Mutant isolation and characterization

Mutant sources

Mutagen-sensitive mutants (mus)

Meiotic mutants (met)

Mutations producing gene and chromosome instability

Genetic and cytological analyses

Mutant influences on meiosis

Mutant influences on mutation

Identification of defects in DNA repair

Photorepair

Excision repair

Postreplication repair

Additional repair processes

Molecular cloning of DNA repair genes

Cloning strategy

Mutation induction by transposon tagging

Progress report

Enzymology related to DNA repair

In the analysis of eukaryotic DNA repair Drosophila provides a valuable link between studies being conducted in unicellular organisms and mammals. On the one hand, many of the refined genetic tools available in fungi for identifying and characterizing repair-related genes have been employed with Drosophila (see Generoso et al. 1980). Like mammals, however, Drosophila is a complex multicellular organism with separate somatic and germ cell lines. This combination of properties permits a detailed genetic analysis of repair functions and their influence on mutagenesis and recombination in both cell types. In addition to its genetic strengths, Drosophila provides a number of unique experimental advantages for the molecular analysis of DNA repair. Because embryonic cells are readily introduced into tissue culture, mutant repair deficiencies can be compared directly with related mammalian deficiences (see Boyd et al. 1983). The capacity of embryos to replicate the entire Drosophila genome within 3 ·4min (Rabinowitz, 1941), furthermore, makes this tissue an extraordinary source of DNA metabolic enzymes (see section on Enzymology Related to DNA Repair), many of which undoubtedly play a role in DNA repair as well as replication. The utility of Drosophila as a model for mammalian repair is also strengthened by observations that it uses similar mechanisms to activate procarcinogens to mutagens (Baars et al. 1980; Waters et al. 1982; Porsch-Hallstr öm et al. 1983).

Current attempts to clone repair-related genes in Drosophila are greatly facilitated by its small genome size (Laird, 1973). The high resolution of in situ hybridization with giant polytene chromosomes has furthermore made this one of the best eukaryotes for performing chromosomal walking (Bender et al. 1983). Finally, Drosophila is one of the most accessible multicellular organisms for cloning by transposon tagging (Bingham et al. 1981) and for testing cloned functions by transformation (Rubin & Spradling, 1982). Application of these experimental systems to DNA repair in Drosophila is summarized in this article. Emphasis is placed on work that has appeared since this subject was last reviewed (Boyd et al. 1983). We start with a discussion of the available mutants and the contribution that their analysis has made to our understanding of the role of DNA repair in mutagenesis and recombination (in the following section). In the third section we describe strategies currently being employed in our laboratories to define the functions of key repair genes through their molecular cloning. Finally, an emerging body of enzymological analysis, which is relevant to DNA repair in Drosophila, is summarized in the last section. As an overview of the mutant analysis, the properties of the major known repair related genes in Drosophila are presented in Table 1. The individual entries in that table are discussed in the body of this review.

Table 1.

Properties of the major* repair-related genes in Drosophila

Properties of the major* repair-related genes in Drosophila
Properties of the major* repair-related genes in Drosophila

Mutant sources

Mutagen-sensitive mutants (mus)

Genes in prokaryotes that are required for DNA repair have been identified through an analysis of mutants that increase the mutation frequency, decrease recombination, or are hypersensitive to mutagens (see Friedberg, 1985). In Drosophila the same three phenotypes have been used to identify genes potentially involved in repair. The most direct search for such functions has been performed by selecting mutants that are conditionally lethal upon exposure to moderate doses of mutagens (Smith et al. 1980). An example of a commonly employed selection scheme is presented in Fig. 1. In this approach DNA in spermatids and spermatozoa is modified by exposure of males to a chemical mutagen. Treated males are mated with virgin females carrying an attached X chromosome. This cross ensures that each son carries a unique mutagenized X chromosome. By next crossing individual F] males to attached X females, separate stocks are established in which the males of each stock carry the same mutagenized X chromosome. Those stocks in which the male larvae prove to be hypersensitive to a mutagen potentially carry a lesion in an X-linked repair function. Analogous schemes have been employed to recover mutants on the major autosomes (Boyd et al. 1981; Snyder & Smith, 1982).

Fig. 1.

Isolation of X-linked mutants that are hypersensitive to mutagens.

Fig. 1.

Isolation of X-linked mutants that are hypersensitive to mutagens.

Mutants recovered by this procedure are termed mutagen-sensitive or mus mutants. The designation for a typical allele is musl01D1;in which the allele is distinguished by a superscript that includes a letter indicating the city of origin (Davis). Genetic loci identified by such mutants are given numbers starting with 101, if they are on the X chromosome; 201 for the second chromosome; etc.

