Nucleotide excision repair in yeast

Mutation Research 451 Ž2000. 13–24 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres

Review

Nucleotide excision repair in yeast Satya Prakash ) , Louise Prakash Sealy Center for Molecular Science, UniÕersity of Texas Medical Branch, 6.103 Medical Research Building, GalÕeston, TX 77555-1061, USA Received 21 September 1999; received in revised form 15 November 1999; accepted 24 November 1999

Abstract In nucleotide excision repair ŽNER. in eukaryotes, DNA is incised on both sides of the lesion, resulting in the removal of a fragment ; 25–30 nucleotides long. This is followed by repair synthesis and ligation. The proteins encoded by the various yeast NER genes have been purified, and the incision reaction reconstituted in vitro. This reaction requires the damage binding factors Rad14, RPA, and the Rad4–Rad23 complex, the transcription factor TFIIH which contains the two DNA helicases Rad3 and Rad25, essential for creating a bubble structure, and the two endonucleases, the Rad1–Rad10 complex and Rad2, which incise the damaged DNA strand on the 5X- and 3X-side of the lesion, respectively. Addition of the Rad7–Rad16 complex to this reconstituted system stimulates the incision reaction many fold. The various NER proteins exist in vivo as part of multiprotein subassemblies which have been named NEFs Žnucleotide excision repair factors.. Rad14 and Rad1–Rad10 form one subassembly called NEF1, the Rad4–Rad23 complex is named NEF2, Rad2 and TFIIH constitute NEF3, and the Rad7–Rad16 complex is called NEF4. Although much has been learned from yeast about the function of NER genes and proteins in eukaryotes, the underlying mechanisms by which damage is recognized, NEFs are assembled at the damage site, and the DNA is unwound and incised, remain to be elucidated. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Nucleotide excision repair; Yeast; Transcription factor

Nucleotide excision repair ŽNER. is characterized by the incision of the damaged DNA strand on both sides of the lesion, resulting in the removal of damage in an oligonucleotide fragment. In both prokaryotes and eukaryotes, NER represents the most important repair system that is uniquely adapted to remove a large variety of DNA lesions, particularly those that distort the DNA helix; thus, it functions in the removal of damage induced by ultraviolet ŽUV. light, DNA intrastrand and interstrand crosslinks, and a

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Corresponding author.

variety of other DNA lesions. In humans, a defect in NER results in xeroderma pigmentosum ŽXP.; individuals with XP are extremely sensitive to UV light and the incidence of sunlight induced skin cancers is ; 2000 fold higher in XP individuals than in the general population. Genetic and biochemical studies in the yeast Saccharomyces cereÕisiae have made major contributions in elucidating the mechanism of dual incision of damaged DNA in eukaryotes, and they have also yielded important insights into the diverse functions of NER proteins. In this review, we summarize the roles of yeast NER genes and proteins in the incision step and in transcription and recombination, and we

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discuss the contribution that these studies have made to the understanding of NER in humans. 1. Genes required for the incision step of NER in yeast Early genetic studies indicated that yeast genes involved in NER could be grouped into two classes. Class 1 consists of the RAD1, RAD2, RAD3, RAD4, RAD10, RAD14, and RAD25 genes, and class 2 contains the RAD7, RAD16, RAD23, and MMS19 genes. Mutations in class 1 genes confer a very high degree of sensitivity to UV light and to other DNA damaging agents, such as crosslinking agents, and they cause a high level of defect in the incision of UV damaged DNA and crosslinked DNA w1–5x. Mutations in class 2 genes cause a moderate degree of sensitivity to these DNA damaging agents, and they have a less adverse effect on incision than do mutations in class 1 genes w6–8x. These genetic observations suggested that class 1 genes were essential for all the steps leading to the incision of damaged DNA, and that class 2 genes affected the proficiency of this reaction. The requirement of such a large number of genes for incision had also raised the possibility that this process would involve a large multi-protein complex w9x. Homologues of all the above mentioned yeast genes, except for RAD7, RAD16, and MMS19, have been identified in humans, and mutations in these human genes affect NER in a similar fashion as they do in yeast, with the exception of XPC, the human counterpart of yeast RAD4. Deletion of RAD4 causes the same high level of UV sensitivity as do mutations in the other class 1 genes w10x, and rad4 mutants are completely defective in incision w1–3x. By contrast, XPC is required for the repair of nontranscribed regions of the genome but not for the repair of the transcribed DNA strand w11x. In yeast, the repair of the nontranscribed DNA strand and of transcriptionally inactive regions has a specific dependence on the RAD7 and RAD16 genes, as repair of these genomic regions is severely impaired in the rad7D and rad16 D mutants, whereas the repair of the transcribed strand is not affected w12,13x. Additionally, extracts from rad7 and rad16 mutant cells are highly deficient in incision and repair synthesis w14–16x.

NER proceeds at a faster rate on the transcribed strand than the non-transcribed strand, and this phenomenon of transcription-coupled repair ŽTCR. has been documented in Escherichia coli, yeast, and humans w17–19x. In humans, individuals with CockayneX s syndrome ŽCS. suffer from growth retardation and mental retardation w20x, and cells from CS patients are defective in TCR w21,22x. Two genes, CSA and CSB function in TCR in humans w23,24x. RAD26 and RAD28 represent the yeast counterparts of CSB and CSA, respectively, and mutations in RAD26 w25x, but not in RAD28 w26x, affect TCR in yeast. 2. Structure and function of yeast NER proteins Incision of UV damaged DNA is a multi-step process involving damage recognition, followed by DNA unwinding, which provides discrimination of the damaged strand from the non-damaged DNA strand, and dual incision, which in eukaryotes, results in the excision of damage in the form of a DNA fragment approximately 25–30 nucleotides long w27x. Table 1 summarizes the biochemical properties of the yeast NER proteins. The damage binding protein factors in yeast include Rad14, RPA, the Rad4– Rad23 complex, and the Rad7–Rad16 complex. Both Rad14 and its human homologue XPA contain a 4-cysteine ŽC 4 . zinc finger motif w4,28x, and they both bind one zinc atom w29,30x. Rad14, however, binds to UV damaged DNA with a higher affinity and specificity than XPA w29,31x. RPA, a trimeric protein, binds single stranded DNA and is essential for DNA replication w32x. RPA is also required for the incision step of NER w33,34x, and human RPA has been shown to have affinity for damaged DNA w35–37x. RPA interacts with XPA and the XPA–RPA complex shows higher affinity for damaged DNA than either protein alone w36,38,39x. The Rad4– Rad23 complex of yeast w40,41x and the XPC– HR23B complex of human w42,43x also have affinity for UV-damaged DNA. Of the various damage-binding factors, the properties of the yeast Rad7–Rad16 complex would suggest that it plays a crucial role in the initial recognition of DNA lesions in non-transcribed regions. The Rad7 and Rad16 proteins form a complex of 1:1 stoichiometry, and this complex binds UV damaged DNA in an ATP-dependent man-

