DNA repair

DNA repair Deborah

E. Barnes, Tomas Lindahl and Barbara ICRF, Clare Hall Laboratories,

South

Mimms,

Sedgwick

UK

Multiple DNA repair processes are required to maintain the integrity of the cellular genome. Recent advances, including elucidation of three-dimensional structures of DNA repair enzymes, and the cloning and characterization of DNA repair genes implicated in human inherited disease, have given new insights into the surprising complexity of cellular responses to DNA damage. Current

Opinion

in Cell Biology

Introduction Counteracting the multitude of different types of spontaneous and environmentally induced DNA damage is clearly a major challenge to organisms, and a cell with out any DNA repair capacity would not be viable. The excision repair of covalently altered nucleotides that cause major helical distortion requires at least as many proteins as act at DNA replication forks and the two processes have many factors in common. A completely separate excision repair pathway is employed to remove more subtle forms of DNA damage. In addition, direct enzyme-catalyzed reversal of some forms of DNA alteration occurs, and anomalous types of DNA replication and recombination (largely beyond the scope of this review) can be mobilized to reduce potentially lethal or mutagenic effects of structurally impaired DNA templates. Furthermore, interaction between DNA repair processes and cell cycle control prevents DNA synthesis or mitosis without prior repair of DNA damage. With regard to investigation of human cells, DNA repair has a unique advantage over the replication and recombination fields in that mutant cell lines defective in various key components are available. These cells are derived from patients with serious inherited disorders, often associated with immunosuppression and elevated cancer frequency. This review focuses on major developments in the field of DNA repair that have been reported in the literature over the last year. Human DNA repair syndromes

genes and inherited

Fanconi’s anaemia (FA), ataxia telangiectasia (AT), xerodem-ra pigmentosum (XP), Cockayne syndrome (CS), and Bloom syndrome (BS) comprise a heterogeneous group of genetic disorders characterized by genome in-

1993, 5:424-433

stability and predisposition to cancer. They are known collectively as DNA repair syndromes on the basis of hypersensitivity to one or more DNA-damaging agents or mutagens: cells from FA and AT patients are uniquely sensitive to DNA crosslinking agents and ionizing radiation, respectively; XP and CS to ultraviolet (UV) light; while BS cells are moderately hypersensitive to several mutagens. Identification of the genes involved, through complementation of mutagen-hypersensitive cell lines from affected individuals, has made slow progress, largely due to the recalcitrance of human cells to the stable integration of exogenous DNA Rodent cells are more amenable in this respect, and genes corresponding to several genetic complementation groups of XP have been identified by complementation of W-hypersensitive rodent cell lines. However, recent reports have demonstrated the successful use of cDNA expression libraries constructed in vectors derived from Epstein-Barr virus (that replicate extrachromosomally and so circumvent the problems inherent in the transfection of human cells with genomic DNA), and notable advances have been made in characterizing genes involved in several DNA repair syndromes

[VI. The first FA cDNA has been cloned by complementation of the hypersensitivity to DNA cross-linking agents of an FA group C cell line. The cDNA sequence gives no clues as to the function of the encoded protein, but mutations in the coding sequence of both an FA group C cell line, as well as two previously unclassified FA patients, would seem to point to the FA group C complementing (FACC) gene as the primary defect [3=*]. How the FACC gene product acts in the process whereby the cell removes DNA interstrand crosslinks is still unclear. As cell fusion experiments have now extended the number of FA complementation groups from two to four [ 41, a considerable molecular compiexity of the repair pathway(s) is indicated.

Abbreviations AT-ataxia

424

telangiectasia; BS-Bloom syndrome; C%Cockayne Syndrome; FRCC--excision repair cross-species complementing; FA-Fanconi’s anaemia; 3-meA-3-methyladenine; Os-meC-Os-methylguanine; NER-nucleotide excision repair; 8-ohC-8-hydroxyguanine; PCNA-proliferating cell nuclear antigen; SSL-suppressor of a stem-loop structure; W-ultraviolet; XP-xeroderma pigmentosum.

