Cellular responses to DNA damage in Drosophila melanogaster

DNA Repair

ELSEVIER

Mutation Research 364 (1996) I33- 145

Minireview

Cellular responses to DNA damage in Drosophila melanogaster

1

Ruth L. Dusenbery ‘, P. Dennis Smith b3* ADepartment b Depurtment

Keywords:

Drosophila;

of Chemistry.

of Biological

DNA repair; Mutagen

Wayne State Unicersity,

MI 48202. USA

Detroit,

MI 48202, USA

sensitivity

1. Introduction Correct expression of cellular genomes and their faithful transmission to the next generation are critically dependent on efficient mechanisms of DNA repair in all organisms. DNA repair mechanisms can be classified into those that identify DNA alterations and restore the normal DNA structure by reversing the alteration and those that identify the alteration and excise it, restoring the DNA structure by resynthesizing the excised region (for a comprehensive review of these topics, see [l]). In the last several years, a veritable explosion of information concerning cellular responses to DNA damage in eukaryotic organisms has both deepened our knowledge of the mechanistic aspects of DNA repair processes, including direct coupling to the transcription complex, and broadened our understanding of how control of the cell cycle by the tumor suppressor gene p.53 is used to assist repair of moderate amounts of DNA damage or eliminate

* Corresponding author. Tel.: (313) 577-3214; Fax: (313) 577. 6891; E-mail: [email protected] ’ This review is dedicated to the memory of Professor James B. Boyd, University of California-Davis, a pioneer in the study of DNA repair in Drosophila. 0921.8777/96/$15.00 Copyright PII SO921-8777(96)00026-2

Detroit,

Sciences, Wayne State University,

highly damaged cells from the population through programmed cell death (apoptosis). The power of genetic systems to elucidate not only the nature of the genes involved in DNA repair pathways, but also the control of gene activity, the interactions between gene products, and the impact of these processes on the physiology of the organism has been amply demonstrated by investigations using the budding yeast, Succharomyces cerevisiae [ 11. Investigations using the fruitfly, Drosophda melanogaster, can extend our understanding of how these processes function in a sexually reproducing, multicellular animal system, which may provide more direct insight into how the human system works. Analysis of DNA repair functions in Drosophila have generally followed two experimental approaches: a genetic approach, initially focused on the isolation of mutagen-sensitive strains [2], which led to the identification of genes involved in DNA repair, and a complementary biochemical approach, which sought to define the mechanistic aspects of DNA repair processes 131. The advent of cloning technology provided even more powerful approaches to isolating and analyzing the genes involved in DNA repair. The purpose of this short review is to highlight the progress made in understanding DNA repair processes in Drosophila since the last major review by Boyd et al. in 1987 [4] and to emphasize the

0 1996 Elsevier Science B.V. All rights reserved.

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R.L. Dusenhrr,~. P.D. Snlith / Mutation Reseurch 364

extraordinary degree of both functional and structural homology that Drosophila repair genes share with both bacterial and other eukaryotic counterparts.

2. DNA damage reversal

2. I. DNA damage reversal by photoreactivation UV irradiation induces two principal types of DNA helix distorting alterations: cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). Early biochemical studies in Drosophila demonstrated photoreactivation of CPDs [4]. Boyd and Harris [5] reported that inactivation of a gene on the second chromosome, phr. at genetic map position 56.8 abolished this photorepair activity. The phr cDNA has been cloned and localized to cytogenetic position 44C-D. It encodes a translation product of 62 kDa, which exhibits a 60% amino acid identity with the CPD photolyase from goldfish [6]. CPD photolyase genes have been isolated from 12 other organisms, and have been grouped into two classes based on deduced amino acid sequence homology: Class I includes genes from microorganisms, except an archaebacterium, which is included in Class II, along with genes from higher eukaryotes [7]. Drosophila CPD photolyase activity is high in embryos and adult ovary, but Northern analysis indicates that phr mRNA is abundant only in the ovary. These data suggest that CPD photolyase is a maternal product specifically elaborated to provide protection to embryos prior to the development of the more UV-resistant adult cuticle. CPD photolyase does not repair (6-4) PPs, and for a number of years no direct evidence existed as to the photoreversal of this lesion in any organism. This was not surprising, since a molecular rearrangement takes place in formation of the 6-4 PP, and breaking the sigma bond between the linked pyrimidines does not restore their original structure [l]. In 1993, Todo and co-workers [8] made what Sancar [9] referred to as “Tl~e ,first unexpected and exciting discovery in the ‘New Age’ history of photolyase”, when they detected a photolyase activity in Drosophila cell-free extracts that was specific for the repair of UV-induced (6-4) PPs. This activity was highest in ovary