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Table 1 Yeast and human nucleotide excision repair proteins

ner w44x. The ATP dependence of the Rad7–Rad16 complex for damage binding distinguishes this factor from Rad14, RPA, and the Rad4–Rad23 complex, which show no such dependence on ATP for damage binding. Rad7 contains no sequence motifs indicative of a DNA binding activity, whereas Rad16 contains two potential zinc binding motifs — a C 4 motif and a C 3 HC 4 ring finger motif, either or both of which could function in DNA binding. Rad16 is a member of the SWIrSNF family and it contains the seven conserved sequence motifs indicative of ATPase activity w8x. Accordingly, the Rad7–Rad16 complex displays a DNA-dependent ATPase activity, and, interestingly, this activity is inhibited by the presence of UV damage in DNA w45x. This observation has suggested a model in which the Rad7–Rad16 complex would track along DNA utilizing the energy from ATP hydrolysis, and inhibition of ATPase activity at the site of the DNA lesion would result in stable binding of this complex to the damage site. According to this scenario, the Rad7–Rad16 complex would be the first to arrive at the damage site in non-transcribed regions of the genome, and the damage-bound Rad7–Rad16 complex would serve as the nucleation site for the recruitment of the other NER factors.

Rad3 and Rad25, and their respective human counterparts XPD and XPB, are DNA helicases which translocate on single-stranded DNA in the 5X 3X and 3X 5X direction, respectively w46–50x. In fact, Rad3 was the first eukaryotic NER protein for which a biochemical activity was identified w46,51x. Rad3 and Rad25 constitute two of the six subunits of core transcription factor TFIIH w52x. TFIIH associates with TFIIK, which consists of the KIN28 kinase and a cyclin subunit, and TFIIK, in association with TFIIH plays an essential role in transcription w53x. Both in yeast and humans, however, TFIIK is dispensable for the incision of damaged DNA, and only the six-subunit core TFIIH is sufficient for the incision reaction w54,55x. Incision of damaged DNA in both yeast and human utilizes the function of two endonucleases — Rad1–Rad10 and Rad2 in yeast, and XPF-ERCC1 and XPG in humans. Rad1 and Rad10 form a tight complex w56,57x, and genetic and biochemical studies with a rad1 mutant allele, whose encoded protein fails to interact with Rad10, provided strong evidence that complex formation was essential for the biological function of these proteins w56x. The Rad1– Rad10 complex exhibits single-strand DNA endonuclease activity w58,59x that acts in a structure specific

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manner and cleaves 3X-ended single stranded DNA at its junction with the duplex DNA w60x. A similar structure-specific activity was subsequently identified in the XPF–ERCC1 complex w61x. Yeast Rad2 also shows a single-strand DNA endonuclease activity w62x. Subsequent studies indicated that Rad2 shares strong sequence similarity with mammalian FEN-1 and with the 5X 3X exonuclease domain of E. coli DNA polymerase I w63,64x. Both FEN-1 and the 5X 3X nuclease of PolI function in the removal of RNA primers formed during lagging strand DNA synthesis w65x, and they both exhibit a structure specific endonuclease activity that cleaves the 5Xended single stranded DNA at its junction with the duplex DNA w63,66x. A similar structure-specific endonuclease activity is also found in Rad2 w67x and its human counterpart XPG w68x. The structurespecific activity of Rad1–Rad10 ŽXPF–ERCC1. and Rad2 ŽXPG. nucleases correctly predicted that they would incise DNA on the 5X and 3X side of the damage, respectively w60,63,68x.

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3. Role of the Rad3 and Rad25 helicases in transcription and NER The initial observations that deletion of RAD3 resulted in lethality w69,70x and that missense mutations in this gene conferred a high degree of incision defect w1,2x, suggested that in addition to its role in the incision step of NER, RAD3 plays an indispensable role in some essential cellular function. Later, RAD25 was also found to be essential for viability w5,71x. The dilemma of the involvement of these two genes in an essential cellular function as well as in NER was solved when their encoded proteins were shown to be components of TFIIH w49,50,52,72x. Additionally, studies with the temperature sensitive Ž ts . mutant alleles of RAD3 and RAD25 revealed the requirement of these two genes for the transcription of a vast majority of mRNAs w48,73,74x. Genetic and biochemical analyses of mutations in the nucleotide-binding motif of Rad3 and Rad25 have disclosed important differences in the role of these DNA helicases in transcription versus NER. A mutation of lysine 48 to arginine in the Walker type A nucleotide binding motif, GKT, of Rad3 abolishes

the ATPase and DNA helicase activities without affecting the ability to bind DNA or ATP. Since the rad3 Arg48 mutation has no effect on viability, the Rad3 helicase function is not necessary for transcription w75x. The rad3 Arg48 mutation, however, confers the same high level of UV sensitivity as that imparted by the completely incision defective rad3 mutant alleles, and UV damage is not removed in the rad3 Arg48 mutant w75x. Unlike the rad3 Arg48 mutation, a mutation of the lysine 392 residue to arginine in the GKT box of Rad25 confers lethality w5x. This indicated that the Rad25 helicase function was essential for transcription, and consistent with this, the Rad25 Arg392 protein does not correct the transcriptional defect of the rad25 ts extract w48x. The rad25 Arg392 mutant allele also does not complement the UV sensitivity of the rad257 9 9 a m mutation that deletes 45 carboxyl terminal residues of the protein w73x; this indicated the indispensability of Rad25 helicase function in NER. Studies with the purified reconstituted system have provided direct evidence for the requirement of Rad3 and Rad25 DNA helicase activities in the incision phase of NER w54x. These studies were made possible because a subassembly of TFIIH that contains the SSL1, TFB1, TFB2, and TFB3 subunits but lacks the Rad3 and Rad25 subunits, and which is referred to as TFIIHincomplete ŽTFIIHi., could be purified w54x. When the six-subunit TFIIH is combined with the various NER factors ŽRad1–Rad10, Rad2, Rad4–Rad23, Rad14, and RPA., UV damaged DNA is incised, but no incision occurs when either TFIIHi or the mixture of Rad3 and Rad25 is combined with the same set of NER factors. When Rad3, Rad25, and TFIIHi are added together with the other NER factors, UV damaged DNA is incised in an ATP-dependent manner and with the same efficiency as when the sixsubunit TFIIH is used. However, little or no incision of UV damaged DNA occurs if wild type Rad25 is replaced with the Rad25 Arg392 mutant protein in the reaction mix, or if the wild type Rad3 protein is replaced with the Rad3 Arg48 mutant protein w54x. Thus, in summary, even though both the Rad3 and Rad25 proteins are essential for transcription, the DNA helicase activity of only the Rad25 protein is required for this function, whereas the DNA helicase activities of both Rad3 and Rad25 are needed for the incision of UV damaged DNA.