@ Current

Biology

Ltd ISSN 0955-0674

Barnes, Lindahl and Sedgwick

DNA repair

This situation is also true for AT, where a defect in a gene on chromosome llq22-23, or possibly two to four closely linked genes, may give rise to the disease. In addition, there are apparently two complementation groups of the closely related ionizing radiation sensitive ‘Nijmegen breakage syndrome’ where patients do not display all the clinical symptoms of AT. A candidate gene for AT (group D) has been isolated that gives partial correction of the cellular phenotype [ 51. It remains to be established whether mutations in this gene correlate with the disease phenotype and indeed, whether AT gene product(s) are involved directly in the repair of DNA damage or some other aspect of the cellular response to ionizing radiation. A patient (46BR), with clinical symptoms in common with BS, was shown to have a biochemical defect in an extensively studied enzyme of DNA repair and replication, DNA ligase I [6]. Two deleterious mutations were identilied in different alleles of the DNA ligase I gene of 46BR cells [ 7**]. One of the mutant alleles produces an inactive protein, while the other encodes a partially active enzyme. As DNA ligase I most likely has an essential function, this leaky mutation would account for viability, as well as the cellular phenotype of retarded joining of Okazaki fragments during lagging-strand DNA synthesis a and hypersensitivity to DNA damage that is counteracted by excision repair pathways (Fig. 1). (a)

425

No coding mutations were found in the DNA ligase I gene of cell lines forming a single complementation group of BS [7**]. The precise molecular basis of an observed DNA ligation defect in these cells remains elusive, but could be due to mutational change in another DNA ligase [6], or in a protein that either directly or indirectly modulates DNA-joining processes. In the latter respect, a specific inhibitor of DNA ligase I has been detected in extracts of human cells [8], and DNA ligase I activity is regulated by the phosphorylation state of the enzyme [p] . The rather modest mutagen hypersensitivity of BS cells precludes complementation of this phenotype as a basis for gene cloning. However, the elevated sister chromatid exchange level of BS cells has been successfully corrected by microcell-mediated transfer of human chromosome 15 [lo*], prompting the search for a candidate gene that maps to this chromosome. Xeroderma pigmentosum, and nucleotide excision

Cockayne repair

syndrome

XP can be due to mutations in any one of at least seven different genes (AC) affecting the DNA incision step of nucleotide excision repair (NER; Fig. 1). The genetic complexity underlying the NRR defect of XP clearly demonstrates the concept that DNA repair processes involve the concerted action of multiple proteins (c)

(b) XP ladOR

XP hdorn

(XP-cl hdluu7)

GlyCOSyhM 1

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AP .ndonus!us* I

I’ a’- 111111111111111111;11111 ‘ c

6’

Phoaphodi.d.r... ‘bI’

I’

I’

*. Pd l/B

Pd c +PCNA(+RFC?)

Pd I

’ 3. 111111111111111 “

I

Lipa**

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Fig. 1. Schematic diagram of DNA excision repair pathways. co/i and (b) human ceils. k) Ubiquitous base excision repair

Nucleotide (x represents

excision repair of a cyclobutane an altered base).

pyrimidine

dimer

(a)

in

(a) E.

426

Nucleus

and gene expression

and also illustrates the enzymatic complexityltequired for the incision of damaged DNA An eighth ‘variant’ complementation group of clinical XP has no clear defect in NER, but may be deficient in ‘bypass’ replication of damaged nucleotides [ 111. The picture is further complicated by the coincidence of two other diseases with certain complementation groups of XP; CS in XP groups B, D and G, and trichothiodystrophy in group D 1121. This latter disease is primarily characterized by altered sulphur metabolism, with an NER defect in some patients apparently coriesponding to mutations in the W-D gene. This might indicate that such apparently unrelated diseases as trichothiodystrophy, CS and XP-D could arise from mutations at different sites in the same (Xp-D) gene. It is also clear that CS can occur without associated XP, in which case individuals do not show a predisposition to skin cancer. Furthermore, overall NER appears normal in CS cells, but preferential repair of the transcribed strand of active genes by a distinct sub-pathway of NER is affected. The human excision repair cross-species complementing ERCCG gene, isolated by complementation of a UV-hypersensitive rodent cell line, specifically corrects the preferential repair defect of CS group B cells and is mutated in a CS-B patient [ 13**]. These results will provide further insights into the inter-relationship of DNA repair with transcription, and the phenomenon of preferential repair. Four of the seven XP NER genes, corresponding to complementation groups A-D, have now been cloned; the XPCC gene is the latest to be identified [ 14*]. Although XF’CC cDNA specifically corrects an XP-C cell line, the molecular basis of the XP-C defect is not clear. Similarly, the human ERCW gene has been shown to confer normal UV resistance to XP-D cells [ 15.1, but again, deleterious mutations in the ERCC2gene of XP-D cells have not yet been reported. However, the primary defect in each case is likely to reside in genes directly involved in the repair of DNA damage and indeed, both XPCC and ERCCZ continue an emerging pattern in which XP cDNAs share homology with the RAD proteins known to be required for NER in yeast,

