced by cellular DNA repair mechanisms is to distinguish potentially deleterious lesions from transient ligands and unusual structural features of some normal nucleotide sequences. Repair enzymes must avoid mistaking a normal DNA-protein complex for a lesion. Furthermore, some types of lesion block transcription, and a blocked RNA polymerase might encumber access of the repair machinery to the damage. Although such inaccessibility has been documented, the arrested RNA polymerase, with the aid of other proteins, can actually target repair enzymes to the offending lesion, thereby facilitating the resumption of transcription. Recently there has been a remarkable convergence of the fields of nucleotide-excision repair and transcription. This association was catalyzed by the discovery that expressed genes are preferentially repaired [1], followed by the revelation that this so-called transcriptioncoupled repair is selective for the transcribed DNA strand within an expressed gene [2]. Transcriptioncoupled repair was originally reported in mammalian cells [3], but has also been documented in bacteria [4] and in yeast [5-71, so it is probably ubiquitous. In Escherichia coli, a transcription-repair coupling factor has been identified and characterized as the product of the mfd gene [8]. In human cells, the product of the ERCC6 gene - ERCC standing for excision repair cross complementing - corrects the DNA-repair deficiency associated with Cockayne's syndrome complementation group B (CSB), and this protein has been implicated in transcription-coupled repair [9-12]. Cockayne's syndrome is a hereditary disease characterized by sunlight sensitivity, neurological dysfunction and severe developmental abnormalities, but not a predisposition to cancer. A rare form of Cockayne's syndrome (CS) results from mutation in the uncloned CSA gene, which also affects transcription-coupled repair. Several additional genes are also implicated in Cockayne's syndrome, as described below. This convergence of nucleotide-excision repair and transcription is bolstered by the growing number of reports that essential protein components of eukaryotic transcription initiation factors are needed in protein complexes that are required for nucleotide-excision repair. Most of these proteins are products of genes previously shown to be defective in another autosomal recessive hereditary disease, xeroderma pigmentosum 518
(XP), in which sufferers are sunlight-sensitive but, unlike Cockayne's syndrome patients, they readily develop cancer in sun-exposed skin. The product of the ERCC3 gene corrects the DNA-repair deficiency in cells from patients with xeroderma pigmentosum complementation group B (XPB), and has also been shown to be a component of the basal transcription initiation factor TFIIH (also called BTF2) [13,14]. Not only are XPB patients deficient in nucleotide-excision repair, but this defect also results in the symptoms of Cockayne's syndrome in the same individuals. The ERCC3 gene product may participate independently in both nucleotide-excision repair (including transcriptioncoupled repair) and transcription. We shall consider the various activities and substrates required for the process of nucleotide-excision repair, and then explore possible ways by which transcription factors and/or their components might contribute to the process. It is not really surprising to find that some rather basic enzymatic activities are shared between different DNA transactions. DNA polymerases and E, proliferating cell nuclear antigen (PCNA), single-strand binding proteins and DNA ligase are all required both for replication and for the later steps in DNA repair [15]. Helicases are also required in both DNA replication and repair, but additionally they are needed in transcription. The ERCC3 component of TFIIH is a helicase that may provide the localized melting needed for open-complex formation in the promoter region, which is required for the initiation of transcription. The product of the ERCC2 gene, which corrects the nucleotide-excision repair deficiency in cells from xeroderma pigmentosum complementation group D (XPD) patients, could also be involved in this process, as it is also a helicase component of TFIIH [16]. Interestingly, some XPD patients, but not all, exhibit the symptoms of Cockayne's syndrome. Some other XPD patients have the symptoms of a third genetic disease, trichothiodystrophy, which is characterized by brittle hair and nails. The products of genes implicated in trichothiodystrophy have also been localized to TFIIH, and it has been suggested that some clinical features of Cockayne's syndrome and trichothiodystrophy may represent 'transcription syndromes' rather than nucleotide-excision repair defects [12]. We shall now consider the mechanism of nucleotideexcision repair. The process is best understood in E. coli, but the same essential steps occur in eukaryotes. The classic damaged-DNA substrate for nucleotideexcision repair studies is the cyclobutane pyrimidine dimer,
© Current Biology 1994, Vol 4 No 6