S. Prakash, L. Prakashr Mutation Research 451 (2000) 13–24

The human XPD gene complements the inviability of the rad3D mutation w47x. This observation offered the opportunity to determine if, like Rad3, the DNA helicase activity of XPD was also dispensable for transcription. The sequences in the Walker type A nucleotide binding motif are highly conserved in S. cereÕisiae Rad3, Schizosaccharomyces pombe rhp3 q , and human XPD proteins, and a mutation of the equivalent lysine 48 to arginine in rhp3 q has no adverse effect on viability w76x. Since the XPD Arg48 mutation restores viability to the rad3D strain w77x, the XPD DNA helicase activity also is dispensable for RNA PolII transcription.

4. The Rad1–Rad10 pathway of genetic recombination In addition to their requirement in NER, the RAD1 and RAD10 genes function in genetic recombination. Deletion of either RAD1 or RAD10 lowered mitotic intrachromosomal recombination of a his3 duplication in which one copy of the gene is deleted at the 3X end and the other is deleted at the 5X end Ž his3D 3X his3D 5X . w78,79x. These two his3 genes share homology of ; 400 base pairs and are separated by the LEU2 and pBR322 sequences. In this system, a functional HIS3 gene is formed by the loss of intervening sequences between the two mutant his3 alleles. The rate of HIS3 intrachromosomal recombination was reduced by ; six-fold in the rad1D , rad10D, and rad1D rad10D strains. Deletion of RAD52 also lowered HIS3 recombination by ; six-fold, and a synergistic decline in recombination occurred in the rad1D rad52 D and rad10D rad52 D strains. These observations indicated that RAD1 and RAD10 function in a mitotic recombination pathway that is distinct from the RAD52 pathway. The incidence of homologous integration of DNA molecules into genomic sequences is also reduced in the rad1D and rad10D strains. The effects of RAD1 on mitotic recombination of direct and inverted repeats have been analyzed in many other studies and mutations in RAD1 reduce recombination stimulated by RNA polymerase I- or RNA polymerase II-dependent transcription w80–86x. Subsequent studies revealed the specific involvement of RAD1 and RAD10 in the single strand annealing

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ŽSSA. pathway of recombination w87,88x. In this role, the Rad1–Rad10 nuclease removes the two non-homologous 3X-ended single stranded DNAs that are formed as an intermediate. The MSH2, MSH3, MSH6, MLH1, and PMS1 genes function in DNA mismatch repair ŽMMR., and genetic and biochemical studies have indicated the involvement of the Msh2–Msh3 and Msh2–Msh6 complexes in the recognition of DNA mismatches. The first evidence of a functional connection between NER and MMR came from genetic studies in yeast in which MSH2 and MSH3 were shown to be involved in the RAD1–RAD10 pathway of recombination w89x. Subsequently, MSH2 and MSH3 were also found to be involved in SSA w90x. In addition, genetic studies in yeast have provided evidence for the involvement of RAD1 and MSH2 in the repair of a 26-base loop from a heteroduplex during meiotic recombination w91x. ERCC1 mutant mice are runted at birth, their life span is greatly reduced, and they also suffer from liver failure and kidney malfunction w92,93x. ERCC1 mutant cells exhibit abnormal replicative senescence, which could be the underlying cause of runted growth. Since mutations in the other NER genes, such as XPA, do not affect viability, growth, or life span, these additional ERCC1 phenotypes must derive from a deficiency in a process other than NER. The involvement of Rad1–Rad10 in recombination might suggest that a deficiency in this process is responsible for the above noted phenotypes of ERCC1 mutant mice. However, these observations also raise the possibility that Rad1–Rad10 and XPF–ERCC1 function in another, as yet unknown, biological process, and that defects in that function underlie the various ERCC1 abnormalities.

5. Nucleotide excision repair factors (NEFs) In yeast, a combination of Rad14, RPA, the Rad4–Rad23 complex, TFIIH, Rad2, and the Rad1– Rad10 complex mediates dual incision of UV damaged DNA w33x. All these protein factors are indispensable for the incision reaction, and no nicking of damaged DNA occurs if either the Rad1–Rad10 endonuclease or the Rad2 endonuclease is absent from the reaction mix. Thus, in addition to providing

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the endonucleolytic activities for dual incision, Rad1–Rad10 and Rad2 are involved in the proper assembly of the NER machinery at the damage site. The human equivalents of the yeast NER proteins also mediate the dual incision of UV damaged DNA w34,94x; but, the human incision ensemble can generate normal levels of 3X-incisions in the absence of XPF–ERCC1 w55x. It has been suggested that all the yeast NER proteins exist together in a protein assembly forming a repairosome w95x. However, there is no good biochemical evidence for such an entity, since most of the repair factors fail to remain associated even under relatively mild purification conditions w96x. However, the various NER proteins in yeast are part of tightly associated multisubunit complexes that can be purified intact. One such protein assembly consists of the damage recognition protein Rad14 and the Rad1–Rad10 endonuclease w96x. These three proteins co-purify through Bio-Rex70, DEAE Sephacel, hydroxyapatite, and Ni-NTA agarose, and they coelute in the Sephacryl S-300 sizing column. Rad2 and TFIIH form another protein assembly w97x. They co-purify through six consecutive chromatographic steps, and in the final Mono Q pool, Rad2 is present with TFIIH in stoichiometric amounts. These and other NER protein assemblies have been named nucleotide excision repair factors ŽNEFs.. Thus, Rad14, Rad1, and Rad10 constitute NEF1, the Rad4–Rad23 complex is NEF2, Rad2 and TFIIH form NEF3, and the Rad7–Rad16 complex constitutes NEF4 w44x ŽTable 2.. The equivalent protein assemblies are likely to exist in humans as well. The XPF–ERCC1 complex binds strongly to the XPA-affinity column Table 2 Yeast nucleotide excision repair factors ŽNEFs. NEFs

Components

Function or Activity

NEF1

Rad1, Rad10, Rad14

NEF2

Rad4, Rad23

NEF3

NEF4

Rad2, Rad3, Rad25, SSL1, TFB1, TFB2, TFB3 Rad7, Rad16

DNA endonuclease, DNA damage recognition DNA damage binding, Tethering of NEF1 with NEF3 DNA endonuclease, DNA helicase

RPA

p69, p36, p13

DNA dependent ATPase, DNA damage recognition DNA damage recognition

w61x, XPG and TFIIH co-purify through several column steps w34x, and XPC forms a tight complex with HR23B w98x.