t I9961133- I45

and embryo, similar to that of CPD photolyase. The cDNA is 1960 bp long, with a single long open reading frame of 540 amino acids, and a calculated molecular mass of 62.9 kDa [7]. The cDNA hybridizes to band 39 B-C on the cytogenetic map. Surprisingly, the Drosophila (6-4) photolyase exhibits greater sequence identity to the microbial Class I CPD photolyases (20-22%) than to the Class II enzymes. including the Drosophila CPD photolyase, and, unexpectedly. 20-24% sequence identity with the blue-light photoreceptors (cryptochrome) of Arabidopsis thaliana and mustard [7]. The sequence data are consistent with binding sites for folate and flavin chromophores, and a mechanism for (6-4) photolyase activity via photo-induced electron transfer was proposed by Sancar [9]. Todo et al. [7] also report identification of a human homolog with 40% sequence identity to the Drosophila (6-4) photolyase, although it is not known whether this gene product possesses either (6-41 photolyase or blue light photoreceptor activity. Kim et al. [IO] report the presence of (6-4) photolyase activity in Xenopus fae~~is and rattlesnake tissues. It will be of interest to determine whether all of these proteins form a superfamily of blue light-activated signal transduction elements.

2.2. DNA damage reversal by alkyltransferases

Although the in vivo source for alkylation damage to DNA is not well understood, alkylation damage at several sites in DNA is efficiently repaired. In E. coli, one of the main pathways involves the action of two gene products classified as 06-methylguanineDNA methyltransferases (06-MGT), which stoichiometrically transfer methyl groups from DNA to a cysteine residue in the repair protein. O”-MGT I, a 39-kDa polypeptide encoded by the ada gene, is responsible for the removal of methyl groups from the O6 position of guanine, the 0’ position of thymine and methylphosphotriesters in the DNA backbone [I]. 06-MGT II, a 19 kDa polypeptide encoded by the ogt gene, removes methyl groups from the 0’ position of guanine and the 0” position of thymine, but not from methylphosphotriesters [l]. A single 2 1.7 kDa 06-MGT activity has been identi-

R.L. Dusenben, P.D. Smirh /Mutation

fied in human cells [l 11. It has a very low sequence homology to the bacterial enzymes and does not act on alkylphosphotriester substrates. In Drosophila, Deutsch and co-workers [12,13] report two polypeptides of 30 kDa and 19 kDa molecular mass, which are present in extracts of adults and other stages, except embryos. and accept radiolabeled methyl groups from N-7 methylguanine, N-3 methyladenine and O”-methylguanine residues by transfer to a cysteine residue in the Drosophila polypeptides. A polyclonal antibody, produced against the 19-kDa methyltransferase protein from E. cc&, also detected in western blots a I9-kDa species from first and third instar larval, pupal and adult extracts. The 30-kDa species could only be detected in western blots when extracts were prepared in the presence of numerous protease inhibitors. Unlike the case of 06-MGT I in E. co/i, neither of the Drosophila activities are induced by pretreatment with alkylating agents. No other 06MGT homologs that have been identified in either prokaryotic or eukaryotic organisms have the capability of dealkylating N-7 methylguanine or N-3 methyladenine residues. Since no protein sequence data have been obtained for the putative Drosophila 06-MGT polypeptides, nor have the gene(s) encoding them been cloned, both the structural relationship between these two polypeptides and the possible relationship to other 06-MGT proteins remain unclear. Evidence obtained in our laboratory does not support a model in which a DNA-alkyltransferase provides the major pathway for repair of alkylation damage in Drosophila. Substantial amounts of unscheduled DNA synthesis (UDS) are observed in DNA repair-proficient control cell lines following treatment with a variety of DNA damaging agents, including ionizing radiation, 254 nm UV. the monofunctional alkylating agents methylmethansulfonate (MMS) and methylnitrosourea (MNU), and the difunctional alkylating agent diepoxybutane (DEB) [14]. Mutants that are hypersensitive to the lethal and mutagenic effects of these agents do not exhibit UDS activity in response to the same dose levels that elicit strong UDS responses in control cells. The observed UDS activity is consistent with an excision repair mechanism for all classes of lesions, including alkylated bases.