Nucleotide eukaryotes

excision

repair

genes

in other

Genes involved in NER are conserved amongst eukaryotes, and insights into the mammalian system may be gained by exploiting laboratory-derived mutant rodent cell lines [l6] and the versatility of the budding and fission yeasts. In Saccbaromyces cerfzvisiae, at least six genes (R4D-4, hHD10 and lUD14) encode proteins required for the incision step of NER They are represented, on the basis of varying degrees of sequence homology, amongst the NER genes of higher eukary otes, where there is only partial overlap between XP and at least 11 complementation groups of NER-defective rodent cell lines. That protein-protein interactions are required for the concerted action of these gene products is exemplified by the specific complex formed between &WI and WIO, which appears essential for the function of both proteins [ 17*,18*].

Several NER genes, including those corresponding to CS group B and XP groups B and D, encode highly conserved domains characteristic of ATP-dependent DNA and DNA/RNA helicases [ 191. The RAD3 protein of S. cerevisiae is the only eukaryotic NER gene product that has been shown experimentally to act as a helicase. This activity is inhibited by several types of damage in the DNA strand to which the RAD3 protein binds, suggesting that it translocates along the DNA until it locates a damaged site [20-l. Both R4D3 and its Scbizosaccharomyces pombe homologue radl5+ are essential for viability, suggesting that they have a role in cell proliferation and not solely in NER [21,22]. This is also the case for the ERCC3 homologues, SSL2 in S. cererukiae [23**,24*] and haylL,ire in Drosophila [25-l.

SSL2 was identified as a post-transcriptional suppressor of a stem-loop structure that blocked ribosome binding/scanning. A mutant ssl2 allele mimicking the mutation in the ERCC. gene of XP-B cells confers UV hypersensitivity but is viable, suggesting that SSLZ(and by inference, ERCCY) has a non-essential role in NER, as well as an essential function in translation initiation [23**], the latter being dependent on ATPase/helicase activity [ 24.1. Alternatively, SSL2/ERCC3 may perform a single essential role in translation initiation, such that mutations in SSL?/ERCC3 lead to poor expression of several genes, including one or more directly involved in NER [ 23**]. This ‘indirect’ model has also been evoked to explain the pleiotropic phenotype conferred by mutant alleles of haywire. The few viable alleles of haywire confer UV hypersensitivity, while the phenotype of mutants compromised for haywire function is reminiscent of XP-B/CS, and may include abnormalities of the central nervous system, ataxia, defective spermatogenesis and reduced lifespan [25*-l. As the first whole animal model for a human DNA repair syndrome, haywire gives novel insights into the relationship between the DNA repair defect and the cellular/clinical phenotype of XP/CS. Essential functions of many DNA repair genes would explain the rarity of associated human syndromes; presumably there would be few mutations resulting in an altered phenotype compatible with cell viability.