6. Order of assembly of NER factors at the damage site Damage recognition is expected to be the ratelimiting step in the incision process since a relatively small number of lesions have to be located within a large background of undamaged DNA. Damage recognition could occur by a diffusion process in which proteins locate the damage by random encounters. Such a process would have no energy requirement, and this is how damage could be located by the damage binding proteins Rad14, Rad4–Rad23, and RPA present in the reconstituted incision system comprised of Rad14, Rad4–Rad23, RPA, TFIIH, Rad1–Rad10, and Rad2, but lacking the Rad7 and Rad16 proteins. In the presence of the Rad7–Rad16 ŽNEF4. complex, however, damage recognition could be a more active process wherein NEF4 would translocate on DNA utilizing the energy derived from ATP hydrolysis, and at the site of the DNA lesion, NEF4 would become stably bound to it, because of the inhibition of its ATPase activity. Rad7 in NEF4 physically interacts with the Rad4–Rad23 complex ŽNEF2., and combining NEF2 and NEF4 results in a synergistic enhancement of damage binding w99x. Since this increased binding is observed only in the presence of ATP, only the ATP bound form of NEF4 may associate with NEF2 on damaged DNA. The order in which the remaining NER factors assemble onto the damage-bound NEF2–NEF4 complex remains to be determined. The ability of Rad23 in NEF2 to interact with Rad14 as well as with TFIIH might suggest that the Rad14– Rad1–Rad10 ŽNEF1. and Rad2–TFIIH ŽNEF3. complexes arrive next, and RPA could be the last factor to join in the assembly. Since the NEF4 equivalent has not yet been identified in humans, the order of assembly of the human incision complex has been deduced in the absence of this factor. One study with partially purified human cell extracts had suggested that XPC–HR23B was the first factor to arrive at the damage site w43x. Results of another, more extensive study, and which

S. Prakash, L. Prakashr Mutation Research 451 (2000) 13–24

utilized purified components w100x, have suggested a different sequence of events in which RPA and XPA bind the damage site first, and that is followed by the binding of TFIIH and XPC w100,101x. XPG then enters this protein assembly, and at that point, XPC departs from the assembly w100,101x. XPF–ERCC1 is the last factor proposed to join in this scheme of assembly. However, the likely association of XPA, XPF, and ERCC1 in one complex and of XPG and TFIIH in another complex, would suggest that these proteins arrive at the damage site as part of these ensembles and not as separate entities. The ATPase activity of Rad26 in yeast w102x and of CSB in humans w103x may function in the removal of RNA polymerase II stalled at a DNA lesion on the transcribed strand. However, in spite of considerable efforts, direct evidence for such a role of these proteins has not been forthcoming w103–105x. rad26 and CSB mutant cells exhibit efficient TCR of lesions present near the transcription initiation site, but they are deficient in the repair of lesions further downstream w106,107x. Since TFIIH is not a part of the elongating RNA PolII complex, the inverse correlation between TFIIH association with RNA PolII and the Rad26rCSB requirement for TCR may suggest that the Rad26rCSB proteins function in recruiting TFIIH to the damage site, which then effects the displacement of RNA polymerase from the damage site w106x. TFIIH would then be the first NER factor to arrive at the damage site on the transcribed strand.

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than 6 h. Only an insignificant fraction of Rad23 molecules is multiubiquitinated, and a change of Lys-49 in the Ub-like domain to Arg has no effect on the level of multibuiqutination of Rad23. This Lys-49 residue is equivalent to Lys-48 in authentic ubiquitin, where attachment of a multiubiquitin chain occurs in the proteolytic substrate, and mutation of Lys-48 to Arg inactivates the formation of multiubiquitin chain and subsequent proteolysis. These and other observations have indicated a non-proteolytic role of the ub-like domain of Rad23 w108x. The 26 S proteasome, which degrades ubiquitinated proteins, is composed of a 20 S core particle which contains the proteolytic activity and a 19 S regulatory particle. The S. cereÕisiae 19 S particle has at least 17 subunits, six of which are ATPases of the AAA family w109x. Rad23 interacts with the Cim3 and Cim5 subunits of the 19 S particle, and this interaction requires the ub-like domain of Rad23 w110x. These results were the first to hint at the possible involvement of the proteasome in NER. Recent studies have confirmed these observations, and they have provided additional evidence that the 19 S particle modulates NER efficiency via its interaction with the Rad23 ub-like domain w111x. Whether the chaperone-like activity of the 19 S particle functions in disassembling the NER complex, as has been suggested w111x, or whether it affects the assembly of this complex remains to be determined.

8. MMS19, transcription, and NER 7. Rad23, proteasome, and NER Rad23 is unique among the NER proteins in having an ubiquitin-like Žub-like. domain at its amino terminus w108x. Deletion of this domain results in a level of UV sensitivity that is intermediate between that of the wild type and the rad23D strain. Importantly, this ub-like domain can be functionally replaced by the authentic ubiquitin sequence, indicating a structural and functional similarity of this domain with ubiquitin. However, by contrast to the well-known role of ubiquitin in protein degradation, this ub-like domain of Rad23 has no role in protein stability w108x. Rad23 is an abundant and highly stable nuclear protein with a half-life much greater

MMS19 was initially identified in a screen for mutations that confer sensitivity to the alkylating agent methyl methanesulfonate ŽMMS. w112x. The mms19-1 mutation thus identified also rendered cells sensitive to UV light and to DNA cross-linking agents, and the removal of pyrimidine dimers and the nicking of DNA containing interstrand cross-links was impaired in the mutant strain w3,6x. Thus, MMS19 affects the incision step of NER. As is characteristic of NER-defective mutants, the frequency of UV-induced mutations is elevated in the mms19-1 mutant w6x. MMS19 encodes a protein of 118 kDa that contains 15 tandem repeats of a leucine-rich motif ŽLRM. w113x. In various other proteins, LRM repeats have

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been shown to function in protein–protein interactions. Deletion of MMS19 affects cell growth adversely. At 308C, growth is retarded, mms19D cells do not grow at 378C, and both the mms19D and mms19-1 mutant strains are methionine auxotrophs. In addition, extract from the mms19D strain exhibits a thermolabile defect in RNA PolII transcription. Addition of purified Mms19 protein does not restore transcription to heat treated mms19D extract, and addition of Mms19 to the reconstituted NER system also has no effect on the incision reaction. Addition of TFIIH to heat treated mms19D extract, however, restores transcriptional activity to the mutant extract w113x. Mms19 is not a component of TFIIH or of RNA PolII holoenzyme. Perhaps Mms19 affects NER and transcription by modulating the activity of TFIIH as an upstream regulatory element; Mms19 may be part of a kinase assembly that affects the activity of TFIIH and of other proteins via phosphorylation. Alternatively, Mms19 may be a component of a multisubunit assembly, which functions directly in NER and transcription.

9. Concluding remarks Studies of yeast and human NER have come a long way, and the roles of many important genes and proteins involved in this process are now well understood. A number of basic issues, however, still remain. First and foremost, is the mechanism of damage recognition. Eventhough many of the NER proteins bind damaged DNA, none of them do so with a high specificity, and none seem to recognize a cyclobutane pyrimidine dimer. How does the NER ensemble, then, recognize base damages which produce little or no helix distortion? Perhaps, we are still missing some of the damage recognition factors. Genetic studies in S. cereÕisiae would seem to support such a notion, as only one allele has been identified for many of the NER genes, and moreover, some of the yeast NER genes were identified subsequent to their identification in human. For example, RAD25, the yeast counterpart of XPB, and RAD26 and RAD28, the yeast counterparts of CSB and CSA, were identified in yeast following their discovery in humans. Second, what is the order by which NER factors assemble at the damage site, and are there

molecular matchmakers involved in this process which deliver protein complexes to the damage site? Third, what is the role of the 19 S proteasome particle in NER? Does it function in the assembly or disassembly of the incision complex? Fourth, what is the role of Mms19 in NER and transcription: does it affect the post-translational modification of TFIIH, or, is Mms19 a part of an as yet unidentified multiprotein complex, which, like TFIIH, functions directly in NER and in transcription? Fifth, how is the DNA packaged in chromatin made accessible to the NER machinery, and which proteins function in this process? And finally, how is the removal of stalled RNA PolII from the damage site coordinated with the assembly of the NER machinery?

