Summary Eukaryotic cells are able to mount several genetically complex cellular responses to DNA damage. The yeast Saccharomyces cerevisiae is a genetically well characterized organism that is also amenable to molecular and biochemical studies. Hence, this organism has provided a useful and informative model for dissecting the biochemistry and molecular biology of DNA repair in eukaryotes. introduction The application of molecular genetics and recombinant DNA technology to the field of DNA repair has led to an appreciation that cellular responses to DNA damage are almost as diverse as the kinds of damage that afflict the genomes of living cells. Thus, for example, the generic reference to excision rcpair has been superceded by the definition of three biochemically distinct modes by which damaged, mispaired or inappropriate bases are excised from DNA. These distinctions are recognized through the more specific designations of nucleotidc excision repair, base excision repair and post-replicative mismatch correction (mismatch repair)('). It is also now generally appreciated that particular cellular responses to DNA damage can involve large numbers of genes whose products presumably participate in complex biochemical reactions. Nucleotide excision repair (NER) of DNA constitutes a notable example of such genetic and biochemical complexity. The purpose of this article is to review selected aspects of excision repair in eukaryotcs, with particular emphasis on recent advances in the yeast Saccharomyces cerevisiae. This organism is especially amenable to both genetic and molecular manipulations and has proven to be an appropriate model system for the stud of numerous aspects of eukaryotic molecular biology(?>3 ) .

Nucleotide Excision Repair of DNA NER defines a DNA repair mode by which helixdistortive base damagc produced by physical agents such as ultraviolet (UVj radiation and bulky chemicals, which arc covalently adducted to nitogenous bases, are

excised from the genome('). The two fundamental elements that are believed to confer most, if not all. of the biochemical specificity associated with NER are damage-specific recognition and damage-specific incision of DNA. In the prokaryote E. coli, the products of three genes designated uvrA, uvrB and uvrC are required for the specific recognition of damaged based4-*). However, the precise mechanism whereby such bases (distributed randomly in the genome and present in concentrations as low as one in ten million nucleotides), are located and discriminated as sites for specific binding of Uvr proteins, has not been deter~nined(~7~). The products of these three uvr genes are also absolutely required for damage-specific incision of DNA('-'). Considerable information has been gleaned about the biochemistry of the latter proccss and it is now established that in E. coli incisions are located on either side of damaged bases on the same DNA strand, and are separated by distances of 12-13 nucleotides("-"). In contrast to the rapidly emerging picture of NER in prokaryotes, essentially nothing is known about damage-specific recognition and damage-specific incision of DNA in eukaryotic cells. Additionally, it is not clear to what extent the extensive packaging of DNA in eukaryotic cells limits the accessibility of NER enzymes to sites of base damage, how such accessibility might vary during different phases of the cell cycle and, to the degree that accessibility is a limiting entity, how this might be overcornc.

Yeast as a Eukaryotic Model The isolation and detailed phenotypic characterization of yeast mutants that are abnormally sensitive to killing by ultraviolet (UVj radiation has led to the elaboration of a detailed genetic framework with which to approach the molecular biology and biochemistry of NER in eukaryotes('"). Despite an extensive search for mutants, however, there is good reason to suspect that this genetic framework is not complete. At least ten genes are known to be involved in NER in S . cere~isiae('.~). but recent studies indicate the cxistencc of an eleventh gene (see below). Additionally, the isolation of UV-sen4itive mutants has demonstrated a significant bias in favor of mutations at a limited number o f genetic loci, suggesting that on statistical grounds other loci involved in NER may be as yet unidentified(9). Based on detailed phenotypic characterization of corresponding mutants, five of the yeast genes which have been associated with NER ( R A D I . RAD2, RAD3. RAD4 and RAf110) are believed to be involved in the primary biochemical events of damage-specific recognition of bulky base damage and damage-specific incision of DNA at or near such sites. As indicated above. a sixth gene is believed to be involved in these

Table 1. Geiierics of niicleotide e.x'.yci.wnrepair

XP group

Rodent group




eukaryotes chromosome

Human gene

9q3.1 2q21



Yeast gene

Polypeptide function


',DNA hclicase

R A L13 (ital)

DNA helicase '?DNA binding protein








ERCCZ (ital)