Complementation analysis between mutagen-sensitive strains has led to the identification of 27 genes in Drosophila whose mutants exhibit this phenotype. Nine of these loci are on the X chromosome (muslOl, musl02, mus!05, musl06, mus 108, mus 109, mus 111, mei-9 and mei-41; see Generoso et al. 1980; Mason et al. 1981; Yamamoto, unpublished observations); seven are on the second chromosome (mus201-mus207; Boyd et al. 1982; Snyder & Smith, 1982); and 11 are on the third chromosome (mus301, mus302, mus304-mus312; Boyd et al. 1981). Mutagens used to select the mus mutants include γ-rays (Nguyen et al. 1978), methyl methane sulphonate (Smith, 1973; Boyd et al. 1976a), A’-acetoxyacetylaminofluorene and nitrogen mustard (Boyd et al. 1981). The isolated mutants exhibit several patterns of mutagen sensitivity (Smith et al. 1980; Snyder & Smith, 1982; Boyd et al. 1983). All mutants, with the exception of those at the mus308 locus, are hypersensitive to methyl methane sulphonate (MMS). The predominant sensitivity of mutants at the mus308 locus is to nitrogen mustard. Since mutants at all tested loci are also sensitive to at least one form of radiation, it is unlikely that the observed sensitivity is due to defects in the metabolism or transport of chemical mutagens. The lack of radiation resistant mutants, therefore, increases the probability that the recovered mutants influence DNA repair.

It is important to note that although the selection schemes are all designed to recover mutants that are conditional lethals, the genes identified by those mutants may be essential for survival. This possibility was demonstrated by the subsequent isolation of lethal alleles at the mus 101, mus105 and musl09 loci (Baker & Smith, 1979). Studies of a temperature-sensitive mus101 allele (Gatti et al. 1983; Smith et al. 1985) have raised the possibility that some of these genes primarily function in the maintenance of chromosome structure rather than in DNA metabolism.

Meiotic mutants (mei)

Genetic investigations of the mutagen-sensitive mutants have revealed that nearly 50% of the tested mus genes are essential for normal meiosis (Snyder & Smith, 1982; see next section). That observation suggested that the large collection of meiotic (mei) mutants that have been isolated in Drosophila over the past 60 years (Baker et al. 1976b) could also be a valuable source of repairdefective mutants. Early genetic studies of the mei-9 mutants (Carpenter & Sandler, 1974) had strongly implicated that locus in DNA metabolism. In confirmation of that suggestion, those mutants were subsequently found to be hypersensitive to methyl methane sulphonate (Boyd et al. 1976b) and new alleles of the mei-9 gene were recovered in screens for mutagen-sensitive strains (Baker & Smith, 1979; Graf et al. 19796; Mason et al. 1981). Allelism has also been demonstrated between a mei-41 mutation and mutations recovered in a search for/raws mutants (Smith, 1976). However, since mutants at only two of several tested meiotic loci exhibit mutagen sensitivity (Mason et al. 1981; Boyd, unpublished observations), a relatively small proportion of the mei mutants are likely to be involved in DNA repair.

Mutations producing gene and chromosome instability

Two additional mutant sources can potentially lead to the identification of repair-related genes. Smith et al. (1985) have recovered a number of temperature-sensitive lethal mutants, 15 of which exhibit mitotic chromosome instability. Since mutants at a majority of the mus loci exhibit similar chromosome instability (Baker et al. 1980; Gatti et al. 1984), complementation tests were performed between the two mutant classes. That analysis revealed that a new muslOl allele had been recovered (Smith et al. 1985). It opened the possibility further for future identification of new mus mutants with this approach. Finally, the mutator strains of Drosophila, which exhibit a high spontaneous mutation frequency, are also a potential source of mutants in repair functions (Green, 1973). Although the correlation of this phenotype with defects in DNA repair is more tenuous, mutationally unstable giant (gt) strains were recently shown to exhibit altered DNA metabolism (Narachi & Boyd, 1985).

Genetic and cytological analyses

Mutant influences on meiosis

As mentioned in the previous section, many mus mutants disrupt meiosis. Mutations at four X-linked loci and in six autosomal mus genes increase the frequencies of meiotic non-disjunction from which it has been inferred that these mutations reduce meiotic exchange (Smith, 1976; Boyd et al. 1976a, 1981). Confirmation of this inference comes from reports that mutations at two loci on the first chromosome, mei-9 and mei-41, two loci on the second chromosome, mus203 and mus204, and at least two loci on the third chromosome, mus312 and mus304, decrease meiotic recombination, as expected (Baker & Carpenter, 1972; Snyder & Smith, 1982; Green, 1981, 1982).