DISPATCH in which adjacent pyrimidines have been fused by ultraviolet (UV) irradiation of the DNA. Both E. coli RNA polymerase and mammalian RNA polymerase II are blocked at a cyclobutane pyrimidine dimer in the transcribed DNA strand [17,18]. Damage recognition may proceed by a scanning mechanism, which in E. coli uses the ATP-dependent helicase activity of the UvrB protein in combination with a homodimer of UvrA to track along the DNA and identify structural distortions [17]. The UvrB protein is loaded onto the DNA at the lesion site, and it then enlists the UvrC protein. UvrC, in turn, stimulates a cryptic endonuclease activity of UvrB to produce an incision in the damaged DNA strand, five phosphodiester bonds 3' of the lesion. This is followed by a second incision, eight phosphodiester bonds 5' of the lesion, produced by an endonuclease activity of UvrC itself. Another helicase, UvrD, then operates in concert with DNA polymerase I to peel away the damaged strand segment and fill the gap with a repair patch. The patch is ultimately ligated at its 3' end to the contiguous, pre-existing DNA strand by DNA ligase. In the yeast Saccharomyces cerevisiae, the damage recognition step probably employs the RAD14 protein, which has been shown to have a high affinity for UVdamaged DNA, particularly when the damaged DNA is negatively superhelical [19]. The RAD3 protein is an ATPase and DNA helicase that selectively binds UVdamaged DNA, and may also function in the recognition step [20]. RAD3 is a component of transcription initiation factor b, the yeast counterpart of TFIIH [211. The dual incisions required to initiate repair are probably made by the RAD1/RAD10 endonuclease complex and by the RAD2 endonuclease. Formation of the incision complex in yeast requires association of the RAD2 and RAD1/RAD10 endonucleases with RAD14 and RAD4 proteins, in addition to factor b. There is no DNA repair in the absence of any one of these activities. There is also no detectable incision without both RAD2 and RAD1/RAD10 endonuclease activities, which is reminiscent of the situation in E. coli in which both UvrB and UvrC must be present for the two incisions. In human cells, the XPA protein is a homologue of RAD14 and, not surprisingly, it too binds damaged DNA. The XPE protein also binds some types of lesions, and may interact with XPA. The yeast homologue of ERCC3 is encoded by the RAD25 (SSL2) gene, originally characterized by an apparent role as a suppressor of RNA stem loops in translation [22,231. Although it has been suggested that this protein may help to couple nucleotide-excision repair to transcription, it is clear that it isn't merely a coupling factor, as rad25 mutants are totally deficient in overall nucleotide-excision repair as well as in transcription-coupled repair [24]. RAD3 is the yeast homologue of the human ERCC2 (XPD) protein. Putative homologues of the yeast incision nucleases are the XPF and ERCC1 proteins, in the case of RAD1 and RAD10, and the XPG protein in the case of RAD2 [15]. The XPC protein shows sequence homology to RAD4 and appears to operate only in nucleotide-excision repair events that are not coupled to transcription
[25]. Although the helicase activity of the RAD25 protein might be involved in damage recognition, it could also participate in the excision of the damaged stretch of DNA, as UvrD does in E. coli. Just as the human proteins ERCC2 and ERCC3, and the product of the gene implicated in trichothiodystrophy, are components of TFIIH, their yeast counterparts are associated with factor b. It is particularly interesting that much of the yeast factor b, containing RAD3 and associated with RAD25, may be used as an intact unit in nucleotide-excision repair, rather than as individual proteins that carry out their respective roles [26]. In the course of evolution, the repair machinery may have been assembled from existing protein complexes as required. It is clear, however, that not all proteins associated with TFIIH are involved in both overall genomic repair and transcriptioncoupled repair. Although there is evidence that the XPC protein is associated with TFIIH [271, XPC mutations do not affect transcription-coupled repair, as noted above. In fact, the XPC gene product is implicated in yet another complex (with the human homolog of RAD23) that has been proposed to uncouple the repair machinery from the basal transcription process so that it may then scan the overall genome for lesions [281. One can imagine a situation in which there would be competition between the transactions of transcription and repair for the limited number of TFIIH complexes. In contrast to the effect of XPC deficiency, mutations in the Cockayne's syndrome genes CSA and CSB predominantly affect the transcription-coupled repair response, with no known effect on transcription. Although ERCC6 (CSB) may be the transcriptionrepair coupling factor, it has not been demonstrated to associate with any transcription factors [12]. Given the large number of proteins implicated in nucleotide-excision repair and in transcription-coupled repair, it is a challenge to understand how so many proteins can be coordinated with one another during the repair process. As discussed above, several of the proteins that interact during recognition and incision of damaged DNA evidently use similar interactions to effect the initiation of transcription. In order to couple nucleotide-excision repair to transcription, there are presumably protein-protein contacts made between the coupling factor and the elements needed for initiating nucleotide-excision repair. ERCC3 and/or ERCC2 mutations may disrupt interactions between their products and the ERCC6 protein, and consequently interfere with transcription-coupled repair as well as with nucleotide-excision repair in general. Transcription-coupled repair entails particular problems that are not encountered in overall genomic repair. It is assumed that the RNA polymerase must be blocked to initiate the process, and it was initially suggested that "the arrest of transcription at lesions and release of polymerase from the template could serve as a specific signal to accelerate repair in active domains" [29]. The process clearly requires an actively elongating RNA
519
520