15 RCG-1 KCG-4 RCG-5 RCG-6 RCG-7 RCG-8

19q13.2 I6 13q 10ql1


KA D l RA 0 2 R A0 4

processes, based on the identification of a yeast homolog of a recently cloned human NER gene (designated ERCC3). This gene complements the incision-defective phenotype of cells from genetic complementation group B of the human hercditary disease xerodei-ma pigmentosum (XP)(") (Table 1) (see later discussion). In addition to the five (or six) genes that are required for NER in yeast (Table 1). a further five genes (RAD7, RADI4. RAD16. RAD23, RAD24), have been identified that confer a henotype of partially defective NER in yeast mutantsi2.?). Nothing is known about the role of these genes in NER. It has been suggested that they may be regulatory genes, or that they niay participate in secondary reactions that h a w quantitative rather than qualitative implications for repair('33). hence the phenotype of deficient rather than defective repair of DNA in mutants. Recent studies suggest that the HAD7, RAL116 and RAD24 genes may participate in NER events associated with the repair of transcriptionally inactive genes uniquely during the G2 phase of the cell cycle, Specifically. the transcriptionally inactive H M L a locus is repaired as efficiently as the transcriptionally active MATulocus, following UV irradiation of cells in the G2 phase. However, in other phases of the cycle, repair of the H M L a locus is less efficient(*'). It has been suggested that a more open conformation of the chromatin structure of the H M L a gene in G3may facilitate NER. However, duri~igother phases of the cell cycle the closed conformation of this gene reduces the efficiency of NER. The removal of bulky base damage from the H M L a ene is defective in uud7, md16 and md24 mutants(' ). These results raise the possibility that these genes (and possibly other genes in this group) are involved in a NER pathway that includes the products of at least some of the R A D l . etc. group of genes, but which is biochemically distinct from that which operates in other phases of the cell cycle.


The Cloned RAD Genes Involved in NER Why are so many genes involved in NER in yeast? In an attempt to provide answers to this fundamental question, we and others have isolated a number of these genes by molecular cloning in order to study their functions and those of the proteins they encodc(2"i. Sequencing of the R A I I I . ctc. group of genes and computer-assisted comparison of their predicted amino acid sequences with those of other polypeptides has been informative only with respect to RAD3, which contains consensus nucleotide binding and DNA helicase domains('' "1, Purification and biochemical characterization of Rad3 protein following overexpression of the cloned KAD3 gene in yeast cells has provided direct verification that the protein is indeed a DNA-dependent ATPase /DNA heli~ase('~-~').in vituo, this DNA helicase operates over a sharply limited p H range. with an optimuin at pH 5.6. The helicase initiates unwinding of duplex regions of partially duplex DNA'" 17). For thk reaction, sin Tle stranded regions 4-20 nucleotides long are requiredk1"). The enzyme has a strict 5'+3' polarity with respect to the single strand to which it binds(" l6 . Rad3 protein can unwind duplex regions that are hundreds of nucleotides long('"). The translated sequence of the human ERCC3 gene mentioned above suggests that it (and. by inference, the homologous yeast gene) also encodes a DNA helicase('"). At this time one can only speculatc on the role(s) of and requirement for multiple DNA helicases in NER. One obvious possibility is that such an enzyme could facilitate the displacement of damage-containing oligonucleotide fragments following incision of DNA on either side of damaged nucleotides (Fig. 1). Alternatively or additionally. unwinding of DNA may be required to generate a particular conformation which facilitates productive binding of other proteins involved in NER at or near sites of DNA damage (Fig. 1). Finally, DNA helicases might facilitate the







I -7 Rad3 helicase




I -

DNA polymerase



accurate semi-conservative DNA synthesis, and/or post-rcplicative mismatch correction. Implicit in both hypotheses is the conclusion that Rad3 protein may participate in as many as three different multiproteiri complexes associated with D N A metabolism, i.e. nucleotide excision repair, post-replicative mismatch correction and DNA replication. Recent studies (A. Sugino, I. Harosh, H. Naegcli and E. C. Friedberg, unpublished observations) have demonstrated that purified Rad3 protein stimulates the strand exchange activity of a yeast recombinase designated [email protected](for sti-and transfer protein)(19) in v i t ~ u ,thus potentially defining a fourth biochemical function for the protein. Remarkably. this stimulation takes place under experimental conditions which do not support the DNA helicase activity, i.e., in the absence of ATP and at pH 7.0.