Three recombination-defective meiotic mutants have been examined in detail for their effects on the structure of the synaptonemal complex (SC) and recombination nodules (Carpenter, 1979). Recombination nodules have been inferred to be intimately associated with meiotic recombination because: (1) their numbers and distribution at pachytene correspond closely with the frequency and distribution of crossovers observed genetically; (2) they are closely associated with the central element of the SC (Carpenter, 1975); and (3) they occur at or are very close to the sites of DNA synthesis during meiosis (Carpenter, 1981). Two of the three mutants examined, mei-9 and mei-41, have been shown to be defective in DNA repair (Boyd & Setlow, 1976; Boyd et al. 1976b), whereas the third, mei-218, exhibits normal sensitivity to mutagens and is probably not repair-defective (Boyd et al. 1976a). None of these mutants affects the structure, continuity or temporal behaviour of the SC. Mutants at the mei-218 and mei-41 loci, however, reduce the number of recombination nodules as well as change their morphology. In a mei-218 mutant the nodule abnormality involves patchy, irregular condensation or formation of nodule material, whereas in a mei-41 mutant the nodules are uniformly less dense than normal. In both mei-41 and mei-218 mutants the distribution of nodules along a chromosome arm is more uniform than in wild type (Carpenter, 1979). These observations are consistent with the hypothesis proposed by Baker & Carpenter (1972) that these two loci function in setting up the preconditions for exchange, that is, that they are involved in the choice or preparation of sites for exchange.

In contrast, the number, morphology and distribution of recombination nodules in a mei-9 mutant are normal (Carpenter, 1979), consistent with the idea that the wild-type allele functions after the exchange preconditions have been established (Baker & Carpenter, 1972; Carpenter & Sandler, 1974) and after recombination nodules have been formed (Carpenter, 1979). Further evidence that mei-9+ functions in the exchange process per se comes from observations that in homozygous mei-9 females crossing over is reduced to less than 10% of normal levels although gene conversion is normal. Unlike the majority of meiotic mutants, which are postulated to mediate precondition events, the distribution of residual recombination events in mei-9 mutants is normal. Finally, since postmeiotic segregation is elevated, mismatch repair is presumed to be reduced. These results have led Carpenter (1982) to conclude that the repair of heteroduplex mismatches and the isomerization step of recombination are closely related and are under the genetic control of the mei-9gene.

Mutant influences on mutation

Two approaches have been employed to measure the effect of mutagen-sensitive mutants on chromosome stability. In a genetic approach, flies are constructed that carry the mutant of interest and are simultaneously heterozygous for somatic cell markers affecting the colour or morphology of cuticular bristles. The effects of these mutants on the frequencies of mutation, chromosome breakage, somatic recombination, non-disjunction and chromosome loss can be detected by monitoring the frequency and size of cell clones that express the somatic cell marker (Fig. 2). Since clones resulting from each of these processes have distinguishable properties, the particular source of chromosome instability in any mutant may be identified. Using this approach, mutagen-sensitive alleles of six loci (mei-9, mei-41, mus101, mus102, mus105, mus 109) were identified as increasing the frequency of somatic spots (Baker et al. 1978; Baker & Smith, 1979). Analysis of the size distribution of clones produced by mutants at these loci suggests that most of the chromosome instability in each of the mutants is the consequence of chromosome breakage.

Fig. 2.

Examples of mitotic events generating cells homozygous or hemizygous for one or more cell markers in heterozygous genotypes. (From Smith et al. (1985).)

Fig. 2.

Examples of mitotic events generating cells homozygous or hemizygous for one or more cell markers in heterozygous genotypes. (From Smith et al. (1985).)

In addition, the effects of mutagen-sensitive mutations on chromosome stability have been assessed by examining somatic chromosomes cytologically (Gatti, 1979; Gatti et al. 1984). Mutants at 12 loci (mei-9, mei-41, mus101l, mus102, mus105, mus100, mus301, mus304, mus305, mus308, mus309, mus312) cause significant increases in the frequency of aberrations. In X-linked mutants the aberrations detected were predominantly chromatid and isochromatid breaks that had arisen during the previous cell cycle. In mutants at four X-linked loci, close to one half of the aberrations involve both chromatids, whereas in mei-9 mutants the majority of aberrations are single chromatid breaks (Gatti, 1979).

After X-irradiation, neuroblasts from mei-9 or mei-41 mutants exhibit a large increase in the frequency of chromosome aberrations, compared with the mei+ control (Gatti et al. 1980). As was seen with unirradiated cells, chromatid and isochromatid breaks were recovered equally frequently in mei-41 -bearing cells, but in mez-9-bearing cells most aberrations are chromatid breaks. Furthermore, chromatid interchanges were slightly reduced in mei-41 cells and could not be induced in mei-9 cells.