Nucleotide

excision

repair

in cell-free

extracts

The availability of an in vitro system that mediates NER in human cell extracts permits direct functional analysis of NER gene products. Fractionation of extracts from human cells has allowed the NER reaction to be resolved into discrete steps (Fig. 1): first, incision of the DNA backbone at the site of damage has been shown to require the XP-A protein and a human single-stranded DNA-binding protein, RPA; second, proliferating cell nuclear antigen (PCNA) is required for DNA polymerization or repair synthesis to fill in the gapped strand after removal of damaged nucleotides, implying that DNA polymerase E or 6 is involved [26**]. The involvement of PCNA in NER is supported by in vivo experiments demonstrating the formation of PCNA-DNA complexes correlating with un-

DNA repair

scheduled DNA synthesis upon UV irradiation of normal, but not XP-A, fibroblasts [27]. The identification of both damage-specific DNA-binding proteins [28,29] and putative damage-locating DNA helicases [19] as the products of eukaryotic NER genes, would indicate that there is functional, if not structural, overlap with the well characterized Escherichiu coli NER pathway (Fig. 1). Most striking is the observed removal of pyrimidine dimers in human cell extracts by a nucleotide excision mechanism that incises at precise sites both 5’ and 3’ to the damage and excises an oligonucleotide in an analogous, but not identical manner, to that of the E. coli NER enzymes [30**]. This property might be used to assay for NER gene products involved in excision and subsequent polymerization/ligation steps; as XP mutants are thought to be defective either at or before incision, they will not be informative for later steps in the NER pathway. The in vitro NER system has also been used to investigate

the action of the chemotherapeutically important DNAplatinating agent c&-diamminedichloroplatinum(II) (cisplatin). The clinical efficacy of this drug may be explained by the inefficient repair of the major DNA adduct that it generates [31*]. The high mobility group protein, HMGl 1321, and the HMG-box cisplatin-DNA structure-specific recognition protein, SSRPl [33], bind to structural distortions caused by the major cisplatin-DNA adducts. Binding of these proteins might interfere with repair of the cisplatin lesions and so potentiate the effect of this drug in the inhibition of DNA replication and resulting cell death. Base excision

repair

Enzymatic removal of damaged bases by DNA glycosylases, spontaneous depurination of DNA and exposure of DNA to ionizing radiation all result in the formation of non-instructional abasic (AP) sites. They are mutagenic and cytotoxic and must be efficiently repaired. Crude cell extracts of both E. coli and human cell lines repair AP sites mainly by replacing a single nucleotide [34*]. The major pathway of repair involves the sequential action of several DNA repair activities (Fig. 1). AP endonucleases rapidly incise the AP site on the 5’side of the damage and a DNA deoxyribophosphodiesterase activity will excise the 5’ deoxyribosephosphate remaining in the AP site. The single nucleotide gap is Iilled by a DNA polyrnerase, most likely polymerase-P in mammalian cells, and a DNA ligase. Ubiquitous DNA glycosylases excise various altered bases from DNA including uracil, 3-methyladenine (3-meA), 8hydroxyguanine (8-ohG) and thymine glycol. They are relatively small proteins and thus particularly amenable to structural analysis. Atomic structure determination combined with site-directed mutagenesis has recently allowed the identification of the catalytic centre and other functionally important regions of two DNA glycosylases. T4 ‘endonuclease V’ and ‘endonuclease III’ of E. coli, two inappropriately named enzymes, are both DNA glycosylases with associated chain-cleaving AP lyase activity. T4 endonuclease V cleaves one of the two sugar-phosphate bonds at cyclobutane pyrimidine dimers in DNA This sin-