********** Damage-Specific Incision

The Essential Function of the RAD3 Gene 5' 3'

3' 5'


Rad3 helicase

5' 3'


5' Other Rad proteins

3' 5'




Fig. 1. Hypothetical roles for a D N A helicase such as Rad3 protein, during NER in yeast. Following damapspecific incision of D N A on citlicr side of a pyriinidinc dimcr (top). release of an oligonucleotide fragment could be facilitated by unwinding of DNA: leav-ing a gap which is repaired by repair synthesis of DNA. If Rad3 protein binds to a single stranded region on the strand opposite the pyrimidine dimer. its 5'-3' polarity requires that oligonucleotide displaccmcnt and rrpair synthesis would occur in opposite dircctions (light arrows). A D N A helicase might also bind to D N A at sites of distortion generated by damage such as pyriinidinc dimers (bottom) and locally unwind D N A , thereby producing a conformational state which facilitates the hinding of other protciiis rcquircd for damage-specific incision. Once again the direction of unwinding shnwn presumes that Kad3 protein binds t o a single stranded regioii on the D N A strand opposite the dimer.

translocation of NER proteins along the genome from initial sitev o f non-specific binding to sites of stable specific binding, a s has been suggested for the uvrAB DNA helicase complex of E. coli('>'). There are indications that the Rad3 helicase activity participate5 in other metabolic transactions of DNA. Several rad3 mutant alleles confer a markedly increased rate of spontaneous mutagenesis(" "). A majority of the mutants with this phenotype that have been examined carry mutations that map to conserved D N A helicase domains("). Albeit rather indirect, this observation suggests that the hclicase activity is required for

It has been known for some years that R A D 3 is an essential gene, which participates in a vital cellular function(s) unrelated to DNA repair(',". Based on the observation that Rad3 protein is a D N A helicase, it is intuitively compelling to associate an essential role of the hclicase with DNA replication. However, a m d 3 mutant known to be defective in the ATPase/helicase activity is viable('"), suggesting that the essential function is not determined by the DNA helicase activity. Clearly, a definitive approach to identification of the essential function of the RAD3 gene is to isolate and characterize conditional-lethal mutants. Attempts to isolate temperature-sensitive, cold-sensitive, osmotic-sensitive and even heavy water-sensitive mutants in niy laboratory have proven largely unsuccessful. A single temperature-sensitive mutant that was isolate~i('.~) is unstable and has an uninformative phenotype at elevated temperatures"~". We have also been unable to isolate extragenic second-site mutants that suppress the lethality confcrrcd by inactivation of the RA 0.3 gene.

Other RAD Genes Involved in PIER At the time of writing, there are no clear indications about the nature and function of the proteins encoded by the other RAD genes required for NER. The translated amino acid sequence of the RAD2 gene shows limited homology over a 237 amino acid stretch with the yeast RADSO gene, a qene known to be involved in meiotic recombinationb'). The significance (if any) of this weak sequence similarity i s not clear. It is of interest that the RA DSO O R F predicts a protein that contains heptad repeat sequences characteristic of helical coiled coil proteins(221.This feature has invited speculation that RadSO protein may be part of a contractile system that brings homologous chromosomes together during pairing, or like kinesin, RadSO protein may be involved in translocation of DNA molecules along microtubules(22).Hydropathy profiles of the predicted Rad2 polypeptide indicate that it is

quite hydrophilic relative to other Rad proteins so analyzed(’). However, analysis of the predicted Rad2 amino acid sequence does not reveal the presence of heptad repeats (A. J. Cooper and E. C. Friedberg, unpublished observations). In addition to their role in NER, the R A D l and RADIO genes have been shown to bc involved in specialized forms of mitotic recornbination. Thus, for example recombination in the ribosomal repeat unit, which is stimulated in wild-type cclls by the ribosomal DNA promoter HOTl, is reduced in a radl mutant(23). Additionally, mutations in the R A D I or RADIO genes result in reduced levels of intrachromosomal recombiHence, it is apparent that in addition to Rad3, some of the other Rad proteins may participate in the formation of alternative multiprotein complexes. Such multifunctionality provides a possible mechanism for co-ordinating potentially competing metabolic functions of DNA by limiting the formation of more than one type of multi-protein complex at any given time. Thus, for example, the sequestration of Rad3 protein by a NER complex might limit its participation in a DNA replication complex, thereby reducing the extent of DNA synthesis during NER. DNA Damage inducibility of RAD Genes A number of yeast genes potentially involved in multiple and diverse cellular responses to DNA damage are induced following exposure of cells to DNAdamaging agents (Table 2). Among these are several of the R A D genes involved in NER (RAD2(”’28), RAD7(l9)and RAD23(”’)).Induction of the RAD2 gcne has been extensively studied. Expression of increased steady-state levels of RAD2 mRNA accompanies treatment of cells with a variety of DNA-damaging agents, including several to which rad2 mutants are not abnormally sensitive. Induction requires that cells be actively traversing the cell cycle. Hence: increased