Acknowledgements A large number of colleagues, Paul Reynolds, Veronique Bailly, John Watkins, Michael Bankmann, ´ David Higgins, Kiran Madura, and many others, have contributed to the study of NER in our group, and we are grateful to them all. In particular, we acknowledge the important contributions of Patrick Sung in the development of biochemical studies of yeast NER and in the elucidation of functions of NER proteins, and of Sami Guzder and Yvette Habraken to the studies of NER proteins. This work has been supported by research grants CA41261 and CA35035 from the National Institutes of Health.

References w1x D.R. Wilcox, L. Prakash, Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cereÕisiae, J. Bacteriol. 148 Ž1981. 618– 623. w2x R.J. Reynolds, E.C. Friedberg, Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cereÕisiae: incision of ultraviolet-irradiated deoxyribonucleic acid in vivo, J. Bacteriol. 146 Ž1981. 692–704. w3x R.D. Miller, L. Prakash, S. Prakash, Genetic control of excision of Saccharomyces cereÕisiae interstrand DNA cross-links induced by psoralen plus near-UV light, Mol. Cell. Biol. 2 Ž1982. 939–948. w4x M. Bankmann, L. Prakash, S. Prakash, Yeast RAD14 and human xeroderma pigmentosum group a DNA-repair genes encode homologous proteins, Nature 355 Ž1992. 555–558.

S. Prakash, L. Prakashr Mutation Research 451 (2000) 13–24 w5x E. Park, S.N. Guzder, M.H. Koken, D.I. Jaspers, G. Weeda, J.H. Hoeijmakers, S. Prakash, L. Prakash, RAD25 Ž SSL2 ., the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability, Proc. Natl. Acad. Sci. U. S. A. 89 Ž1992. 11416–11420. w6x L. Prakash, S. Prakash, Three additional genes involved in pyrimidine dimer removal in Saccharomyces cereÕisiae: RAD7, RAD14 and MMS19, Mol. Gen. Genet. 176 Ž1979. 351–359. w7x R.D. Miller, L. Prakash, S. Prakash, Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cereÕisiae, Mol. Gen. Genet. 188 Ž1982. 235–239. w8x S. Prakash, P. Sung, L. Prakash, DNA repair genes and proteins of Saccharomyces cereÕisiae, Ann. Rev. Genet. 27 Ž1993. 33–70. w9x R.H. Haynes, B.A. Kunz, DNA repair and mutagenesis in yeast, in: The Molecular Biology of the Yeast Saccharomyces, J. Strathern, E.W. Jones, J.R. Broach ŽEds.., Life Cycle and Inheritance Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981, pp. 371–414. w10x R.D. Gietz, S. Prakash, Cloning and nucleotide sequence analysis of the Saccharomyces cereÕisiae RAD4 gene required for excision repair of UV-damaged DNA, Gene 74 Ž1988. 535–541. w11x J. Venema, A. van Hoffen, A.T. Natarajan, A.A. van Zeeland, L.H.F. Mullenders, The residual repair capacity of xeroderma pigmentosum complementation group C fibroblasts is highly specific for transcriptionally active DNA, Nucl. Acids Res. 18 Ž1990. 443–448. w12x R. Verhage, A.-M. Zeeman, N. de Groot, F. Gleig, D.D. Gang, P. van de Putte, J. Brouwer, The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cereÕisiae, Mol. Cell. Biol. 14 Ž1994. 6135–6142. w13x J.P. Mueller, M.J. Smerdon, Repair of plasmid and genomic DNA in a rad7D mutant of yeast, Nucl. Acids Res. 23 Ž1995. 3457–3464. w14x Z. He, J.M.S. Wong, H.S. Maniar, S.J. Brill, C.J. Ingles, Assessing the requirements for nucleotide excision repair proteins of Saccharomyces cereÕisiae in an in vitro system, J. Biol. Chem. 271 Ž1996. 28243–28249. w15x Z. Wang, S. Wei, S.H. Reed, X. Wu, J.Q. Svejstrup, W.J. Feaver, R.D. Kornberg, E.C. Friedberg, The RAD7, RAD16, and RAD23 genes of Saccharomyces cereÕisiae: requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products, Mol. Cell. Biol. 17 Ž1997. 635–643. w16x S.H. Reed, Z. You, E.C. Friedberg, The yeast RAD7 and RAD16 genes are required for postincision events during nucleotide excision repair, J. Biol. Chem. 273 Ž1998. 29481–29488. w17x I. Mellon, V.A. Bohr, C.A. Smith, P.C. Hanawalt, Preferential DNA repair of an active gene in human cells, Proc. Natl. Acad. Sci. U. S. A. 83 Ž1986. 8878–8882. w18x I. Mellon, G. Spivak, P.C. Hanawalt, Selective removal of