Barnes, Lindahl

and Sedgwick

gle compact domain protein comprises three cr-helices but no P-sheet structure, and has an accessible concave surface with many positive charges that may make electrostatic contact with DNA The negatively charged Glu23 surrounded by basic residues forms the catalytic centre for both N-glycosylase [35**,36] and AP lyase activity [37]. Endonuclease III excises thymine glycol and cytosine glycol (cytotoxic oxidised pyrirnidines) from DNA and is the first ubiquitous DNA repair enzyme for which the three-dimensional structure has been determined. This iron-sulphur enzyme has a deep cleft separating two similarly sized domains. The 4Fe-4.S &and of novel pattern in a four-helix domain may position basic residues for interaction with DNA A seven residue phairpin may have a role in recognition of thymine glycol. Another DNA repair enzyme, the MutY glycosyiase, which excises adenine from mispaired 8-ohG:adenine base pairs in DNA, is also an Fe.5 protein with sequence similarities to endonuclease III and complete conservation of the g-hairpin and 4Fe-4S ligand spacing. Involvement of a conserved glutamic acid residue (Glu112) surrrounded by several basic residues in the six-helix domain of endonuclease III is implicated in the N-glycosylase reaction [38**] and suggests that T4 endonuclease V and endonuclease III may have evolved similar mechanisms of N-glycosylase activity. A pyrimidine dimer specific DNA glycosyktse activity related to T4 endonuclease V, previously described only in Micrococcus luteus, has now been found in the lower eukaryote, S. cerevtitie. This is the first organism in which both direct reversal and base excision repair, in addition to nucleotide excision repair, of pyrimidine dimers in DNA has been observed [3P]. Although such an activity has not been detected in higher eukaryotes, the T4 enzyme applied to UV-irradiated mouse skin in liposomes removes pyrimidine dimers and prevents radiation-induced immunosuppression. The generation of pyrimidine dimers in DNA is thus implicated as the primary event in W-induced immunosuppression. [40]. The ubiquitous uracil-DNA glycosylase prevents elevated mutation rates caused by spontaneous hydrolytic dearnination of cytosine residues in DNA The E. coIi, Streptococcus pneumoniae, herpes virus, yeast and human enzymes exhibit strong sequence conservation. In fact, uracil-DNA glycosylase is one of the most highly conserved proteins (56 % identity) between E. coli and human cell nuclei [41,42]. There has been considerable confusion in the literature with regard to the mammalian enzyme, because of reports on apparent cryptic uracil-DNA glycosylase activities of two other proteins. It has been claimed that the 37kDa subunit of glyceraldehyde-3-phosphate dehydrogenase is able to cleave uracildeoxyribose bonds in DNA [43]. However, the authentic mammalian uracil-DNA glycosylase [ 41,421 has a l-10 million-fold higher specific activity than the subunit of glyceraldehyde-3-phosphate dehydrogenase. It is difficult to evaluate from these data whether preparations of glyceraldehyde-3-phosphate dehydrogenase possess an intrinsic, extremely weak activity on uracil-contaming DNA or are contaminated with approximately 0.0001% uracil-DNA glycosylase. In a similar vein, an apparent weak activity on uracil-containing DNA has been

427

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and gene expression .

reported in preparations of a mammalian protein belong ing to the cyclin A family 1441. Neither glyceraldehyde3phosphate dehydrogenase nor cyclin A show any detectable sequence similarity with uracil-DNA glycosylase. It seems unfortunate that the latter proteins are referred to as potential uracil-DNA glycosylases, as these cryptic reactions are unlikely to be of any biological significance, and possibly only reflect in vitro artefacts. Major cellular AP endonucleases from several mammalian, Drasophilu and bacterial sources show conservation of primary sequence and function. They all possess AP endonuclease and 3’ phosphodiesterase activity and some (but not the mammalian enzymes) have associated exonuclease activity [45,46]. X-ray diffraction analysis of recently crystallized exonuclease III, the major AP endonuclease of E. coli, should define structural features that determine these different DNA repair activities [47]. An intriguing finding was that the human AP endonuclease (the HAPl/APE protein) is identical to the ubiquitous nuclear Ref-1 protein (redox factor). Ref-1 may regulate the activity of the AP-1 transcription factor (Jun-Fos heterodimer), which in turn controls expression of a large number of genes. Reduction by Ref-1 of a conserved cysteine in the DNA-binding domain of AP-1 stimulates its binding to regulatory elements. Thus, Ref-l/HAPl/APE could perhaps be a component of signal transduction processes that regulate eukaryotic gene expression in response to cellular oxidative stress. In this regard, it might be expected that an AP endonuclease should exhibit reducing activity as it is important to avoid the risk of DNA-protein cross-linking at AP sites due to conversion of the base-free deoxyribose residue to a reactive free aldehyde form. The exact nature of the connection (if any) between oxidative signaling, DNA repair and transcriptional control is not yet clear [48*]. A comparison may be made here with the oxidative stress response of E. coli in which the DNA-binding capacity of the OxyR transcription factor is determined by its redox state [49]. Multiple cellular defences mutagenic 8-hydroxyguanine