expression is not observed in non-cycling (Go) cells maintained in the stationary hase, nor in cells arrested in the GI phase of the cycle(‘). These, as well as other observations (see below), suggest that events associated with DNA replication may be necessary for induction of the RAD2 gene. Several cis-acting sequences required for induction of RAD2 have been identified by deletion analysis of the RAD2 promoter(31).Two of these are believed to be yeast upstream activator sequences (enhancers) and are designated DREl and DRE2 (for damage responsive element). Sequences closely related to that in DREl have been identified in the yeast RAD7 and the PHRl promoter rcgions; however, as yet there is no direct evidence that these sequences are specifically required for induction of the RAD7 and PHRl genes. Band shift assays using a radiolabclled oligonucleotide with sequences from D R E l and DRE2 have identified specific protein/oligonucleotide complexes in extracts of unirradiated cells (W. Siede and E. C. Friedberg, unpublished observationiiic encodes a DN A-dependent ATPase. Proi.. Narl Amd. S r r . ( I S A 84, h045-604V 16 SiWG, p.. P R A K A S H . L,., M A I S O N W . AND PKAKASH. S. (1987). RAD3 pi-otrin crf Sacchni.omyi.~scerevisiae 15 it DNA helitase. Proc. Narl A c i d Sci. &iSA 84, X951-8YS5 17 HAKUSH. 1.. N A U M ~ VL.~.AND ~ I .F R I E D B EE. R ~C~.. (I9S9). Purification and characterization of thc Rad3 ATPascIDNA hclicaw trom Srrrchurnrriyrm cerelbiae. .I. B i d . chert^ 264. 20 532-205.79. 18 MOI\TFI.ONE. B. A . . H O F K S T R A , hf. F. AND b l A L O N L K. E. (1988). Spontaneous mitotic rewmbination in yeast: the hyper-recombinational reml mniaiionh ,ire alleles of the RAD3 gene. Gmufic&119. 189-301. 19 L)YKSTR.\. C. C:., N i i i b s , .I.. RHNICK. M. A. A N D SUGINO, A. (1988). ATPindependent DKA strand transfer catalyzed by proteins fi-om thc yeast Sacchnvo?nyces cercvisioa. In: Mechnnisnrr arid Coriseqiiuvfres of D N A Danqqe Pvorrs7iiig. edited by Friedberg. E. C. and Haiiaaalt. P. C., Alan R. Liss Inc.. hew York. p.231-235. 20 sc~ ( iP.. . HI WIN^. D.. PRAKASH. L. AND PRMASH. S. (1988). Mutatlor1 {jr ly\in(i-48 to nrginine in the yeast RAD3 protcin abolishes its ATPase and DNA helicase activities but not the ability to bind ATP. ERIRO .I. 7. 3263-3269. 21 H . ~ \ Y N R .~H. s ..AND K u ~ z B. . .A. (1981). DNA repair and mutagcnciii in yeast. I n : 71w Molwfrlar Biology of rhi, Yrwr S;icch;iromycea (edited by Strathci-n. J. N . , h i e . ; . E. W. and Broach, J . K.). Cold Spring Harbor Labvralur) Preas. Cold Spnng Harbor. hew York. p . 371-314. 22 ALANI, E.. SIJBBIAH. s. AND KI F.CKNF.R. N.(1989). The yeabl RAD50 gene encodes a predicted 153kD protcin containing a purine nuclentide-binding domain and two large heptad repeat regions. Generics 122. 47-57. 23 VOLI.KEL-MEIM\N. K.. KEIL. R. L. AND ROFDER, S. (19x7). Reconihination-

stimulating sequences in yeast ribosomal DKA correspond to Fequences regulating transcription by RNA polyiiierasc I. Cell 48. 1071-1079. 24 S r H r E s T I , R.H. AND PRALASH, L. (1988). RADI. an excision repair gene of Sacchnromyces ccrevisiae, is also involvcd in recombination. Mol. Cell. Biol. 8,