Since lethal alleles of mus101, mus 105 and mus 109 have been found, the functions defined by these loci are essential for survival (Baker et al. 1982; Gatti et al. 1983). Larvae carrying lethal alleles of mus105 and mus109 have degenerate imaginai discs and die at the time of pupation. Mitotically dividing cells in those larvae exhibit high frequencies of chromosome aberrations, which probably lead to high levels of cell death (Baker et al. 1982). An allele of mus101 has been shown to abolish the amplification of chorion genes that normally occurs during oogenesis (Orr et al. 1984; Baker, personal communication). Data derived from a temperature-sensitive allele of mus101 suggest that this locus is required for proper condensation of heterochromatin and that the mutagen sensitivity and inhibition of DNA repair found in viable alleles of mus101 are secondary consequences of a primary defect in chromatin condensation (Gatti et al. 1983).

Chromatin structure may play an important role in mutagenesis by determining which DNA repair pathways are operative. This statement is supported by observations that in mus105 and mus109 mutants the distribution of chromosome breaks is altered. In mus 105 mutants about 80 % of the breaks are euchromatic and in mus109 mutants about 80 % of the breaks occur at the heterochromatic-euchromatic junctions (Gatti, 1979). Mutants of mus101, a locus controlling chromatin condensation, decrease the frequency of nitrogen mustard-induced mutations (Graf et al. 1979a, 1982). Further support for this hypothesis is derived from studies of chromosome aberrations induced by X-rays in the presence of the mu-2 mutation. Mutations at this locus potentiate the recovery of X-ray-induced terminal deficiencies from homozygous females (Mason et al. 1984), but do not cause hypersensitivity to killing by either MMS or X-rays. Homozygous mu-2 males are indistinguishable from wild-type males in potentiating the recovery of partial Y losses (Mason, unpublished), In mu-2 females, partial Y loss can be induced at a high frequency when the Y chromosome is irradiated in mature oocytes but not when the Y chromosome is irradiated in mature sperm, suggesting that mu-2 can distinguish between maternally derived and paternally derived chromosomes. Furthermore, conformation of partial Y losses in progeny of mu-2 females suggests that mu-2 can distinguish between lesions in heterochromatin and those in euchromatin. X-ray - induced breaks in euchromatin give rise primarily to terminal deficiencies, whereas breaks in heterochromatin give rise to two break rearrangements (Mason, unpublished). Rather than controlling DNA repair directly, it is therefore more likely that the mu-2 gene modulates the activity of DNA repair systems at the chromatin level.

The phenomenon of ribosomal DNA magnification in Drosophila reverses the loss of deleted ribosomal cistrons. Magnification has been shown by Polito et al. (1982) to be dependent upon the mei-9 function under specified experimental conditions. Hawley & Tartof (1983), using a somewhat different assay, were subsequently able to show that this process can also be altered by mutants at the mei-41 locus. A similar dependence has also been reported for the mus101 and mus!08 loci (Hawley et al. 1985). Each of these repair-related genes has therefore been implicated in yet another aspect of DNA metabolism.

The repair-defective mutants have been incorporated into experiments designed to examine their effect on mutagenesis during germ cell development. Early evidence for the timing of repair functions during spermatogenesis was based on the lack of a dose fractionation effect for radiation-induced mutations in mature sperm (Muller, 1940) and spermatids (Sobels, 1972). These observations suggested the absence of repair of X-ray-induced lesions in postmeiotic male germ cells. The repair-defective mutants provide a more direct test of this hypothesis. Using two different alkylating agents, Smith et al. (1983) were able to show that alkylation-induced mutation frequencies in meiotic and postmeiotic germ cells are similar for mei-9 and wild-type males, but that in premeiotic cells the mutation frequency falls to a low level in wildtype, but not mei-9 males. This result strongly suggests that the low induced mutation frequency normally seen in premeiotic cells is due to repair of the alkylation-induced lesions. Similar experiments using a mus201 mutant in treated females indicate that excision repair is normally functional throughout oogenesis (Badaruddin et al. 1984). These results confirm earlier work on unscheduled DNA synthesis during oogenesis (Kelley & Lee, 1983; and section on Excision Repair, below).