Current Biology 1994, Vol 4 No 6 polymerase II transcription complex, as it is eliminated by the transcription elongation inhibitor, cx-amanitin, or by incubation at the non-permissive temperature of yeast carrying temperature-sensitive RNA polymerase II mutations [5,6,301. Ribosomal DNA, which is transcribed by RNA polymerase I, is not subject to transcriptioncoupled repair [30]. Using an E. coli in vitro system with purified proteins, it was shown that the RNA polymerase and the incomplete transcript are released from the DNA template during transcription-coupled repair [17]. It is necessary to get the polymerase out of the way, both to permit access to the lesion site by the repair enzymes and to allow reannealing of the DNA strands so that repair replication can take place. Eukaryotes generally have larger genes than E. coli, and the release of the incomplete transcript could present a problem. For example, the human dystrophin gene - defective in Duchenne muscular dystrophy - is 2.4 megabases long and should require nearly eight hours to be transcribed. It would seem inefficient to abort nearly complete transcripts as long as this. An alternative to transcript abortion is provided by the phenomenon of transcript shortening. The eukaryotic transcription factor SII (TFIIS) facilitates elongation by RNA polymerase II through transcriptional pause sites formed by particular DNA structures, DNA-bound drugs or DNA-bound proteins [31]. SII appears to induce transcript shortening so as to allow the polymerase to reelongate, thereby increasing the number of chances a polymerase molecule has of bypassing the block [31]. Recent studies have documented SII-dependent transcript shortening at the site of a cyclobutane pyrimidine dimer [18]. SII may induce a conformational change in the polymerase that also requires interaction of the repair complex in order to uncover and repair the cyclobutane pyrimidine dimer on the transcribed strand of the DNA template. Alternatively some other proteins may enable repair at the site of the blocked RNA polymerase II, and SII may be essential for the resumption of transcription after the template has been repaired. It has recently been proposed that when RNA polymerases are arrested at pause sites, the 3' ends of the transcripts dissociate from the catalytic site in the polymerase [31]. Nascent transcript cleavage, mediated by SII in eukaryotes, or by the GreA and GreB proteins in E. coli, restores the association of the 3' end of the transcript with the catalytic site in the polymerase. In like manner, the 3' end of a transcript stalled at a cyclobutane pyrimidine dimer may lose contact with the catalytic site in the polymerase. In order to resume transcription after repair, the transcript would then have to be cleaved in an SII dependent manner. A possible sequence of steps in transcription-coupled repair is outlined in Figure 1. For simplicity of presentation, many details have been omitted and many questions remain unanswered. We do not know whether the blockage of RNA polymerase II at a lesion
Fig. 1. Possible components and steps in human transcriptioncoupled DNA repair. is sufficient to initiate the transcription-coupled repair process, or whether the nucleotide-excision repair protein complex must 'see' the lesion itself. We do not know where the products of other genes implicated in
DISPATCH transcription-coupled repair may operate. Pathways other than nucleotide-excision repair may also be coupled to transcription. Evidence for this comes from the recent report that cells from Cockayne's syndrome patients show a defect in strand-specific repair of DNA damage produced by ionizing radiation that is not subject to nucleotide-excision repair [32]. There are preliminary indications that the process of mismatch repair may also be coupled to transcription [331. Finally, as an indication of greater complexities to come, there are recent reports that, in vitro at least, ERCC3 associates with another transcription factor, TFIIE [34], and with the p53 tumor suppressor gene product [35].
cyclobutane pyrimidine dimer in the DNA template. Proc Natl Acad Sct USA 1994, in press. 19. PRAKASH S, SUNG P, PRAKASH L: DNA repair genes and proteins
of Saccbaromyces cerevlslae. Annu Rev Genet 1993, 27:33-70. 20. SUNG P, WATKINS JF, PRAKASH L, PRAKASH S: Negative superhelic-
ity promotes ATP-dependent binding of yeast RAD3 protein to ultraviolet-damaged DNA. JBiol Cbem 1994, in press. 21.
roles of a multiprotein complex from S. cerevtsiae in transcription and DNA repair. Cell 1993, 75:1369-1387. 22.
23. PARK E, GUZDER SN, KOKEN MHM, JASPERS-DEKKER I, WEEDA G, HOEIJMAKERS JHJ, PRAKASH S, PRAKASH L: RAD23 (SSL2), the
yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability. Proc Natl Acad Sct USA 1992, 89:11416-11420.
BOHR VA, SMITH CA, OKUMOTO DS, HANAWALT PC: DNA repair
in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 1985, 40:359-369. 2.
4.
5.
6.
7. 8.
scription-blocking DNA Damage from the transcribed strand of the mammalian DHFR gene. Cell 1987, 51:241-249. MELLON I, HANAWALT PC: Induction of the Escherlcbta col lactose operon selectively increases repair of its transcribed DNA strand. Nature 1989, 342:95-98. SWEDER KS, HANAWALT PC: Preferential repair of cyclobutane pyrimidine dimers in the transcribed DNA strand in yeast chromosomes and plasmids is dependent upon transcription. ProcNatlAcad Sci USA 1992, 89:10696-10700. LEADON SA, LAWRENCE DA: Strand-selective repair of DNA
SELBY CP, SANCAR A: Molecular mechanism of transcription-
LEGERSKI R, PETERSON C: Expression cloning of a DNA repair
gene involved in xeroderma pigmentosum group C. Nature 1992, 359:70-73. 26. WANG Z, SVEJSTRUP JQ, FEAVER WJ, Wu X, KORNBERG RD,
FRIEDBERG EC: Transcription factor b CTFIIH) is required during nucleotide-excision repair in yeast. Nature 1994, 368:74-76. 27. DRAPKIN R, REARDON J, ANSARI A, HUANG JC, ZAWEL L, AHN KJ, SANCAR A, REINBERG D: TFIIH, a link between RNA polymerase
transcription and DNA excision repair. Nature 1994, 368:769-772. 28. MASUTANI C, SUGASAWA K, YANAGISAWA J, SONOYAMA T, UI M, ENOMOTO T, TAKIO K, TANAKA K, VAN DER SPEK PJ, BOOTSMA D,
ET AL.: Purification and cloning of a nucleotide-excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD 23. EMBOJ 1994, 13:1831-1843. 29.