(1990). Use of in v i i w and in vituo assay for the characterization of mamlilalian excision repair and isolation of repair proteins. Muration Res. 236, 223-238. 41 THOMPSON, L. H . , SHlUMl, r., ShLAZAR. E. P. AND STEWART,s. A. (1988). An eighth complementation group of rodent cells hy7erseusitive to ultraviolet 3619-9626. radiation. Somat. Cell Mol. Genet. 14, 605-612. 25 A~;IJ[LEKA. A.AND KLEIN.H. L. (1989). Genetic and molecular analysis of 42 THOMPSON, L. H . , WEBER,c.A. ANU CAKKANO. A. V. (1988). Human DNA recombination cvciits i i i Surr.harornyres rrrevissiae occurring in the prcsencc of repair genes. h i : Mechonrsms and Consequences of D N A Dumugi, Prowssing. the hyper-rccombination mutation hprl. Genetics 122. 503-517. sdiled by Friedhrrg, L. C. and Hanawalt, P. C.. Alan R . Liss Inc., N e w York. p.28') -293. 26 SCHIESTL,R. H. .ANT) PRAKASH, S. (1990). RADIO. an excision repair gene of 43 HOLIJMAKERS. J . H. J., VAN DUN, M.. WEF.r.4. G , v . 4 ~DEK EB, A. J. Saidiurorn),reY;iw. is involved in the R A DI pathway of recombination. TROELSTRA. C . , EKER,A. P. M.. .TASPF.RS. H C . J., WESILKVELD, A. AND ,Iful. Cell. Biol. 10, 2485-2491. BOOTSI*IA. D . (1900). Analysi? of mammalian excision repair; from mutants to 27 ROBINSON. G . W.. NICOLET, c . M..KALAINOV. D . AND FRIEDBERG, E. c. genes and gene products. In: ,Mechnni,rm, find Conreqrrenres of DA'A Damage (1986). A yeast excision repair gene is inducible by DNA damaging agents. Proce.ising, edited by Friedberg, E. C. and fianawalt. P. C., Alan R.Liss Inc.. I'ruc. Nad A c i d . Sci. USA. 83, 1842-1846. New York. p. 281-287. 28 MADURA,K . A N D PKAKASH, s. (1486). Nucleotide sequence, transcript 44 THOMPSON. L. H . AND Uoorsun, D. (1988). Designation of mammalian mapping, and regulation of the IC/IL)S gene of Saccharomyes cerei~isiue..I. complementation groups and repair genes. In: Mecliunisnis and Con~eyuencec Uacteriol. 166. Y14-923. ofD,VA Damugr, Processing. edited by Friedberg, E. 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AVD convtituthe damage-specific DNA hiding protein is synthesized at bizher WESTERVELD. A. (1989). The cloned human DNA excision repair gene E'RCC-I levels in LJV irradiated primate crlli. Mol. Cell. B i d . 10, 2031-2048. fails to correct xeroderma pigmentosum complementation groups A through I. 35 PATTERSON, M. A N D CHI!, G. (1989). Evidence that xeroderrna Mutation RES 217, 83-92. pigmentusuiri cells from complementation group E are deficient in a homolog of 49 MORRISON, A , , C H K I S I ~ N SR. EN B., . ALLEY. J . . BECK, A. K., BERNSTINE. E. yeast photolyaae. Mol. C d B i d . 9,5105 5112. G.. LEMONTI., J . F. AND LAWRENCE. C. W. (lrBO).REV3, a yeast gene whose 36 C L ~ V EJ. RE . . (1990). Do w e know the cause of xcrodcrma pigmentosum? function is required for induced mutagcncsis. is predicted to cncodc a now Chrcinogenrsis 11. 875-882. esiential DNA polymerase. J. Bncteriol. 171. 5659-5667. 37 C h , G. AKT) CHANG. E. (1990). Cisplatin-resistatit cclli cxprcss increased 50 ABOUSSEKHRA. A , . CHANET. R.. Zcr4cr.4, Z.. CASSIER-CHKYAT. C.. HEUTJF, levels of a factor that recognizes damaged DNA. Proc. Null Acud. Sri. 1K-l.87. M. AND F~ABRF. F. (1989). R A D H , a gene or Su

Eukaryotic DNA repair: glimpses through the yeast Saccharomyces cerevisiae.

Eukaryotic cells are able to mount several genetically complex cellular responses to DNA damage. The yeast Saccharomyces cerevisiae is a genetically w...
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