Studies of the genetic control of mutagenesis in Drosophila have been conducted by crossing mutagenized males to repair-defective females to provide evidence of repair after fertilization (Graf et al. 1979a). Using this technique with eight different alkylating agents, Vogel et al. (1985) found that the hypermutability in the excision deficient mutants mei-9L1 and mus201D1 correlates well with the Swain-Scott constant s (Swain & Scott, 1953). They interpreted this result to mean that excision repair has a significant effect on mutation induction at alkylated N atoms in DNA (induced by chemicals with high s values), but not at alkylated O atoms in DNA (more prominent lesions induced by chemicals with low s values). They further suggested that mutagenesis due to nitrogen alkylation in DNA occurs primarily through error-prone repair whereas mutagenesis due to base oxygen alkylation occurs predominantly by mispairing during replication (Smith & Dusenbery, 1985). These conclusions are consistent with the observation that base nitrogen alkylation in repair-proficient Drosophila is correlated with high frequencies of chromosome breakage while base oxygen alkylation produced primarily point mutations (Vogel & Natarajan, 1979a,b). That conclusion is not consistent, however, with the observation that two compounds with low Swain-Scott constants (DEN, ENU) induce high frequencies of partial Y loss when treated Y-bearing males are crossed with mei-9a females (Zimmering, 1983).

Very little is known about the role that cellular DNA-metabolizing enzymes play in controlling the frequency or precision of transposition in Drosophila. Rasmuson (1984) demonstrated that both mei-9 and mei-41 mutants increase the spontaneous mutation frequency of an unstable white mutation that contains an insertion sequence (IS) element. The MMS-induced somatic mutation rate was, however, reduced by a mei-41 allele. That approach has been extended to additional mus mutants and alternate alkylating agents by Fujikawa & Kondo (1986). It has been suggested that the frequencies of germ line mutations induced by P element mutagenesis at the sn and ras loci are increased by the presence of mutations at mei-9 and mei-41 (Eeken & Sobels, 1981). Slatko et al. (1984), however, were unable to detect any effect of mei-9, mei-41, muslOl or musl02 mutations on the frequencies of P- element-induced sex-linked recessive lethals or recombination in the male germ line. Similarly, Voelker et al. (1984) found that mutations at mei-9 and mei-41 have no influence on the frequency of reversion of a lethal caused by the insertion of a P element but suggested that these mutations may increase the proportion of imprecise excision. If such imprecise excision results in large deletions, they could account for the observation that mei-41 and muslOl mutations decrease the recovery of P-bearing chromosomes from hybrid dysgenic males (Slatko et al. 1984).

Identification of defects in DNA repair

Mutants at 24 of the 31 loci associated with mutagen sensitivity have been tested for defects in at least one form of DNA repair. That analysis, which has recently been reviewed (Boyd et al. 1983), has revealed one locus that is required for photorepair, two that are necessary for excision repair and four whose mutants are strongly deficient in postreplication repair (Table 1). Although less extensive defects have been identified in mutants at 11 additional loci, those genes are presently accorded less emphasis because, with the exception of mus101, it is not known whether the moderate defects are due to leaky mutants in key functions or to mutants in genes whose functions are ancillary to the repair process under study. The following discussion accordingly emphasizes the seven loci identified in that table.

Photorepair

Early experiments demonstrating the existence of photorepair in Drosophila have been reviewed (Boyd et al. 1980). Because of the difficulty of selecting mutants in Drosophila with u.v. treatment (P. D. Smith & J. B. Boyd, unpublished observations), however, defects in this process have not been recovered in mutant screens. Recently, Boyd & Harris (1985) identified a photorepair-deficient mutant (phr) in a standard laboratory stock. Embryonic cells, which are homozygous for this second chromosomal mutation, are entirely devoid of photorepair. Analyses of this mutant have demonstrated that the phr+ allele is essential for the enhanced survival of u.v.-treated larvae that is afforded by irradiation with visible light (unpublished observation). Because similar mutants in Escherichia coli (Sancar et al. 1983) have been shown to fall in the structural gene for a photolyase, it is likely that the phr gene in Drosophila codes for the photorepair enzyme recently characterized by Beck & Sutherland (1979) and Beck (1982). Both the photorepair defect and a partial defect in excision repair have been mapped genetically to position 57 on the 2nd chromosome. Since coincident defects in excision and photorepair have also been observed in E. coli (Yamamoto & Shinagawa, 1985), Neurospora (Inoue & Ishii, 1985) and man (Sutherland et al. 1975), it is becoming increasingly likely that those two repair systems do not function independently of one another in vivo. Further characterization of such mutants should serve to illuminate the molecular basis of that interaction. A partial defect in photorepair observed by Ferro (1985) has not been characterized thoroughly enough to know if it represents an allele of the phr gene.