MELLON I, BOHR VA, SMITH CA, HANAWALT PC: Preferential DNA
HOEIJMAKERS JHJ: ERCC6, a member of a subfamily of putative
repair of an active gene in human cells. Proc Natl Acad Sci USA 1986, 83:8878-8882. 30. HANAWALT PC: Transcription-dependent and transcriptioncoupled DNA repair responses. In Proceedings, Alfred Benzon Symposium No 35 on DNA Repair Mechanisms. Edited by Bohr V, Wasserman K, Kraemer KH. Copenhagen: Munksgaard; 1992:231-242. 31. REINES D: Nascent RNA cleavage by transcription elongation complexes. In Transcription:Mechanisms and Regulation. Edited by Conaway RC, Conaway JW. New York: Raven Press; 1994:263-278.
helicases, is involved in Cockayne's syndrome and preferential
32.
LOMMEL L, HANAWALT PC: The genetic defect in the Chinese
VAN HOFFEN A,
NATARAJAN AT, MAYNE LV, VAN ZEELAND AA,
MULLENDERS LHF: Deficient repair of the transcribed strand of
active genes in Cockayne's syndrome cells. Nucleic Acids Res 1993, 21:5890-5895. 11.
25.
damage in the yeast GAL7 gene requires RNA polymerase II. JBiol Chem 1992, 267:23175-23182. SMERDON MJ, THOMA F: Site-specific DNA repair at the nucleosome level in a yeast minichromosome. Cell 1990, 61:675-684.
hamster ovary cell mutant UV61 permits moderate selective repair of cyclobutane pyrimidine dimers in an expressed gene. Mutat Res: DNA repair 1991, 255:183-191. 10.
of stem loop (SSL2/RAD25) in yeast is essential for overall genomic excision repair and transcription-coupled repair. JBiol Chem 1994, 269:1852-1857.
MELLON I, SPIVAK G, HANAWALT PC: Selective removal of tran-
repair coupling. Science 1993, 260:53-58. 9.
24. SWEDER KS, HANAWALT PC: The COOH-terminus of suppressor
HANAWALT PC, MELLON I: DNA repair: stranded in an active
gene. CurrBlol 1993, 3:67-69. 3.
GULYAS KD, DONAHUE TF: SSL2, a suppressor of a stem-loop
mutation in the H1S4 leader encodes the yeast homolog of human ERCC-3. Cell 1992, 69:1031-1042.
References 1.
FEAVER WJ, SVEJSTRUP JQ, BARDWELL L, BARDWELL AJ, BURATOWSKI S, GULYAS KD, DONAHUE TF, FRIEDBERG EC, KORNBERG, RD: Dual
TROELSTRA C, VAN GOOL A, DE WIT J, VERMEULEN W, BOOTSMA D,
repair of active genes. Cell 1992, 71:939-953. 12. BOOTSMA D, HOEIJMAKERS JHJ: DNA repair: engagement with transcription. Nature 1993, 363:114-115. 13. SCHAEFFER L, ROY R, HUMBERT S, MONCOLLIN V, VERMEULEN W, HOEIJMAKERS JHJ, CHAMBON P, EGLY J-M: DNA repair helicase: a
component of BTF2 (IIH) 1993, 260:58-63.
basic transcription factor. Science
LEADON SA, COOPER PK: Preferential repair of ionizing
radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome. Proc Natl Acad Sci USA 1993, 90:10499-10503. 33. SKANDALIS A, FORD BN, GLICKMAN BW: Strand bias in mutation involving 5-methylcytosine deamination in the human bprt gene. Mutat Res 1994, 314:21-26. 34.
MAXON ME, GOODRICH JA, TJIAN R: Transcription factor
VAN VUUREN AJ, VERMEULEN W, MA L, WEEDA G, APPELDOORN E, JASPERS NGJ, VAN DER EB AJ, BOOTSMA D, HOEIJMAKERS JHJ, HUMBERT S, SCHAEFFER L, EGLY J-M: Correction of xeroderma
IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance. Genes Dev 1994, 8:515-524.
pigmentosum repair defect by basal transcription factor BTF2 (TFIIH). EMBOJ1993, 13:1645-1653. 15. ABOUSSEKHRA A, WOOD RD: DNA repair. Curr Opin Genet Dev 1994, 4:212-220. 16. DRAPKIN R, SANCAR A, REINBERG D: Where transcription meets repair. Cell 1994, 77:9-12. 17. SELBY CP, SANCAR A: Transcription-repair coupling and mutation frequency decline. JBacteriol1993, 175:7509-7514.
35. WANG XW, FORRESTER K, YEH H, FEITELSON MA, GU J-R, HARRIS
14.
18.
DONAHUE
BA, YIN S, TAYLOR JS, REINES D, HANAWALT PC:
Transcript cleavage by RNA polymerase II arrested by a
CC: Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci USA 1994, 91:2230-2234.