Resear& 364 (lYY6) 133-135

135

3. Excision repair

3.1. Base

excision

repair

Base excision repair (BER) is an important pathway for the removal of modified bases which have structures that do not extensively distort the DNA duplex, but may be mutagenic through miscoding events during replication. BER involves: removal of the modified base, incision of the resulting abasic site. repair synthesis, and religation of the DNA backbone. 3.2. DNA glycosylases Two classes of enzymes which initiate BER have been identified: glycosylases that remove the modified base and leave a free abasic site and glycosylases with associated apurinic/apyrimidinic (AP) lyase activity, which remove the modified base and go on to catalyze cleavage of the phosphodiester backbone at the AP site [I]. The literature contains conflicting reports concerning the presence of DNA glycosylase activities in Drosophila. Deutsch and co-workers 115,161 have reported an inability to detect either uracil DNA glycosylase or glycosylase activity for methylated bases in extracts from a number of developmental stages. In contrast, using two separate assay methods, Morgan and Clebek [ 171 found evidence for uracil-DNA glycosylase activity in extracts from Drosophila embryos and third instar larvae. To date. this enzyme has not been purified nor have genes encoding uracil DNA glycosylase activity been identified. The only other report of glycosyiase activity in Drosophila was made by Breimer [18]. An activity specific for oxidized thymine residues, associated with a 40-kDa protein, was partially purified from embryos. Unfortunately, no further characterization of this enzyme has been published. Caution must be exercised in drawing conclusions about the importance of the base excision repair pathway in Drosophila from these limited in vitro assays. 3.3. AP endonucleases Abasic (apurinic/apyrimidinic; AP) sites, one of the most frequently formed lesions in cellular DNA

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P.D. Smith/Mutation

[ I I, pose a danger to the readout of genetic information from DNA with the potential to arrest the replication complex or act as non-instructive lesions to cause mutations through replication errors. In the absence of DNA replication, AP sites can lead to errors in the transcriptional readout of cellular genes. The primary repair process for AP sites involves hydrolytic cleavage of the phosphodiester bond 5’ to the AP site by an AP endonuclease, leaving a normal 3’-OH and a S-terminal phosphate. However, the abasic residue must be removed by a 3’-phosphodiesterase. In E. coli, nearly all measurable AP endonuclease activity is due to the 28kDa Exo III enzyme, encoded by the xth gene (90%), and the 33-kDa Endo IV enzyme, encoded by the nfo gene (10%). Exo III also exhibits robust 3’-phosphodiesterase, 3’-phosphomonoesterase and 3’-S-exonuclease activities, as well as ribonuclease H activity [I]. Eukaryotic members of the Exo III family are generally larger proteins with the highly conserved AP endonuclease domain in the C-terminal portion, and unique activities associated with the variable N-terminal portion of the molecule. Drosophila Rrp 1 protein exhibits 33% sequence identity with Exo III in the 278 amino acid C-terminal region [19]. In addition, Rrpl exhibits a higher ratio of AP endo to 3’-repair activities compared to Exo III. The values for Rrpl relative to Exo III are: 2.70 for S-AP endonuclease, 0.41 for 3’-phosphodiesterase, 0.30 for 3’-5’ exonuclease, and 0.018 for 3’-phosphatase activities [20]. However, Gu et al. [21] demonstrated that expression of the cloned Drosophila Rrpl protein in E. coli xth nfo double mutant cells can functionally rescue these cells from extreme sensitivity to killing by both oxidative and alkylation-induced DNA damage, confirming a conserved cellular role for the Rrpl enzyme in base excision repair. Detailed structural information from X-ray crystallographic analysis of both E. coli Exo III and the human HAP1 proteins has provided insight into the mechanism of phosphodiester bond cleavage by these enzymes and the residues that are important for catalytic activity [22,23]. The ternary complex of Exo III with Mn’+ and dCMP reveals the position of catalytic residues: Asp-229, His-259 and a water molecule form the catalytic triad, and Glu-34 and Asp-258 stabilize the metal ion. These critical