protect

against

8-ohG (also called 7,8 dihydro-8-oxoguanine) in DNA mispairs with adenine resulting in G:C +TA transversions, and is one of many DNA modifications that results from oxidative damage and y-irradiation. Three mechanisms of excluding 8-ohG from DNA maintain a low spontaneous mutation frequency in E. coli [50]. One mechanism of protection (analogous to the degradation of dUTP by a specific dUTPase) is the MutT dGTPase, which degrades 8-ohdGTP much more efficiently than dGTP in the nucleotide pool, preventing its incorporation into DNA [51**]. When 8-ohG arises in DNA as a result of direct DNA damage and is paired with cytosine, it is excised by the MutM or Fpg DNA glycosylase [ 52,531. This enzyme also excises cytotoxic formamidopyrimidines (imidazole ring opened purines) from DNA [ 541. When 8-ohG is paired with adenine as a result of adenine misincorporation instructed by an oxidized DNA template, the adenine residue is excised by the MutY glycosylase 155.1. MutY is also active on AG mispairs but its primary function

in viva appears to be the repair of 8-ohG:A mismatches

[ 561. Thus, any ad enine incorporated opposite an 8-ohG will be removed; ultimately a correct cytosine will be incorporated, whereupon the MutM glycosylase can excise the oxidized guanine residue. An activity similar to MutT has been reported in mammalian cells [57*], which also have a formamidopyrimidine-DNA glycosylase, indicating a conserved system of defence against mutagenic 8-ohG residues. Repair and tolerance damage

of DNA methylation

Methylating agents alkylate cellular DNA at many sites. The major hamlful modified bases are 3-meA and OGmethylguanine (06-meG). Adaptive responses induced specifically by methylating agents occur widely in bacterial species and several DNA repair enzymes including 3meA-DNA glycosylase, 06-meG-DNA methyltransferase and methylphosphotriester-DNA methyltransferase are induced. The conservation of these responses in bacteria suggests the widespread occurrence of methylating agents particularly in the soil environment [58]. A specific response to methylation damage is not apparent in yeast or mammalian cells but has been detected in Rspergillta nidttlam, a soil organism. This is the first demonstration of a specific adaptive response and DNA-methylphosphotriester repair in a eukaryote [ 59.1. The adaptive response of E. coli is known to be regulated by the Ada protein, which is converted into a strong transcriptional activator by self-methylation on repair of methylphosphotriesters in DNA. The repair of these innocuous lesions in A nidulans indicates that a similar induction mechanism also exists in this ftlamentous fungus. The regulatory amino-terminal domain of the E. coli Ada protein contains a single essential zinc atom. Four conserved cysteines are proposed to serve as the zincbinding site, including Cys69, which accepts the methyl group from a methylphosphotriester. A specific role for the zinc ligand in conformational switching and activation of this protein as a transcriptional activator has been proposed [GO*]. 06-meG-DNA methyltransferases of bacteria, yeast and mammalian cells directly demethylate 06.meG in DNA and in so doing protect cells against the mutagenic and toxic effects of this methylated base. Moreover, they reduce the frequency of spontaneous mutations (G:C +A:T transitions) in all these organisms indicating the occurrence of a natural metabolite that alkylates DNA [61,62]. Epigenetic control of gene expression may result in the absence of the methyltransferase from certain mammalian cells and an accompanying lack of repair of 06-meG in DNA; this could be a critical determinant in cancer initiation by alkylating agents. Recently, this prediction has been substantiated by the targeted overexpression of the human alkyltransferase gene in the thymus of transgenic mice, which strikingly reduced the incidence of lymphomas induced by N-methyl-N-nitrosourea [63**] : 06-meG in DNA is both cytotoxic and mutagenic but it is unclear how it kills cells. In the absence of repair by 06-meG-DNA methyltransferase, repeated attempts