P.C. Hanawalt, B.A. Donahue and K.S. Sweder, Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
521
REPAIR AND TRANSCRIPTION
Collision or collusion? While some proteins have distinct responsibilities in both transcription and DNA repair, additional proteins are needed to couple these essential DNA transactions in expressed genes. Unrepaired damage in genomic DNA can interfere with the essential transactions of replication and transcription. The formidable task faced by cellular DNA repair mechanisms is to distinguish potentially deleterious lesions from transient ligands and unusual structural features of some normal nucleotide sequences. Repair enzymes must avoid mistaking a normal DNA-protein complex for a lesion. Furthermore, some types of lesion block transcription, and a blocked RNA polymerase might encumber access of the repair machinery to the damage. Although such inaccessibility has been documented, the arrested RNA polymerase, with the aid of other proteins, can actually target repair enzymes to the offending lesion, thereby facilitating the resumption of transcription. Recently there has been a remarkable convergence of the fields of nucleotide-excision repair and transcription. This association was catalyzed by the discovery that expressed genes are preferentially repaired [1], followed by the revelation that this so-called transcriptioncoupled repair is selective for the transcribed DNA strand within an expressed gene [2]. Transcriptioncoupled repair was originally reported in mammalian cells [3], but has also been documented in bacteria [4] and in yeast [5-71, so it is probably ubiquitous. In Escherichia coli, a transcription-repair coupling factor has been identified and characterized as the product of the mfd gene [8]. In human cells, the product of the ERCC6 gene - ERCC standing for excision repair cross complementing - corrects the DNA-repair deficiency associated with Cockayne's syndrome complementation group B (CSB), and this protein has been implicated in transcription-coupled repair [9-12]. Cockayne's syndrome is a hereditary disease characterized by sunlight sensitivity, neurological dysfunction and severe developmental abnormalities, but not a predisposition to cancer. A rare form of Cockayne's syndrome (CS) results from mutation in the uncloned CSA gene, which also affects transcription-coupled repair. Several additional genes are also implicated in Cockayne's syndrome, as described below. This convergence of nucleotide-excision repair and transcription is bolstered by the growing number of reports that essential protein components of eukaryotic transcription initiation factors are needed in protein complexes that are required for nucleotide-excision repair. Most of these proteins are products of genes previously shown to be defective in another autosomal recessive hereditary disease, xeroderma pigmentosum 518
(XP), in which sufferers are sunlight-sensitive but, unlike Cockayne's syndrome patients, they readily develop cancer in sun-exposed skin. The product of the ERCC3 gene corrects the DNA-repair deficiency in cells from patients with xeroderma pigmentosum complementation group B (XPB), and has also been shown to be a component of the basal transcription initiation factor TFIIH (also called BTF2) [13,14]. Not only are XPB patients deficient in nucleotide-excision repair, but this defect also results in the symptoms of Cockayne's syndrome in the same individuals. The ERCC3 gene product may participate independently in both nucleotide-excision repair (including transcriptioncoupled repair) and transcription. We shall consider the various activities and substrates required for the process of nucleotide-excision repair, and then explore possible ways by which transcription factors and/or their components might contribute to the process. It is not really surprising to find that some rather basic enzymatic activities are shared between different DNA transactions. DNA polymerases and E, proliferating cell nuclear antigen (PCNA), single-strand binding proteins and DNA ligase are all required both for replication and for the later steps in DNA repair [15]. Helicases are also required in both DNA replication and repair, but additionally they are needed in transcription. The ERCC3 component of TFIIH is a helicase that may provide the localized melting needed for open-complex formation in the promoter region, which is required for the initiation of transcription. The product of the ERCC2 gene, which corrects the nucleotide-excision repair deficiency in cells from xeroderma pigmentosum complementation group D (XPD) patients, could also be involved in this process, as it is also a helicase component of TFIIH [16]. Interestingly, some XPD patients, but not all, exhibit the symptoms of Cockayne's syndrome. Some other XPD patients have the symptoms of a third genetic disease, trichothiodystrophy, which is characterized by brittle hair and nails. The products of genes implicated in trichothiodystrophy have also been localized to TFIIH, and it has been suggested that some clinical features of Cockayne's syndrome and trichothiodystrophy may represent 'transcription syndromes' rather than nucleotide-excision repair defects [12]. We shall now consider the mechanism of nucleotideexcision repair. The process is best understood in E. coli, but the same essential steps occur in eukaryotes. The classic damaged-DNA substrate for nucleotideexcision repair studies is the cyclobutane pyrimidine dimer,
© Current Biology 1994, Vol 4 No 6