w19x

w20x w21x

w22x

w23x

w24x

w25x

w26x

w27x

w28x

w29x

w30x

w31x

21

transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene, Cell 51 Ž1987. 241–249. I. Mellon, P.C. Hanawalt, Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand, Nature 342 Ž1989. 95–98. M.A. Nance, S.A. Berry, Cockayne syndrome: review of 140 cases, Am. J. Med. Genet. 42 Ž1992. 68–84. J. Venema, L.H.F. Mullenders, A.T. Natarajan, A.A. van Zeeland, L.V. Mayne, The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA, Proc. Natl. Acad. Sci. U. S. A. 87 Ž1990. 4707–4711. A. van Hoffen, A.T. Natarajan, L.V. Mayne, A.A. van Zeeland, L.H.F. Mullenders, J. Venema, Deficient repair of X the transcribed strand of active genes in Cockayne s synŽ . drome cells, Nucl. Acids Res. 21 1993 5890–5895. C. Troelstra, A. van Good, W.J. de Wit, W. Vermeulen, D. Bootsma, J.H. Hoeijmakers, ERCC6, a member of a subX family of putative helicases, is involved in Cockayne s syndrome and preferential repair of active genes, Cell 71 Ž1992. 939–953. K.A. Henning, L. Li, N. Iyer, L.D. McDaniel, M.S. Reagan, R. Legerski, R.A. Schultz, M. Stefanini, A.R. Lehmann, L.V. Mayne, E.C. Friedberg, The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH, Cell 82 Ž1995. 555–564. A.J. van Gool, R. Verhage, S.M.A. Swagemakers, P. van de Putte, J. Brouwer, C. Troelstra, D. Bootsma, J.H.J. Hoeijmakers, RAD26, the functional S. cereÕisiae homolog of the Cockayne syndrome B gene ERCC6, EMBO J. 13 Ž1994. 5361–5369. P.K. Bhatia, R.A. Verhage, J. Brouwer, E.C. Friedberg, Molecular cloning and characterization of Saccharomyces cereÕisiae RAD28, the yeast homolog of the human Cockayne syndrome A ŽCSA. gene, J. Bacteriol. 178 Ž1996. 5977–5988. J.C. Huang, D.L. Svoboda, J.T. Reardon, A. Sancar, Human nucleotide excision nuclease removes thymine dimers from X DNA by incising the 22nd phosphodiester bond 5 and the X 6th phosphodiester bond 3 to the photodimer, Proc. Natl. Acad. Sci. U. S. A. 89 Ž1992. 3664–3668. K. Tanaka, N. Miura, I. Satokata, I. Miyamoto, M.C. Yoshida, Y. Satoh, S. Kondo, A. Yasui, H. Okayama, Y. Okada, Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc finger domain, Nature 348 Ž1990. 73–76. S.N. Guzder, P. Sung, L. Prakash, S. Prakash, Yeast DNA repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet damaged DNA, Proc. Natl. Acad. Sci. U. S. A. 90 Ž1993. 5433–5437. E.H. Morita, T. Ohkubo, I. Kuraoka, M. Shirakawa, K. Tanaka, K. Morikawa, Implications of the zinc-finger motif found in the DNA-binding domain of the human XPA protein, Genes Cells 1 Ž1996. 437–442. C.J. Jones, R.D. Wood, Preferential binding of the xero-

22

w32x

w33x

w34x

w35x

w36x

w37x

w38x

w39x

w40x

w41x

w42x

w43x

w44x

w45x

S. Prakash, L. Prakashr Mutation Research 451 (2000) 13–24 derma pigmentosum group A complementing protein to damaged DNA, Biochemistry 32 Ž1993. 12096–12104. M.S. Wold, RPA: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism, Ann. Rev. Biochem. 66 Ž1997. 61–92. S.N. Guzder, Y. Habraken, P. Sung, L. Prakash, S. Prakash, Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH, J. Biol. Chem. 270 Ž1995. 12973–12976. D. Mu, C.-H. Park, T. Matsunaga, D.S. Hsu, J.T. Reardon, A. Sancar, Reconstitution of human DNA repair excision nuclease in a highly defined system, J. Biol. Chem. 270 Ž1995. 2415–2418. C.K. Clugston, K. McLaughlin, M.K. Kenny, R. Brown, Binding of human single-stranded DNA binding protein to DNA damaged by the anticancer drug cis-diamminedichloroplatinum ŽII., Cancer Res. 52 Ž1992. 6375–6379. Z. He, L.A. Henricksen, M.S. Wold, C.J. Ingles, RPA involvement in the damage-recognition and incision steps of nucleotide excision repair, Nature 374 Ž1995. 566–569. J.L. Burns, S.N. Guzder, P. Sung, S. Prakash, L. Prakash, An affinity of human replication protein A for ultravioletdamaged DNA: implications for damage recognition in nucleotide excision repair, J. Biol. Chem. 271 Ž1996. 11607–11610. T. Matsuda, M. Saijo, I. Kuraoka, T. Kobayashi, Y. Nakatsu, A. Nagai, T. Enjoji, C. Masutani, K. Sugasawa, F. Hanaoka, A. Yasui, K. Tanaka, DNA repair protein XPA binds replication protein A ŽRPA., J. Biol. Chem. 270 Ž1995. 4152–4157. L.X.L. Li, C.A. Peterson, R.J. Legerski, An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair, Mol. Cell. Biol. 15 Ž1995. 5396–5402. S.N. Guzder, P. Sung, L. Prakash, S. Prakash, Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA, J. Biol. Chem. 273 Ž1998. 31541–31546. L.E.T. Jansen, R.A. Verhage, J. Brouwer, Preferential binding of yeast Rad4–Rad23 complex to damaged DNA, J. Biol. Chem. 273 Ž1998. 33111–33114. J.T. Reardon, D. Mu, A. Sancar, Overproduction, purification, and characterization of the XPC subunit of the human DNA repair excision nuclease, J. Biol. Chem. 271 Ž1996. 19541–19546. K. Sugasawa, J.M.Y. Ng, C. Masutani, S. Iwai, P.J. van der Spek, A.P.M. Eker, F. Hanaoka, D. Bootsma, J.H.J. Hoeijmakers, Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair, Mol. Cell. 2 Ž1998. 223–232. S.N. Guzder, P. Sung, L. Prakash, S. Prakash, Yeast Rad7– Rad16 complex specific for the nucleotide excision repair of the nontranscribed DNA strand, is an ATP-dependent DNA damage sensor, J. Biol. Chem. 272 Ž1997. 21665– 21668. S.N. Guzder, P. Sung, L. Prakash, S. Prakash, The DNA-

w46x

w47x

w48x

w49x

w50x

w51x

w52x

w53x

w54x

w55x

w56x

w57x

w58x

w59x

dependent ATPase activity of yeast nucleotide excision repair factor 4 and its role in DNA damage recognition, J. Biol. Chem. 273 Ž1998. 6292–6296. P. Sung, L. Prakash, S.W. Matson, S. Prakash, RAD3 protein of Saccharomyces cereÕisiae is a DNA helicase, Proc. Natl. Acad. Sci. U. S. A. 84 Ž1987. 8951–8955. P. Sung, V. Bailly, C. Weber, L.H. Thompson, L. Prakash, S. Prakash, Human xeroderma pigmentosum group D gene encodes a DNA helicase, Nature 365 Ž1993. 852–855. S.N. Guzder, P. Sung, V. Bailly, L. Prakash, S. Prakash, RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription, Nature 369 Ž1994. 578– 581. R. Drapkin, J.T. Reardon, A. Ansari, J.C. Huang, L. Zawel, K. Ahn, A. Sancar, D. Reinberg, Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II, Nature 368 Ž1994. 769–772. L. Schaeffer, V. Moncollin, R. Roy, A. Staub, M. Mezzina, A. Sarasin, G. Weeda, J.H.J. Hoeijmakers, J.M. Egly, The ERCC2rDNA repair protein is associated with the class II BTF2rTFIIH transcription factor, EMBO J. 13 Ž1994. 2388–2392. P. Sung, L. Prakash, S. Weber, S. Prakash, The RAD3 gene of Saccharomyces cereÕisiae encodes a DNA-dependent ATPase, Proc. Natl. Acad. Sci. U. S. A. 84 Ž1987. 6045– 6049. W.J. Feaver, J.Q. Svejstrup, L. Bardwell, A.J. Bardwell, S. Buratowski, K.D. Gulyas, T.F. Donahue, E.C. Friedberg, R.D. Kornberg, Dual roles of a multiprotein complex from S. cereÕisiae in transcription and DNA repair, Cell 75 Ž1993. 1379–1387. W.J. Feaver, J.Q. Svejstrup, N.L. Henry, R.D. Kornberg, Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIHrTFIIK, Cell 79 Ž1994. 1103– 1109. P. Sung, S.N. Guzder, L. Prakash, S. Prakash, Reconstitution of TFIIH and requirement of its DNA helicase subunits, Rad3 and Rad25, in the incision step of nucleotide excision repair, J. Biol. Chem. 271 Ž1996. 10821–10826. D. Mu, D.S. Hsu, A. Sancar, Reaction mechanism of human DNA repair excision nuclease, J. Biol. Chem. 271 Ž1996. 8285–8294. V. Bailly, C.H. Sommers, P. Sung, L. Prakash, S. Prakash, Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10, Proc. Natl. Acad. Sci. U. S. A. 89 Ž1992. 8273– 8277. L. Bardwell, A.J. Cooper, E.C. Friedberg, Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro, Mol. Cell. Biol. 12 Ž1992. 3041–3049. A.E. Tomkinson, A.J. Bardwell, L. Bardwell, N.J. Tappe, E.C. Friedberg, Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endonuclease, Nature 362 Ž1993. 860–862. P. Sung, P. Reynolds, L. Prakash, S. Prakash, Purification