Research 364 f 19961 133-145

residues are absolutely conserved among all of the members of the Exo III family, including Rrp 1, and other residues predicted to be important for substrate binding also show absolute sequence conservation in all members of the Exo III family analyzed to date. In keeping with the high degree of amino acid conservation, mutation of the putative metal binding Glu-461 in Rrpl, which is the homolog of Glu-34 in Exo III, decreases the AP endonuclease activity of the mutant Rrpl protein, expressed in an E. coli xrh cfo background, by 30-fold relative to the wild-type enzyme, and also decreases the 3’-phosphodiesterase and 3’-phosphatase activities of the mutant protein 1241. The Glu-461 mutation fails to provide resistance to either oxidative or alkylation-induced DNA damage to the host cells. Mutations at three other amino acids in Rrp 1, T462A. K463Q, and L484P. provide resistance to alkylation damage, but not oxidative damage when expressed in the xth nfo background. Based on this extraordinary conservation of catalytically important residues, it is predicted that mutations in Rrpl residues Asp-644 and His-670 would abolish enzymatic activity associated with the C-terminal AP endo segment, and that mutations in Asp-669 would also produce a significant reduction in all these activities. The extended N-terminal domain of Rrpl has two activities: an ATP- and single-strand binding protein-independent DNA strand transfer activity, which is unique among DNA strand-exchange proteins, and a single-strand DNA renaturation activity [25]. The biological functions for these catalytic activities are not known, but Rrpl appears to participate in recombinational repair and homologous recombination [26]. Genetic mapping of the Rrpl locus and generation of mutant strains are critical steps in further understanding of the biological role of the multifunctional Rrpl protein. Sander and co-workers have recently determined that Rrpl is located within the region covered by Df (2L) JS17, corresponding to the cytogenetic region 23Cl-2 to 23El-2 on the second chromosome (M. Sander, personal communication). No previously identified mus or mei genes are located in this region of the genome. A second unusual class of Drosophila proteins which exhibit AP endonuclease activity have been cloned recently. Two proteins, AP3/PO [27,28] and

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S3 [29,30], function as ribosomal proteins, but have AP lyase activity in vitro, specifically cleaving AP sites by a p-elimination reaction. AP3 was isolated on the basis of its cross reactivity with an antibody raised against a human AP endonuclease, but it has 66% amino acid sequence identity with the human PO gene, which is a putative ribosome-associated protein, 66% identity with rat and mouse PO proteins, and 52% identity with the yeast A0 homolog. Association of the human PO protein with a DNA damage response is inferred because it is induced by a variety of antitumor agents, and is overexpressed in Mer- tumor cell lines deficient in O”-MGT [30]. AP3 is located at 79CD on the third chromosome near several mus genes [4]. The S3 protein was cloned using the cDNA from the rat S3 gene [29]. The cloned gene encodes a putative protein of 27.5 kDa, which contains a nuclear localization signal KKRK [29]. Antibody localization indicates that it is 70% cytoplasmic and 30% nuclear [30]. It cytogenetic location is 94F/95A on the third chromosome [30]. Andersson et al. [31] have determined that the gene encoding S3 is the Minute gene M(3)95A, which is homozygous lethal, and not one of the mus or mei genes. Early reports of two other AP endonuclease activities partially purified from tissue extracts, referred to as APl and AP2, exhibiting AP lyase activity,

Table 1 Correlations

between NER genes in representative

have not been confirmed by isolation of the structural gene for these putative activities [32]. One of them may be related to the AP3 gene product [30].

3.4. Nucleotide

excision repair

Nucleotide excision repair (NER) is used to remove most types of bulky lesions from genomic DNA. Mutations leading to UV sensitivity identified E. coli UUY genes, S. cereuisiae RAD genes belonging to the RAD3 epistasis group, and human genes associated with the xeroderma pigmentosum (XP) syndrome. Only a few mus and mei genes exhibit significant UV sensitivity: mei-9, mus201 and mus310 [4]. In vitro reconstitution experiments with purified proteins have demonstrated that six E. coli proteins are sufficient to complete the process for CPD lesions: UvrA, UvrB, UvrC, UvrD, DNA polymerase I, and ligase [33]. Similar reconstitution experiments using human proteins [34,35] demonstrate that twelve proteins ( * 30 polypeptides) are required: XPA. XPG, TFIIH (including XPB and XPD), XPC, ERCC 1/XPF, IF7, replication protein-A (RP-A), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), DNA pol E, DNA ligase I, and possibly XPE.

eukaryotes

H. sapiens

S. cerevisiae

Function of gene product

D. melanogaster

XPA XPB XPC XPD XPE XPF(ERCC4) XPG ERCC 1

RAD14 SSL2 (RAD25) RAD4 RAD3

DNA damage recognition 3’.5’ DNA helicase Unknown 5’-3’ DNA helicase DNA damage recognition? Endonuclease. 5’ of lesion Endonuclease, 3’ of lesion Endonuclease. 5’ of lesion Pol II basal transcription Pol II basal transcription Unknown WD-repeats Complex with subunits TFIIK Repair transcribed strand Repair silenced chromatin Repair silenced chromatin Repair silenced chromatin

DmXPA DmXPB(hayf DmXPC DmXPD

P44 p62 ~52 MAT1 CSB(ERCC6)

HHR23A HHR23B

RAD 1 RAD2 RADIO SSLl TFBI TFB2 TFB3 RAD26 RAD7 RAD16 RAD23

DmXPF (mei-9)

lodestar?