Excision repair

Of the two identified loci in Drosophila that are absolutely required for excision repair, the mus201 gene most closely resembles excision defective mutants in other organisms (Boyd et al. 1982). Since that mutant is hypersensitive to u.v. and several carcinogens but is relatively insensitive to X-rays (Boyd et al. 1982; Dusenbery et al. 1983; Todo et al. 1985), it is likely to be defective in repair of lesions that distort the DNA helix as is true of excision-defective mutants in other organisms (see Cerutti, 1975). This expectation is born out by observations that cells homozygous for mus201D1 quantitatively retain pyrimidine dimers in their DNA after incubation times that permit wild-type cells to remove 80% of those lesions (Boyd et al. 1982). In addition, the incision event associated with normal excision repair is not detected in 201D1 cells following treatment with either u.v. or N-acetoxy-N-acetyl-2-aminofluorene, agents that generate bulky DNA lesions. Cytological studies of unscheduled DNA synthesis in mus201D1 have, however, revealed a deficiency in repair of at least a portion of the lesions generated by alkylating agents (Dusenbery et al. 1983) in addition to a defect in u.v.-induced unscheduled DNA synthesis (Boyd et al. 1982). Since mus201D1 cells lack most of the X-ray-induced unscheduled DNA synthesis but are relatively insensitive to X- rays, the lesions being repaired by the mus201 pathway are probably not the primary lethal lesions induced by X-rays. The mus201m allele fails to exhibit any detectable effect on meiotic chromosome segregation or recombination. That mutant also exhibits a normal capacity for postreplication repair following u.v. treatment and the capability to seal single-strand breaks induced by X-rays. A mus201D1 mutant has recently been introduced into permanent cell culture by Todo et al. (1985).

Mutants at the mei-9 locus also exhibit an absolute block in excision repair. Repair replication is abolished (Nguyen & Boyd, 1977) and pyrimidine dimers are retained (Boyd et al. 19766) due to a block in the initial incision event (Harris & Boyd, 1980). The mei-9 mutants are unique among excision-defective mutants in any organism in that they are hypersensitive to all classes of mutagens including X-rays (Baker et al. 1976a; Nguyen et al. 1979; Smith et al. 1980) and they exhibit a strong meiotic effect (section on Mutant Sources). In a molecular analysis of DNA repair in Drosophila oocytes, Kelley & Lee (1983) demonstrated that the mei-9a mutation reduces repair replication induced by alkylation damage. Analysis of unscheduled DNA synthesis has also revealed a block in the repair of alkylation-induced lesions in somatic cells of mutant mei-9 cells (Dusenbery et al. 1983). Although the molecular mechanisms of alkylation repair that are interrupted in either the mei-9 or mus201 mutants have not been investigated directly, repair of bases modified at nitrogen atoms is strongly implicated as the relevant repair target (section on Genetic and Cytological Analyses).

Utilization of the excision-deficient mutants has permitted the identification of a previously undetected step in excision repair that can occur in the absence of excision (Boyd et al. 1983; Harris & Boyd, unpublished observations). This step includes an apparent alteration in chromatin structure, which renders DNA lesions more accessible to repair. This function is more easily detected in excision-defective cells because of the lack of competing excision repair. The increase in accessibility is apparently not necessary for initial repair involving a subset of the chromatin; rather this process is postulated to permit repair of initially less-accessible regions through a topoisomerase-mediated alteration of chromatin structure.

Postreplication repair

The term postreplication repair refers to those mechanisms that permit DNA synthesis to proceed on a damaged template (Lehmann & Karran, 1981). Although the mechanism of this process in eukaryotes is not understood, the classical recombination mechanism in E. coli (Ganesan, 1974) is not a major component of postreplication repair in Drosophila (Boyd et al. 1983) or mammals (Lehmann & Kirk-Bell, 1978). Mutants defective in this process produce abnormally small polynucleotide chains during DNA synthesis following mutagen treatment (Lehmann et al. 1977). In Drosophila mutations in four different genes (Table 1) exhibit strong defects in this process (Boyd & Setlow, 1976; Boyd & Shaw, 1982). The deficiency in these mutants is similar to that exhibited by human xeroderma pigmentosum variant patients with the exception that caffeine does not have as strong a potentiating effect in Drosophila as it does in man (Lehmann et al. 1977; Boyd & Shaw, 1982).

The mei-41 locus is unique among the four strong post-replication repair-deficient genes in that most of its mutants are hypersensitive to all tested mutagens (Smith et al. 1980; Nguyen et al. 1979) and they disturb meiosis (Baker et al. 1980; Mason et al. 1981). Recently, a strong hypersensitivity of mei-41 mutants to hydroxyurea has been correlated with the generation of excessive chromosomal aberrations by that compound (Banga, Shenkar & Boyd, unpublished observations). Since the primary target of that inhibitor is thought to be ribonucleotide reductase (Tyrsted, 1982), it is possible that alterations in nucleotide pools play a role in the mei-41 defect. Either the pools themselves may be altered or the replication complex may be particularly sensitive to variations in pool levels. Sensitivity to hydroxyurea is not a general feature of strong postreplication-deficient mutants, however, because mutants at the other three loci are not hypersensitive to that compound (Table 1).