S. Prakash, L. Prakashr Mutation Research 451 (2000) 13–24

w60x

w61x

w62x

w63x

w64x

w65x

w66x

w67x

w68x

w69x

w70x

w71x

w72x

w73x

w74x

and characterization of the Saccharomyces cereÕisiae RAD1rRAD10 endonuclease, J. Biol. Chem. 268 Ž1993. 26391–26399. A.J. Bardwell, L. Bardwell, A.E. Tomkinson, E.C. Friedberg, Specific cleavage of model recombination and repair intermediates by the yeast Rad1–Rad10 DNA endonuclease, Science 265 Ž1994. 2082–2085. C.-H. Park, T. Bessho, T. Matsunaga, A. Sancar, Purification and characterization of the XPF–ERCC1 complex of human DNA repair excision nuclease, J. Biol. Chem. 270 Ž1995. 22657–22660. Y. Habraken, P. Sung, L. Prakash, S. Prakash, Yeast excision repair gene RAD2 encodes a single-stranded DNA endonuclease, Nature 366 Ž1993. 365–368. J.J. Harrington, M.R. Lieber, Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair, Genes Dev. 8 Ž1994. 1344–1355. P. Robins, D.J. Pappin, R.D. Wood, T. Lindahl, Structural and functional homology between mammalian DNase IV X and the 5 -nuclease domain of Escherichia coli DNA polymerase I, J. Biol. Chem. 269 Ž1994. 28535–28538. R.A. Bambara, R.S. Murante, L.A. Henricksen, Enzymes and reactions at the eukaryotic DNA replication fork, J. Biol. Chem. 272 Ž1997. 4647–4650. V. Lyamichev, M.A.D. Brow, J.E. Dahlberg, Structurespecific endonucleolytic cleavage of nucleic acids by eubacterial polymerases, Science 260 Ž1993. 778–783. Y. Habraken, P. Sung, L. Prakash, S. Prakash, Structurespecific nuclease activity in yeast nucleotide excision repair protein Rad2, J. Biol. Chem. 270 Ž1995. 30194–30198. X A. O Donovan, A.A. Davies, J.G. Moggs, S.C. West, R.D. X Wood, XPG endonuclease makes the 3 incision in human DNA nucleotide excision repair, Nature 371 Ž1994. 432– 435. D. Higgins, S. Prakash, P. Reynolds, R. Polakowska, S. Weber, L. Prakash, Isolation and characterization of the RAD3 gene of Saccharomyces cereÕisiae and inviability of rad3 deletion mutants, Proc. Natl. Acad. Sci. U. S. A. 80 Ž1983. 5680–5684. L. Naumovski, E.C. Friedberg, A DNA repair gene required for the incision of damaged DNA is essential for viability in Saccharomyces cereÕisiae, Proc. Natl. Acad. Sci. U. S. A. 80 Ž1983. 4818–4821. K.D. Gulyas, T.F. Donahue, SSL2, a suppressor of a stemloop mutation in the HIS4 leader encodes the yeast homolog of human ERCC-3, Cell 69 Ž1992. 1031–1042. L. Schaeffer, R. Roy, S. Humbert, V. Moncollin, W. Vermeulen, J.H.J. Hoeijmakers, P. Chambon, J.-M. Egly, DNA repair helicase: a component of BTF2 ŽTFIIH. basic transcription factor, Science 260 Ž1993. 58–63. H. Qiu, E. Park, L. Prakash, S. Prakash, The Saccharomyces cereÕisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II, Genes Dev. 7 Ž1993. 2161–2171. S.N. Guzder, H. Qiu, C.H. Sommers, P. Sung, L. Prakash, S. Prakash, DNA repair gene RAD3 of S. cereÕisiae is

w75x

w76x

w77x

w78x

w79x

w80x

w81x

w82x

w83x

w84x

w85x

w86x

w87x

w88x

w89x

23

essential for transcription by RNA polymerase II, Nature 367 Ž1994. 91–94. P. Sung, D. Higgins, L. Prakash, S. Prakash, Mutation of lysine-48 to arginine in the yeast RAD3 protein abolishes its ATPase and DNA helicase activities but not the ability to bind ATP, EMBO J. 7 Ž1988. 3263–3269. P.R. Reynolds, S. Biggar, L. Prakash, S. Prakash, The Schizosaccharomyces pombe rhp3q gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cereÕisiae, Nucl. Acids Res. 20 Ž1992. 2327–2334. S.N. Guzder, P. Sung, S. Prakash, L. Prakash, Lethality in yeast of triochothiodystrophy ŽTTD. mutations in the human xeroderma pigmentosum group D gene, J. Biol. Chem. 270 Ž1995. 17660–17663. R.H. Schiestl, S. Prakash, RAD1, an excision repair gene of Saccharomyces cereÕisiae, is also involved in recombination, Mol. Cell. Biol. 8 Ž1988. 3619–3626. R.H. Schiestl, S. Prakash, RAD10, an excision repair gene of Saccharomyces cereÕisiae, is involved in the RAD1 pathway of mitotic recombination, Mol. Cell. Biol. 10 Ž1990. 2485–2491. H.L. Klein, Different types of recombination events are controlled by the RAD1 and RAD52 genes of Saccharomyces cereÕisiae, Genetics 120 Ž1988. 367–377. A. Aguilera, H. Klein, Yeast intrachromosomal recombination: long gene conversion tracts are preferentially associated with reciprocal exchange and require the RAD1 and RAD3 gene products, Genetics 123 Ž1989. 683–694. B.J. Thomas, R. Rothstein, The genetic control of direct-repeat recombination in Saccharomyces cereÕisiae: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene, Genetics 123 Ž1989. 725– 738. B.R. Zehfus, A.D. McWilliams, Y.H. Lin, M.F. Hoekstra, R.L. Keil, Genetic control of RNA polymerase I stimulated recombination in yeast, Genetics 126 Ž1990. 41–52. A.M. Bailis, L. Arthur, R. Rothstein, Genome rearrangement in top3 mutants of Saccharomyces cereÕisiae requires a functional RAD1 excision repair gene, Mol. Cell. Biol. 12 Ž1992. 4988–4993. B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 Ž1995. 1199–1211. A.J. Rattray, L.S. Symington, Multiple pathways for homologous recombination in Saccharomyces cereÕisiae, Genetics 139 Ž1995. 45–56. J. Fishman-Lobell, J.E. Haber, Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1, Science 258 Ž1992. 480–484. E.L. Ivanov, J.E. Haber, RAD1 and RAD10, but not other excision repair genes, are required for double-strand breakinduced recombination in Saccharomyces cereÕisiae, Mol. Cell. Biol. 15 Ž1995. 2245–2251. M. Saparbaev, L. Prakash, S. Prakash, Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1–