138

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The current model for mammalian NER includes the following steps: (1) recognition of the lesion by XPA, RP-A and possibly XPE; (2) recruitment of proteins to the site of the lesion to open the duplex, including the XPB and XPD subunits of TFIIH, which unwind the structure in opposite directions; and (3) incision 5’ to the lesion by the XPF/ERCCl and 3’ to the lesion by XPG [36]. The gap is then filled and sealed by DNA polymerase E, PCNA, RPC and DNA ligase. Table 1 lists the genes currently recognized to participate in NER in human, yeast and Drosophila cells. To date, only one of the 29 Drosophila mutagen-sensitive genes, mei-9, has been shown to have structural homology to NER genes. It is anticipated that cloning of additional Drosophila genes will identify more. The Drosophila XPA/RAD14 homolog, DmXPA. has been cloned [37] and shown to be capable of partially rescuing human XPA cells [38]. The putative protein product has 296 amino acids, with a calculated molecular mass of 34 kDa. Homology to XPA in the N-terminal portion of the protein is limited to the nuclear localization signal region (3 I%> and a short polyglutamate region. A more extensive homology of 56% is found in the C-terminal 173 amino acids. This region contains 7 precisely located cysteine residues, four of which are predicted to form a zinc-finger motif in all homologs cloned to date, including the S. cereLisiae RADl4 gene. Its role in NER appears to be in damage recognition, as determined by the in vitro reconstitution experiments [34,35] DmXPA has been mapped to 3F6-8 at the distal end of the X chromosome, close to, but distinct from, the mei- mutagen-sensitive locus [38]. No fly strains deficient for the structural gene have been reported. DmXPA+ mRNA is expressed throughout fly development, and is more abundant in the thorax and abdomen than in the head of adult flies. However, immunodetection shows high protein concentrations in the central nervous system (CNS) of both embryos and adults. This is consistent with the CNS defects observed in some XPA patients, which have been linked to accumulation of oxidative damage in DNA in the absence of NER activity [39]. The fly XPB homolog, DmXPB. was identified independently by two groups. Koken et al. [40] used low stringency hybridization with probes from the

Research 364 (19961 133-145

human ERCC3/XPB gene. Mounkes et al. 1411 used genomic clones from a region which uncovered the gene for the neurological mutant, haywire, to screen a cDNA library and found that haywire was the DmXPB homolog. The DmXPB gene product exhibits N 70% amino acid identity with human XPB protein, especially in the DNA binding region and the 7 helicase domains. These regions also have 50% homology to S. cereuisiae Ss12/Rad25 protein, consistent with its role as a 3’-5’ helicase in NER. Despite these similarities, DmXPB DNA did not correct the UV sensitivity of XPB cells [40]. DmXPB maps to 6783/4 on chromosome 3, in a region devoid of known mus mutants. It is constitutively expressed at low levels in all developmental stages [40]. Null alleles of haywire are lethal, but viable heterozygotes exhibit UV sensitivity and display neuromuscular defects and reduced longevity [41] similar to effects observed in XPB patients that manifest features of Cockayne’s syndrome (CS). It has been postulated that many of these characteristics may be related to the defects in transcription coupled-DNA repair [I]. Henning et al. 1421 cloned the XPC homolog by hybridization with XPC cDNA. An open reading frame codes for a 1293 amino acid polypeptide. The C-terminal 346 amino acids are 50% identical to the human XPC protein and 27% identical to the S. cererisiae Rad4 protein. Like the other two genes, the putative DmXPC protein is highly basic and contains three bipartite nuclear localization signals. No definitive function has been determined for either the RAD4 or XPC gene products. Recent studies in our laboratory have localized DmXPC to the second chromosome at cytogenetic position 5 1F (Smith and Risman, unpublished), a region containing no known ELLISor mei mutants. A preliminary report by Zuirta et al. [43] describes identification of the Drosophila homolog of XPD/RAD3 using conserved seqences in an RT-PCR protocol. A clone from an embryonic cDNA library appears to contain a full length sequence for a protein having substantial sequence homology to XPD and Rad3. Further analysis of this gene will be of interest, since the XPD/Rad3 protein is the subunit of holoTFIIH responsible for 5’-3’ unwinding of the duplex at the damage site. Its physiological role seems to be even more complex than that of the

R.L. Dusenbey.