Additional repair processes

Human cells from patients with ataxia telangiectasia fail to reduce DNA synthesis following exposure to X-rays (de Wit et al. 1981). A related phenomenon has been observed in the giant (gt) mutants of Drosophila (Narachi & Boyd, 1985), in which mutant larval cells fail to respond to u.v. treatment with the usual reduction in DNA synthesis. These mutants, which exhibit additional abnormalities in DNA metabolism, should ultimately contribute to an improved understanding of the response of DNA synthesis in eukaryotes to DNA damage. Although mutants at 24 loci have been tested for their capacity to repair single-strand breaks induced by X-rays (Boyd & Setlow, 1976; Oliveri, unpublished observations), all exhibit a normal repair capacity.

Cloning strategy

As this symposium has documented, many of the recent advances in our knowledge of DNA repair in unicellular organisms are due to the application of DNA cloning. An in-depth analysis of repair mechanisms in multicellular eukaryotes is, therefore, likely to require cloning of the corresponding repair genes in these organisms as well. Gene cloning in Drosophila not only offers the potential for elucidating the structure and function of repair genes in this organism, but it may also facilitate parallel analyses in less genetically accessible organisms by providing heterologous probes. In our laboratories we have chosen the mei-9 and mei-41 genes as the initial targets for gene cloning because isolation of these two genes will provide an entry into the mechanisms of both excision (mei-9) and postreplication repair (mei-41).

The current strategy for cloning these genes is based upon a combination of transposon tagging and chromosome walking, which was initially developed by Bingham et al. (1981). An outline of this approach as it applies to the mei-41 gene is depicted in Fig. 3. This method begins with the isolation of mutants that are generated by the insertion of a transposable element. In Drosophila the most convenient means of ‘tagging’ a gene in this way is using dysgenic crosses to mobilize transposable P elements as described below. The presence of a P insert at the locus of interest can then be verified by using a previously cloned P element as a probe for in situ hybridization to polytene chromosomes. In the appropriate mutant that probe will hybridize to the chromosomal region 14B13-14D1,2, which is the cytogenetic map position of the mei-41 gene (Mason et al. 1981). In the second step of this procedure, fusion fragments of the mei-41 gene and the P insert are recovered from a genomic library of the mutant stock by probing with labelled P sequences. Recovery of the correct genomic sequences is verified by in situ hybridization of the clones to wild-type salivary gland chromosomes lacking P elements. Finally, the fusion fragments are used as probes to recover the complete gene sequence from a wild type genomic library.

Fig. 3.

Strategy for cloning the mei-41 gene by transposon tagging.

Fig. 3.

Strategy for cloning the mei-41 gene by transposon tagging.

Mutation induction by transposon tagging

The phenomenon of hybrid dysgenesis in Drosophila has greatly expanded the applicability of transposon tagging because it is associated with a dramatic increase in the mobility of selected transposable elements (Engels, 1983). In the mating scheme depicted in Fig. 4 males carrying up to 50 P elements per genome are crossed to M females lacking such elements. Under these conditions the germ line of the progeny Gi males experiences an elevated mutation frequency primarily due to increased P transposition. Stocks derived from individual G2 males can then be tested for the presence of new mutations, which are usually due to the insertion of a P element into a gene of interest.

Fig. 4.

Use of hybrid dysgenesis to generate mus mutants by transpositional insertion. Any mutant X chromosomes segregated by the dysgenic F1 males are maintained in males, because the females carry attached X chromosomes.

Fig. 4.

Use of hybrid dysgenesis to generate mus mutants by transpositional insertion. Any mutant X chromosomes segregated by the dysgenic F1 males are maintained in males, because the females carry attached X chromosomes.

Progress report

We have used several variations of the basic strategy outlined in Fig. 4 to recover X-linked mus mutants with transposon insertions. These procedures differed primarily in the source of the GQ parents and in the methods used to propagate the recovered mutants. Essentially, the highest mutant yields were obtained when the resulting mutations were rapidly introduced into an M cytotype (a stock with a minimum number of P elements). A total of 10 mutants were recovered from 61 865 X chromosomes tested for hypersensitivity to methyl methane sulphonate and tested for complementation with the previously isolated mutants. Five mutations are mei-41 alleles, four are allelic to mei-9, and one is a musl02 allele. Consistent with their cytological map positions in situ hybridization has revealed the presence of a P insert at chromosomal position 4B in one of the mei-9 mutants and P inserts at 14C in three of the mei-41 mutants. We have, therefore, completed step A shown in Fig. 3 for both the mei-41 and mei-9 genes, and P-containing clones are currently being recovered from genomic libraries derived from those stocks. We are, therefore, confident that a thorough characterization of the structure and function of these two genes is now possible.