DNA repair

to process 06-meG in DNA by other mechanisms, possibly by mismatch correction, may result in cytotoxicity [64]. A thymine-DNA glycoslase function in human cell extracts acts at G:T 1651 and possibly also at 06-meG:T [66] mispairs. DNA replication repeatedly incorporating thymine opposite Oh-meG followed by removal of the poorly paired thymine may initiate the lethal effects of 06. meG in DNA Mammalian cell lines that tolerate 06-meG and 6thioguanine in DNA but not other types of DNA damage have a mutator phenotype [ 641. A human activity that binds to G:T mismatches [67] is absent from two methylation-tolerant cell lines and implies that their resistance to Oh-meG toxicity and mutator phenotype may result from a defect in mismatch repair [68]. The precise role of the G:T binding protein has still to be defined. A separate less-specilic mechanism of tolerance conveys resistance to methylating and other DNA-damaging agents. A cloned mouse gene product allows error-prone DNA replication to proceed on damaged templates providing a common means of tolerating a variety of types of DNA damage [691. Until recently, the harmful effects of methylated bases other than 06-meG had not been evaluated in mammalian cells due to the lack of cell lines specifically deficient in their repair. Of particular interest is 3-meA in DNA, which is known to be a cytotoxic DNA lesion in bacteria. 3-meA-DNA glycosylases including the E. coli Tag and AlkA proteins and the rat APDG enzyme excise 3-meA from DNA Expression of genes encoding these enzymes in various mammalian cell lines has now been shown to convey resistance to cell killing by methylating agents [70,71]. Resistance conferred by the Tag protein, which specifically excises only 3-meA from DNA, clearly demonstrates that this lesion is potentially cytotoxic in mammalian cells as well as in E. coli. The Tag protein also conveys limited protection against mutation induction by methylating agents, suggesting that 3meA is also a premutagenic lesion in mammalian cells. These observations indicate that DNA repair of 3-meA can be a limiting factor in cellular resistance to alkylating agents.

Single- and double-strand mammalian cells

break

repair

in

Single-strand and double-strand breaks are major DNA lesions generated by ionizing radiation and radiomimetic mutagens, and several genes complementing ionizing radiation sensitive mutants of E. coli, S. cerezbiae and S. pombe have been cloned. The genetic study of ionizing radiation hypersensitive rodent cell lines and their overlap with the complementation groups of AT has been less systematic than in the case of the W-hypersensitive lines and XP [I6], and only one human gene affecting ionizing-radiation sensitivity, XRCC1 (X-ray cross-species complementing), has been cloned by this means to date [72], although others have been mapped [73]. The most striking phenotype resulting from defective XRCCl gene function is, in fact, cellular hypersensitivity to methylating agents and anomalous sensitivity to sister chromatid exchange induction, characteristics more reminiscent of the DNA repair defect of BS and 46BR.

Barnes, Lindahl

and Sedgwick

In vitro systems have established that the efficient repair of single- and double-strand breaks generated by ionizing radiation requires functions in addition to DNA ligation 1741 and have demonstrated that DNA repair is temporarily inhibited by the binding of poly(ADP-ribose) polymerase at DNA strand Interruptions [75*]. Singlestrand breaks also arise during the course of excision repair processes (Fig. l), but only base excision repair, and not NER, is inhibited by the polymerase [76 1. It seems likely that an association of multiple gene products at a damaged site or a replication fork may physically exclude poly(ADP-ribose) polymerase from transient strand breaks that arise during NER and laggingstrand DNA synthesis. DNA double-strand breaks also arise as intermediates in recombination pathways such as the V(D)J joining of immunoglobulin and T cell receptor genes. Intriguingly, severely immunodeficient mice homozygous for the scid mutation and defective in V(D)J joining are also hypersensitive to DNA damage induced by ionizing radiation. A recent report has shown that in aborted joining of V and J coding regions, the broken DNA ends are covalently sealed as hairpins [77**]. This indicates a role for such unusual intermediates in V(D)J joining (and possibly double-strand break repair), and that failure to resolve these structures may lead to the scid defect. This promises to be an unexpected source of information on double-strand break repair in higher eukaryotes.