P.D. Smith/Mutation

XPB/SslZ protein, since a defect in its function has been associated trichothiodystrophy as well as XP and CS disorders. It is anticipated that detailed genetic analysis of the Drosophila homolog will provide additional insight into how mutations differentially affect the transcription and NER functions of this essential gene. Drosophila meiwas recently identified as a homolog of the XPF(ERCC4) and RADI genes [44. The mei- mutation was first identified in a genetic screen for meiotic mutants [45], and was later reisolated in the screen for mutagen sensitive mutants 131. It exhibits an extreme sensitivity to a wide variety of DNA damaging agents, including UV. and was shown to be blocked in NER at a pre-incision step [4]. It is located near the tip of the X chromosome at cytogenetic location 4B3/4 and genetic map position 5 [4]. Initial cloning of the mei- gene involved production of a mutation induced by P-element transposition, followed by cloning of the adjacent genomic sequences [44]. This analysis identified a 3.4 kb cDNA with a predicted protein of 946 amino acid residues with 30% identity and 51% similarity to the yeast Radl/XPF protein, which, in conjunction with the yeast RadlO/ERCCl protein functions as the endonuclease 5’ to the site of DNA damage. No Drosophila homolog of the RADIO gene has been identified. The mei- gene was cloned independently by a differential expression screen, in which a Pi clone containing a genomic fragment from the 4B3/4 cytogenetic region was hybridized to immobilized RNA extracted from either wild-type or meifly strains (Araj and Smith, 1996). A 3.4-kb mRNA species identified by the PI clone in wild-type extracts was missing from extracts prepared from the mei-9AT3 strain. The cDNA was isolated and shown to have homology to the RADI gene. Although both meiand radl mutants exhibit extreme sensitivity to a wide variety of DNA damaging agents, differences are observed in their recombination phenotypes. While there appears to be a Radl/RadlO-mediated contribution to mitotic recombination in yeast cells, there is no evidence for the action of the Radl gene product in meiotic processes. In contrast, the severe decrease (90%) in reciprocal exchange events in meiotic recombination with no effect on meiotic gene conversion is a

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(1996)133-145

139

defining feature of meimutations [45-471, discussed below. Mutations in the three genes RAD7, 16 and 23 result in only a slight sensitivity to UV damage. Their functions are not well understood, but it is postulated that they play a role in repair in regions of the genome containing silenced chromatin. The putative Radl6 protein has a degree of amino acid homology to a number of other proteins in a seven helicase repeat segment. One of these proteins is encoded by the Drosophila lodestar gene, which maps to cytological position 84D13- 14. Expression of this gene is cell cycle-dependent, and mutants are characterized by the formation of chromatin bridges at anaphase [48]. Currently, there is no experimental evidence for the participation of lodestar in Drosophila NER. However, its involvement with chromatin structure is intriguing.

3.5. Mismatch

repair

A number of mismatch repair systems have been described in prokaryotes, the most well-studied being that of methyl-directed mismatch repair in E. coli, involving the mutH, mutL and mu6 genes [49]. Modrich and his colleagues have determined that the MutHLS system requires 10 separable protein activities [50]. Moreover, studies of bacterial recombination in m&S and mutL mutant strains have indicated that these proteins affect strand transfer in response to mispairs within recombination heteroduplexes [5 I]. Worth et al. [51] propose that these proteins block branch migration within recombinant heteroduplexes containing mismatched bases. Misincorporation of bases during DNA replication or formation of heteroduplex DNA during meiotic recombination represent the major cellular processes that can give rise to base mismatches in eukaryotes, necessitating DNA mismatch repair. In S. cerer:isiae, recombination studies with mutant strains of MSH2 and PMSI, homologs of E. coli m&S and mutL, demonstrated abnormal 4: 4 meiotic tetrads and aberrant co-conversion events, leading to the suggestion that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA [50].