24

w90x

w91x

w92x

w93x

w94x

w95x

w96x

w97x

w98x

w99x

w100x

S. Prakash, L. Prakashr Mutation Research 451 (2000) 13–24 RAD10 pathway of mitotic recombination in Saccharomyces cereÕisiae, Genetics 142 Ž1996. 727–736. M. Sugawara, F. Paques, M. Colaiacovo, J.E. Haber, Role of Saccharomyces cereÕisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 9214–9219. D.T. Kirkpatrick, T.D. Petes, Repair of DNA loops involves DNA-mismatch and nucleotide-excision repair proteins, Nature 387 Ž1997. 929–931. J. McWhir, J. Selfridge, D.J. Harrison, S. Squires, D.W. Melton, Mice with DNA repair gene Ž ERCC-1. deficiency have elevated levels of p53, liver nuclear abnormalities and die before weaning, Nat. Genet. 5 Ž1993. 217–224. G. Weeda, I. Donker, J. de Wit, H. Morreau, R. Janssens, C.J. Vissers, A. Nigg, H. van Steeg, D. Bootsma, J.H.J. Hoeijmakers, Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence, Current Biol. 7 Ž1997. 427–439. A. Aboussekhra, M. Biggerstaff, M.K.K. Shivji, J.A. Vilpo, V. Moncollin, V.N. Podust, M. Protic, U. Hubscher, J.-M. ¨ Egly, R.D. Wood, Mammalian DNA nucleotide excision repair reconstituted with purified protein components, Cell 80 Ž1995. 859–868. J.Q. Svejstrup, Z. Wang, W.J. Feaver, X. Wu, D.A. Bushnell, T.F. Donahue, E.C. Friedberg, R.D. Kornberg, Different forms of TFIIH for transcription and DNA repair: Holo-TFIIH and a nucleotide excision repairosome, Cell 80 Ž1995. 21–28. S.N. Guzder, P. Sung, L. Prakash, S. Prakash, Nucleotide excision repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome, J. Biol. Chem. 271 Ž1996. 8903–8910. Y. Habraken, P. Sung, S. Prakash, L. Prakash, Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 10718–10722. C. Matsutani, K. Sugasawa, J. Yanagisawa, T. Sonoyama, M. Ui, T. Enomoto, K. Takio, K. Tanaka, P.J. van der Spek, D. Bootsma, J.H.J. Hoeijmakers, F. Hanaoka, Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23, EMBO J. 13 Ž1994. 1831–1843. S.N. Guzder, P. Sung, L. Prakash, S. Prakash, Synergistic interaction between yeast nucleotide excision repair factors NEF2 and NEF4 in the binding of ultraviolet-damaged DNA, J. Biol. Chem. 274 Ž1999. 24257–24262. M. Wakasugi, A. Sancar, Order of assembly of human DNA repair excision nuclease, J. Biol. Chem. 274 Ž1999. 18759–18768.

w101x M. Wakasugi, A. Sancar, Assembly, subunit composition, and footprint of human DNA repair excision nuclease, Proc. Natl. Acad. Sci. U. S. A. 95 Ž1998. 6669–6674. w102x S.N. Guzder, Y. Habraken, P. Sung, L. Prakash, S. Prakash, X The RAD26, yeast homolog of human Cockayne s syndrome group B gene, encodes a DNA dependent ATPase, J. Biol. Chem. 271 Ž1996. 18314–18317. w103x C.P. Selby, A. Sancar, Human transcription-repair coupling factor CSBrERCC6 is a DNA-stimulated ATPase but is not a helicase and does not dirupt the ternary transcription complex of stalled RNA polymerase II, J. Biol. Chem. 272 Ž1997. 1885–1890. w104x C.P. Selby, R. Drapkin, D. Reinberg, A. Sancar, RNA polymerase II stalled at a thymine dimer: footprint and effect on excision repair, Nucl. Acids Res. 25 Ž1997. 787– 793. w105x C.P. Selby, A. Sancar, Cockayne syndrome group B protein enhances elongation by RNA polymerase II, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 11205–11209. w106x M. Tijsterman, R.A. Verhage, P. van de Putte, J.G. Tasseron-de Jong, J. Brouwer, Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of Saccharomyces cereÕisiae, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 8027–8032. w107x Y. Tu, S. Bates, G.P. Pfeifer, Sequence-specific and domain-specific DNA repair in xeroderm pigmentosum and Cockayne syndrome cells, J. Biol. Chem. 272 Ž1997. 20747–20755. w108x J.F. Watkins, P. Sung, L. Prakash, S. Prakash, The Saccharomyces cereÕisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function, Mol. Cell. Biol. 13 Ž1993. 7757– 7765. w109x M.H. Glickman, D.M. Rubin, C.A. Fried, D. Finley, The regulatory particle of the Saccharomyces cereÕisiae proteasome, Mol. Cell. Biol. 18 Ž1998. 3149–3162. w110x C. Schauber, L. Chen, P. Tongaonkar, I. Vego, D. Lambertson, W. Potts, K. Madura, Rad23 links DNA repair to the ubiquitinrproteasome pathway, Nature 391 Ž1998. 715– 718. w111x S.J. Russell, S.H. Reed, W. Huang, E.C. Friedberg, S.A. Johnston, The 19 S regulatory complex of the proteasome functions independently of proteolysis in nucleotide excision repair, Mol. Cell. 3 Ž1999. 687–695. w112x L. Prakash, S. Prakash, Isolation and characterization of MMS-sensitive mutants of Saccharomyces cereÕisiae, Genetics 86 Ž1977. 33–55. w113x S. Lauder, M. Bankmann, S.N. Guzder, P. Sung, L. Prakash, S. Prakash, Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription, Mol. Cell. Biol. 16 Ž1996. 6783–6793.