DISPATCH in which adjacent pyrimidines have been fused by ultraviolet (UV) irradiation of the DNA. Both E. coli RNA polymerase and mammalian RNA polymerase II are blocked at a cyclobutane pyrimidine dimer in the transcribed DNA strand [17,18]. Damage recognition may proceed by a scanning mechanism, which in E. coli uses the ATP-dependent helicase activity of the UvrB protein in combination with a homodimer of UvrA to track along the DNA and identify structural distortions [17]. The UvrB protein is loaded onto the DNA at the lesion site, and it then enlists the UvrC protein. UvrC, in turn, stimulates a cryptic endonuclease activity of UvrB to produce an incision in the damaged DNA strand, five phosphodiester bonds 3' of the lesion. This is followed by a second incision, eight phosphodiester bonds 5' of the lesion, produced by an endonuclease activity of UvrC itself. Another helicase, UvrD, then operates in concert with DNA polymerase I to peel away the damaged strand segment and fill the gap with a repair patch. The patch is ultimately ligated at its 3' end to the contiguous, pre-existing DNA strand by DNA ligase. In the yeast Saccharomyces cerevisiae, the damage recognition step probably employs the RAD14 protein, which has been shown to have a high affinity for UVdamaged DNA, particularly when the damaged DNA is negatively superhelical [19]. The RAD3 protein is an ATPase and DNA helicase that selectively binds UVdamaged DNA, and may also function in the recognition step [20]. RAD3 is a component of transcription initiation factor b, the yeast counterpart of TFIIH [211. The dual incisions required to initiate repair are probably made by the RAD1/RAD10 endonuclease complex and by the RAD2 endonuclease. Formation of the incision complex in yeast requires association of the RAD2 and RAD1/RAD10 endonucleases with RAD14 and RAD4 proteins, in addition to factor b. There is no DNA repair in the absence of any one of these activities. There is also no detectable incision without both RAD2 and RAD1/RAD10 endonuclease activities, which is reminiscent of the situation in E. coli in which both UvrB and UvrC must be present for the two incisions. In human cells, the XPA protein is a homologue of RAD14 and, not surprisingly, it too binds damaged DNA. The XPE protein also binds some types of lesions, and may interact with XPA. The yeast homologue of ERCC3 is encoded by the RAD25 (SSL2) gene, originally characterized by an apparent role as a suppressor of RNA stem loops in translation [22,231. Although it has been suggested that this protein may help to couple nucleotide-excision repair to transcription, it is clear that it isn't merely a coupling factor, as rad25 mutants are totally deficient in overall nucleotide-excision repair as well as in transcription-coupled repair [24]. RAD3 is the yeast homologue of the human ERCC2 (XPD) protein. Putative homologues of the yeast incision nucleases are the XPF and ERCC1 proteins, in the case of RAD1 and RAD10, and the XPG protein in the case of RAD2 [15]. The XPC protein shows sequence homology to RAD4 and appears to operate only in nucleotide-excision repair events that are not coupled to transcription
[25]. Although the helicase activity of the RAD25 protein might be involved in damage recognition, it could also participate in the excision of the damaged stretch of DNA, as UvrD does in E. coli. Just as the human proteins ERCC2 and ERCC3, and the product of the gene implicated in trichothiodystrophy, are components of TFIIH, their yeast counterparts are associated with factor b. It is particularly interesting that much of the yeast factor b, containing RAD3 and associated with RAD25, may be used as an intact unit in nucleotide-excision repair, rather than as individual proteins that carry out their respective roles [26]. In the course of evolution, the repair machinery may have been assembled from existing protein complexes as required. It is clear, however, that not all proteins associated with TFIIH are involved in both overall genomic repair and transcriptioncoupled repair. Although there is evidence that the XPC protein is associated with TFIIH [271, XPC mutations do not affect transcription-coupled repair, as noted above. In fact, the XPC gene product is implicated in yet another complex (with the human homolog of RAD23) that has been proposed to uncouple the repair machinery from the basal transcription process so that it may then scan the overall genome for lesions [281. One can imagine a situation in which there would be competition between the transactions of transcription and repair for the limited number of TFIIH complexes. In contrast to the effect of XPC deficiency, mutations in the Cockayne's syndrome genes CSA and CSB predominantly affect the transcription-coupled repair response, with no known effect on transcription. Although ERCC6 (CSB) may be the transcriptionrepair coupling factor, it has not been demonstrated to associate with any transcription factors [12]. Given the large number of proteins implicated in nucleotide-excision repair and in transcription-coupled repair, it is a challenge to understand how so many proteins can be coordinated with one another during the repair process. As discussed above, several of the proteins that interact during recognition and incision of damaged DNA evidently use similar interactions to effect the initiation of transcription. In order to couple nucleotide-excision repair to transcription, there are presumably protein-protein contacts made between the coupling factor and the elements needed for initiating nucleotide-excision repair. ERCC3 and/or ERCC2 mutations may disrupt interactions between their products and the ERCC6 protein, and consequently interfere with transcription-coupled repair as well as with nucleotide-excision repair in general. Transcription-coupled repair entails particular problems that are not encountered in overall genomic repair. It is assumed that the RNA polymerase must be blocked to initiate the process, and it was initially suggested that "the arrest of transcription at lesions and release of polymerase from the template could serve as a specific signal to accelerate repair in active domains" [29]. The process clearly requires an actively elongating RNA
519
520