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Half-tetrad analyses with Drosophila provided the initial demonstration of gene conversion in this organism [52] and subsequent studies identified genes which displayed defects in intergenic meiotic recombination [45]. Biochemical studies with meidemonstrated a defect in excision repair [3] while genetic studies [46] revealed an increase in events attributed to post-meiotic segregation, suggesting that the recombination defect in meiwas limited to crossover events and did not interfere with gene conversions [47]. These data imply that one function of the mei-9’ gene product involves excising base pair mismatches from heteroduplex DNA formed during meiotic gene conversion events. Biochemical evidence to support these earlier studies demonstrated that Drosophila cells possess the ability to correct single base pair mismatches [53]. Base pair corrections were mismatch-site and strand-specific and in vitro reaction inhibitors suggested the involvement of cx and/or 6 class DNA polymerase(s). More recently, a yeast MSH2 homolog, has been identified in Drosophila. This gene, named spellcheckerl (spell) is viable and fertile, and maps to the second chromosome at 35A (C. Flores and W. Engels, personal communication).

3.6. Excision repair-related

DNA synthesis

Four nuclear DNA polymerases, 01, B. 6 and E, exist in eukaryotic cells. Pol B is the only one that appears to play no role in semiconservative replication. In vitro experiments by Singhal et al. [54] show that Pol B is active in the gap-filling step in uracilinitiated BER. Yoo et al. [55] found that overexpression of Pol B in transgenic Drosophila constructs did not affect the level of repair of UV, MMS or mitomycin C damage, but did increase the spontaneous rate of both mitotic and meiotic recombination in the transgenic strains. Thus, Pol B may play only a specialized role in DNA repair. This supported by the fact that Pol E is the DNA polymerase required in the in vitro reconstitution of NER [34,56]. TO date, the nuclear DNA polymerases (Y, B and 6 have been identified in Drosophila, and some of the subunits cloned [57]. Chiang and Lehman [58] recently cloned the catalytic subunit of Drosophila Pol 6 and found a high homology with other eukaryotic Pal 6

Research 364 (19%) 133-145

proteins. It is presumed that a Drosophila Pol E also exists to complete NER functions. Four additional proteins have been shown to be necessary to complete resynthesis of DNA in NER: PCNA, RP-A, RPC and ligase [56]. The Drosophila homolog of the PCNA gene was cloned by Yamaguchi et al. [59]. The 260 amino acid conceptual protein has 70% sequence identity to the rat and human PCNA proteins, with conserved leucine repeats in the C-terminal portion. The gene is highly expressed in adult ovaries, unfertilized eggs and early embryos, indicating maternal control. Independent cloning of mus209 through transposon tagging by Henderson et al. [60] led to the surprising conclusion that this gene was identical to the DmPCNA gene previously cloned by Yamaguchi [59]. This remarkable finding broadened the biological role of PCNA. Nine lethal mutations, three of them exhibiting temperature-sensitive lethality, showed that: (1) PCNA is required in nearly all stages of fly development; (2) DNA repair-related functions may be distinct from its vital function; and (3) mutations in PCNA suppress position-effect variegation. It is anticipated that additional studies will clarify the molecular basis for these pleiotropic functions of PCNA. The Drosophila homolog of the RP-A protein has been purified from embryo extracts [61]. DmRP-A has subunits of about 66, 31 and 8 kDa, comparable to RP-A proteins from other species. The genes encoding the subunits have not been cloned or mapped, and no mutants have been isolated. A third protein required for excision repair synthesis, RPC. has been identified in Drosophila, and the gene encoding the 40 kDa subunit has been located at 64A10,ll [62]. The conceptual gene product, Rfc40, has 68% amino acid identity to human RPC. Five viable mutant alleles of the Rfc40 gene were isolated, but it is not known whether it is an essential gene [62]. Two distinct DNA ligases have been purified from Drosophila embryos [63]. More recent studies indicate that DNA ligase I activity more closely parallels periods of high replicative activity, while DNA ligase II activity is lower and persists throughout all developmental stages [64]. The similarity of the activity patterns of DNA Pol B and DNA ligase II suggest that ligase 11 may play an important role in

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some form of DNA repair. Unfortunately, no studies to date have reported the occurrence of Drosophila mutant strains for either of the ligases.