Relationship processes

of DNA repair

to other

cellular

An immediate consequence of failure to remove DNA damage can be to stall the cell’s DNA replication, transcription and recombination machinery, with longterm consequences for mutagenesis, gene expression and chromosomal integrity. The inter-relationships between DNA repair and other aspects of DNA metabolism formed the major focus for a previous review in this series [78]. More recent reports have increasingly emphasized a connection between DNA repair and the cell cycle. The importance of DNA repair to cell cycle progression, as first illustrated by the rad9 mutant of S. cede vfiiae, has now been coniirmed by the demonstration that radiation-sensitive mutants of S. porn&e also fail to arrest in Gz after DNA damage. Furthermore, their rediscovery amongst mutants that enter mitosis when DNA synthesis is blocked suggests that there is considerable overlap between the pathway that arrests mitosis upon detection of DNA damage, and the mechanism coupling mitosis to the completion of DNA replication [79*,80*]. In mammalian cells, ionizing radiation transiently inhibits DNA synthesis by G1 and G2 arrests; arrest in G1, but not Gz, apparently being dependent on accumulation of wildtype p53 protein [81*]. An irradiation-induced increase in p53 was not detected in cells from AT patients and apparently accounts for their failure to arrest in G1. Moreover, the growth arrest and DNA damage-inducible GADD45 transcript was not induced, possibly because p53 acts

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directly on the GADD45 gene to which tt binds. The AT gene product(s) apparently acts upstream of ~53 in a pathway that btings about G1 arrest and repair of DNA damage before entry into S phase [82**]. The repercussions of failure to operate the G,-S checkpoint in response to DNA damage, for example in AT or ~53 mutant cells, would have far-reaching consequences in genomic mstability and tumourigenesis 183,841. Loss of this checkpoint function in cells with altered ~53 would also provide a rationale for the observed preferential killing of tumour cebs by ionizing radiation and chemical agents in cancer therapy. Conclusion Evolutionary conservation of DNA repair pathways emphasizes the fundamental importance of cellular defences that maintain the integrity of the genetic material and validates the strategy of relying on well characterized microbial model systems to elucidate DNA repair processes. Recent progress in isolating genes underlying human DNA repair disorders promises further insights into cellular responses to DNA damage. Cloned genes will also allow the generation of animal models by targeted gene disruption, which should provide answers to one of the more intriguing questions posed by the study of DNA repair; how does an inherited defect in a DNA repair enzyme translate into the bewildering array of symptoms (including predisposition to certain cancers> that characterizes the associated clinical disorder? References

and recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1. BARNES DE: Damage-Limitation Exercises. Nature 1992, 359:12-13. 2.

HOEIJ~~AKEW JHJ, Boors~~ D: DNA Repair: Two Pieces of the Puzzle. Nulure Gener 1992, 1:313314.

3.

STRATHDEE CA, GAWH H, SHANNON WR, BUCHWA~D M: Cloning of cDNAs for Fanconi’s Anaemia by Functional Complementation. Nature 1992, 356:763-767. A functional compiementation method was used to clone cDNAs that correct the defect of FA cells from complementatlon group C. The CDNAs encode alternatively processed transcripts of a new gene that is mutated in group C patients. 4. STRATHDEECA, DUNCAN AMV, BUCHV&ID M: Evidence for at Least Four Fanconi Anaemia Genes Including FACC on Chromosome 9. Nafure Genel 1992, 1:196-198. 5. KAPP I.N, PAINTER RB, YU L-C, VAN tiN N, RICHARD CW ILI, JAMESMR, COX DR, MURNANEJP: Cloning of a Candidate Gene for AtaxIa-Telangiectasia Group D. Am J Hum Genet 1992, 51:45-54. 6. LNDAHL T, BDE: Mammalian DNA Ligases. Annu Rev Biocbem 1992, 61:251-281. ..

7.

BARNES DE, TOMKIN~ON AE, LEHMANN AR, WEBSTER ADB, T: Mutations in the DNA Ligase I Gene of an Individual with Immunodeficiencies and Cellular Hypersensitivity to DNA-Damaging Agents. Cell 1992, 69495503. Two mis-sense mutations were identUied in different alleles of the DNA ligase I gene of a mutant human fibroblast strain; these correlate with both a biochemical defect in the enzyme and the cellular phenotype. ..

IINDAHL

8.

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