Current Biology 1994, Vol 4 No 6 polymerase II transcription complex, as it is eliminated by the transcription elongation inhibitor, cx-amanitin, or by incubation at the non-permissive temperature of yeast carrying temperature-sensitive RNA polymerase II mutations [5,6,301. Ribosomal DNA, which is transcribed by RNA polymerase I, is not subject to transcriptioncoupled repair [30]. Using an E. coli in vitro system with purified proteins, it was shown that the RNA polymerase and the incomplete transcript are released from the DNA template during transcription-coupled repair [17]. It is necessary to get the polymerase out of the way, both to permit access to the lesion site by the repair enzymes and to allow reannealing of the DNA strands so that repair replication can take place. Eukaryotes generally have larger genes than E. coli, and the release of the incomplete transcript could present a problem. For example, the human dystrophin gene - defective in Duchenne muscular dystrophy - is 2.4 megabases long and should require nearly eight hours to be transcribed. It would seem inefficient to abort nearly complete transcripts as long as this. An alternative to transcript abortion is provided by the phenomenon of transcript shortening. The eukaryotic transcription factor SII (TFIIS) facilitates elongation by RNA polymerase II through transcriptional pause sites formed by particular DNA structures, DNA-bound drugs or DNA-bound proteins [31]. SII appears to induce transcript shortening so as to allow the polymerase to reelongate, thereby increasing the number of chances a polymerase molecule has of bypassing the block [31]. Recent studies have documented SII-dependent transcript shortening at the site of a cyclobutane pyrimidine dimer [18]. SII may induce a conformational change in the polymerase that also requires interaction of the repair complex in order to uncover and repair the cyclobutane pyrimidine dimer on the transcribed strand of the DNA template. Alternatively some other proteins may enable repair at the site of the blocked RNA polymerase II, and SII may be essential for the resumption of transcription after the template has been repaired. It has recently been proposed that when RNA polymerases are arrested at pause sites, the 3' ends of the transcripts dissociate from the catalytic site in the polymerase [31]. Nascent transcript cleavage, mediated by SII in eukaryotes, or by the GreA and GreB proteins in E. coli, restores the association of the 3' end of the transcript with the catalytic site in the polymerase. In like manner, the 3' end of a transcript stalled at a cyclobutane pyrimidine dimer may lose contact with the catalytic site in the polymerase. In order to resume transcription after repair, the transcript would then have to be cleaved in an SII dependent manner. A possible sequence of steps in transcription-coupled repair is outlined in Figure 1. For simplicity of presentation, many details have been omitted and many questions remain unanswered. We do not know whether the blockage of RNA polymerase II at a lesion
Fig. 1. Possible components and steps in human transcriptioncoupled DNA repair. is sufficient to initiate the transcription-coupled repair process, or whether the nucleotide-excision repair protein complex must 'see' the lesion itself. We do not know where the products of other genes implicated in
DISPATCH transcription-coupled repair may operate. Pathways other than nucleotide-excision repair may also be coupled to transcription. Evidence for this comes from the recent report that cells from Cockayne's syndrome patients show a defect in strand-specific repair of DNA damage produced by ionizing radiation that is not subject to nucleotide-excision repair [32]. There are preliminary indications that the process of mismatch repair may also be coupled to transcription [331. Finally, as an indication of greater complexities to come, there are recent reports that, in vitro at least, ERCC3 associates with another transcription factor, TFIIE [34], and with the p53 tumor suppressor gene product [35].
cyclobutane pyrimidine dimer in the DNA template. Proc Natl Acad Sct USA 1994, in press. 19. PRAKASH S, SUNG P, PRAKASH L: DNA repair genes and proteins
of Saccbaromyces cerevlslae. Annu Rev Genet 1993, 27:33-70. 20. SUNG P, WATKINS JF, PRAKASH L, PRAKASH S: Negative superhelic-
ity promotes ATP-dependent binding of yeast RAD3 protein to ultraviolet-damaged DNA. JBiol Cbem 1994, in press. 21.
roles of a multiprotein complex from S. cerevtsiae in transcription and DNA repair. Cell 1993, 75:1369-1387. 22.
23. PARK E, GUZDER SN, KOKEN MHM, JASPERS-DEKKER I, WEEDA G, HOEIJMAKERS JHJ, PRAKASH S, PRAKASH L: RAD23 (SSL2), the
yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability. Proc Natl Acad Sct USA 1992, 89:11416-11420.
BOHR VA, SMITH CA, OKUMOTO DS, HANAWALT PC: DNA repair
in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 1985, 40:359-369. 2.
4.
5.
6.
7. 8.
scription-blocking DNA Damage from the transcribed strand of the mammalian DHFR gene. Cell 1987, 51:241-249. MELLON I, HANAWALT PC: Induction of the Escherlcbta col lactose operon selectively increases repair of its transcribed DNA strand. Nature 1989, 342:95-98. SWEDER KS, HANAWALT PC: Preferential repair of cyclobutane pyrimidine dimers in the transcribed DNA strand in yeast chromosomes and plasmids is dependent upon transcription. ProcNatlAcad Sci USA 1992, 89:10696-10700. LEADON SA, LAWRENCE DA: Strand-selective repair of DNA
SELBY CP, SANCAR A: Molecular mechanism of transcription-
LEGERSKI R, PETERSON C: Expression cloning of a DNA repair
gene involved in xeroderma pigmentosum group C. Nature 1992, 359:70-73. 26. WANG Z, SVEJSTRUP JQ, FEAVER WJ, Wu X, KORNBERG RD,
FRIEDBERG EC: Transcription factor b CTFIIH) is required during nucleotide-excision repair in yeast. Nature 1994, 368:74-76. 27. DRAPKIN R, REARDON J, ANSARI A, HUANG JC, ZAWEL L, AHN KJ, SANCAR A, REINBERG D: TFIIH, a link between RNA polymerase
transcription and DNA excision repair. Nature 1994, 368:769-772. 28. MASUTANI C, SUGASAWA K, YANAGISAWA J, SONOYAMA T, UI M, ENOMOTO T, TAKIO K, TANAKA K, VAN DER SPEK PJ, BOOTSMA D,
ET AL.: Purification and cloning of a nucleotide-excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD 23. EMBOJ 1994, 13:1831-1843. 29.