4. Double-strand

break repair

The majority of lethal effects associated with ionizing radiation (IR) exposure have been attributed to DNA double-strand breaks 16.51. Cellular responses to IR-induced double-strand breaks in eukaryotes are designed to offset the lethal effects and have been shown to involve activation of DNAdamage signalling pathways which stall transcriptional activity, arrest the cell cycle, and recruit DNA repair activities [66]. DNA-dependent serine/threonine protein kinase (DNA-PK) has been shown to play a primary role in cell response to IR. Human DNA-PK, composed of a 350-kDa catalytic subunit and two regulatory components comprising the 150~kDa heterodimer Ku autoantigen [67], was shown to be strongly activated by low concentrations of double-stranded DNA [68]. Catalytic activation of DNA-PK occurs upon binding specifically to the ends of DNA molecules The absence of Ku DNA binding ability in extracts of hamster xrs-6 cells, which are deficient in DNA double-strand break repair and somatic recombination in V(DJJ immune cells, supports its role in recombination and repair [69]. Double-strand break repair in Drosophila cells was first measured in 1982 [70]. Recently, Finnie et al. 1691 have described an enzymatic assay which detected DNA-PK activity in Drosophila cells, suggesting that DNA-PK will play an important role in double-strand break processing in flies. An alternative approach to the analysis of double-strand breaks in Drosophila has made use of the P element transposon and has led to the suggestion of a new double-strand break repair model, ‘synthesis-dependent strand annealing’ (SDSA) [71], based on an earlier proposal by Szostak [72]. Transposition of the P element appears to involve a conservative cut-and-paste mechanism involving movement of the P element to a new site. Transposition is incomplete, leaving behind several P element derived nucleotides in addition to the 8 base-pair target site duplication [73]. Repair of this site requires the

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action of an exonuclease to remove the P element nucleotides left at the original site. Exonuclease activity widens the spot to a gap, leaving 3’ singlestrand overhangs. The SDSA model envisions potentially extensive widening of the gap, leaving 3’ overhangs of as much as 2 kb. Repair synthesis is postulated to involve a ‘bubble migration’ mechanism, based on the yeast system described by Fishman-Lobe11 et al. [74]. This mechanism can use either one or two templates, leading to eventual single-strand annealing of the resynthesized extensions. These studies have been reviewed by Lanke.nau [75]. Depending upon the nucleotide sequences resynthesized, gene conversions can result from this double-strand break repair activity. Additional studies [76] with the rudl mutant of Saccharomyces suggested that the role of the RAD1 gene involved the removal of nonhomologous DNA from the 3’ ends of the recombining DNA. Studies by Friedberg and his colleagues, using model repair and recombination DNA substrates [77], demonstrated Rad 1-Rad 1Omediated cleavage at DNA duplex-single-strand junctions. More recent studies [78] indicate that purified Radl and RadlO proteins interact with a synthetic bubble similar to that proposed by Engels. A similar reaction. involving the RADI homolog, mei9, could explain the genetic data of strongly reduced recombination but intact gene conversion observed with tnei-9 mutants [46,47].

5. Cellular responses

to DNA damage

Cellular responses to DNA damage involve biochemical steps which identify the damage, activate specific repair pathways and transduce cellular signals to coordinate cell activities. particularly those that regulate cell cycle progression. Studies examining the regulation of these repair responses have been fairly limited. Vivino et al. provided the first evidence for gene activation in response to UV radiation. identifying a 1 kb transcript following treatment of Drosophila cells in culture [79]. Using methyl methanesulfonate as a DNA damaging agent, Howard-Flanders’ lab determined that more than 20 proteins were synthesized in response to treatment of cells by this compound [80]. None of the genes

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encoding these proteins have been identified, nor has their role in DNA repair been elaborated. Employing a band shift electrophoretic assay. Todo and Ryo identified two protein complexes in embryonic cells of the repair-proficient wild-type Drosophila strain, Canton-S [81]. One complex, ‘factor 2’, appears to be missing from extracts of 4 Drosophila repair mutants, mus201, phr, mus308, and mus205. Another complex, referred to as ‘factor I’, was reported as missing in the mus104 gene, now known to be an allele of nzei-41. Hawley and colleagues have recently cloned the meigene and determined that the putative gene product belongs to a family of phosphatidylinositol3-kinases which have been implicated in cell cycle checkpoints [82]. DNA sequence and phenotypic comparisons suggest that meiis a structural homolog of the human ataxia telangiectasia (ATM) gene. Identification of the Drosophila ATM homolog in conjunction with our recent identification and characterization of the functional homolog of the ~53 tumor suppressor gene in Drosophila cells ([83-851, Dusenbery and Yakes, unpublished), provide exciting possibilities for the genetic dissection of the signal transduction pathway leading from DNA damage to cell cycle arrest and apoptosis in the Drosophila system.

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