MELLON I, BOHR VA, SMITH CA, HANAWALT PC: Preferential DNA
HOEIJMAKERS JHJ: ERCC6, a member of a subfamily of putative
repair of an active gene in human cells. Proc Natl Acad Sci USA 1986, 83:8878-8882. 30. HANAWALT PC: Transcription-dependent and transcriptioncoupled DNA repair responses. In Proceedings, Alfred Benzon Symposium No 35 on DNA Repair Mechanisms. Edited by Bohr V, Wasserman K, Kraemer KH. Copenhagen: Munksgaard; 1992:231-242. 31. REINES D: Nascent RNA cleavage by transcription elongation complexes. In Transcription:Mechanisms and Regulation. Edited by Conaway RC, Conaway JW. New York: Raven Press; 1994:263-278.
helicases, is involved in Cockayne's syndrome and preferential
32.
LOMMEL L, HANAWALT PC: The genetic defect in the Chinese
VAN HOFFEN A,
NATARAJAN AT, MAYNE LV, VAN ZEELAND AA,
MULLENDERS LHF: Deficient repair of the transcribed strand of
active genes in Cockayne's syndrome cells. Nucleic Acids Res 1993, 21:5890-5895. 11.
25.
damage in the yeast GAL7 gene requires RNA polymerase II. JBiol Chem 1992, 267:23175-23182. SMERDON MJ, THOMA F: Site-specific DNA repair at the nucleosome level in a yeast minichromosome. Cell 1990, 61:675-684.
hamster ovary cell mutant UV61 permits moderate selective repair of cyclobutane pyrimidine dimers in an expressed gene. Mutat Res: DNA repair 1991, 255:183-191. 10.
of stem loop (SSL2/RAD25) in yeast is essential for overall genomic excision repair and transcription-coupled repair. JBiol Chem 1994, 269:1852-1857.
MELLON I, SPIVAK G, HANAWALT PC: Selective removal of tran-
repair coupling. Science 1993, 260:53-58. 9.
24. SWEDER KS, HANAWALT PC: The COOH-terminus of suppressor
HANAWALT PC, MELLON I: DNA repair: stranded in an active
gene. CurrBlol 1993, 3:67-69. 3.
GULYAS KD, DONAHUE TF: SSL2, a suppressor of a stem-loop
mutation in the H1S4 leader encodes the yeast homolog of human ERCC-3. Cell 1992, 69:1031-1042.
References 1.
FEAVER WJ, SVEJSTRUP JQ, BARDWELL L, BARDWELL AJ, BURATOWSKI S, GULYAS KD, DONAHUE TF, FRIEDBERG EC, KORNBERG, RD: Dual
TROELSTRA C, VAN GOOL A, DE WIT J, VERMEULEN W, BOOTSMA D,
repair of active genes. Cell 1992, 71:939-953. 12. BOOTSMA D, HOEIJMAKERS JHJ: DNA repair: engagement with transcription. Nature 1993, 363:114-115. 13. SCHAEFFER L, ROY R, HUMBERT S, MONCOLLIN V, VERMEULEN W, HOEIJMAKERS JHJ, CHAMBON P, EGLY J-M: DNA repair helicase: a
component of BTF2 (IIH) 1993, 260:58-63.
basic transcription factor. Science
LEADON SA, COOPER PK: Preferential repair of ionizing
radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome. Proc Natl Acad Sci USA 1993, 90:10499-10503. 33. SKANDALIS A, FORD BN, GLICKMAN BW: Strand bias in mutation involving 5-methylcytosine deamination in the human bprt gene. Mutat Res 1994, 314:21-26. 34.
MAXON ME, GOODRICH JA, TJIAN R: Transcription factor
VAN VUUREN AJ, VERMEULEN W, MA L, WEEDA G, APPELDOORN E, JASPERS NGJ, VAN DER EB AJ, BOOTSMA D, HOEIJMAKERS JHJ, HUMBERT S, SCHAEFFER L, EGLY J-M: Correction of xeroderma
IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance. Genes Dev 1994, 8:515-524.
pigmentosum repair defect by basal transcription factor BTF2 (TFIIH). EMBOJ1993, 13:1645-1653. 15. ABOUSSEKHRA A, WOOD RD: DNA repair. Curr Opin Genet Dev 1994, 4:212-220. 16. DRAPKIN R, SANCAR A, REINBERG D: Where transcription meets repair. Cell 1994, 77:9-12. 17. SELBY CP, SANCAR A: Transcription-repair coupling and mutation frequency decline. JBacteriol1993, 175:7509-7514.
35. WANG XW, FORRESTER K, YEH H, FEITELSON MA, GU J-R, HARRIS
14.
18.
DONAHUE
BA, YIN S, TAYLOR JS, REINES D, HANAWALT PC:
Transcript cleavage by RNA polymerase II arrested by a
CC: Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci USA 1994, 91:2230-2234.
P.C. Hanawalt, B.A. Donahue and K.S. Sweder, Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
521