The list of repair-related enzymes that have been identified in Drosophila has roughly doubled since this subject was reviewed three years ago. Although most of the newly recognized enzymes have not been extensively purified, they generally exhibit properties similar to those of more thoroughly purified mammalian enzymes. This correlation suggests that Drosophila will provide a valuable model for evaluating the relative importance of these enzymes in eukaryotic chromosome stability and mutagenesis once the corresponding genes have been identified. Within the same period Drosophila has also played a leading role in the analysis of eukaryotic polymerases and topoisomerases, primarily because of the extraordinary concentration of those enzymes in oocytes and embryos. At present, however, the genetic advantages of this organism have yet to be exploited in analysing the functions of any of these enzymes. In this section DNA metabolic enzymes that have been implicated either directly or indirectly in DNA repair are presented in alphabetical order.

Two classes of endonucleases have been identified that nick damaged DNA. In analogy with mammalian studies, the AP endonuclease activity of Drosophila embryos can be separated into two fractions by phosphocellulose chromatography (Spiering & Deutsch, 1981). The variable levels and low purity of the flow-through fraction, however, indicate further work is needed before the identity and characterization of that activity are secure (Presley, unpublished observations). In parallel with analyses of xeroderma pigmentosum D cells, excision-deficient mutants at the mei-9 and mus201 loci in Drosophila exhibit reduced AP endonuclease activity (Osgood & Boyd, 1982).

An additional nuclease activity which degrades uracil-containing DNA has been detected in third instar larvae of Drosophila (Deutsch & Spiering, 1982). Although this activity may be due to a single enzyme similar to E. coli endonuclease V (Gates & Linn, 1977), the extensive nuclease activity present at this stage of development (Boyd, 1969) can complicate the interpretation of data obtained with impure preparations.

Previous failures to identify glycosylases in Drosophila have led to the suggestion that this organism might lack base-excision repair (Green & Deutsch, 1983). However, a glycosylase from Drosophila embryos that acts on oxidized thymine residues has recently been isolated (Breimer, 1986). This observation does not rule out the possibility that Drosophila repair enzymes may act on alkylated (Green & Deutsch, 1983) or uracil-containing (Deutsch & Spiering, 1982) DNA in a unique fashion.

Deutsch & Spiering (1985) have recently reported the presence of a purine-base insertase activity in Drosophila embryos that shares many properties with its mammalian counterpart (Deutsch & Linn, 1979). Since mutants affecting this activity are not available, its discovery in a genetically tractable eukaryote offers a possible clarification of the role of this activity.

A DNA ligase activity has recently been purified to virtual homogeneity from Drosophila embryos (Rabin & Chase, personal communication). This development should make it possible to identify the ligase structural gene.

The recent identification of a methyltransferase in Drosophila (Green & Deutsch, 1983) begins to fill the gap in our knowledge of alkylation repair in this organism. In view of the difficulty in identifying glycosylases that are specific for alkylated bases (Green & Deutsch, 1983), transferase activity may play a much greater role in the repair of alkylation damage in Drosophila than it does in other organisms.

Since the phr+ gene described in the section on Photorepair is absolutely required for photorepair, there is a strong possibility that it encodes a photorepair enzyme. A probable candidate has been partially purified from Drosophila tissue culture cells by Beck & Sutherland (1979). That enzyme requires an RNA cofactor and exhibits assay requirements similar to those of the E. coli enzyme (Beck, 1982). Proof of the proposed gene-enzyme relationship would permit a refined definition of the biological role of that enzyme.

Although ribosylation of nuclear proteins by poly(ADP-ribose)synthetase has been strongly implicated in the regulation of eukaryotic DNA repair (Shall et al. 1982), no genetic proof of that relationship is available. Recently, however, Nolan & Kidwell (1982) have identified and partially characterized a poly(ADP-ribose)- synthetase in Drosophila tissue culture cells. Smulsonet al. (1983) have furthermore demonstrated the specific accumulation of a protein at puffed regions of Chironomous polytene chromosomes as a result of its cross-reactivity to an antibody directed against poly(ADP-ribose)synthetase from man.

Three polymerases, which appear to be functionally homologous to the α, β and y forms in mammals, have been purified to homogeneity from Drosophila embryos (Kaguni et al. 1983; Sakaguchi & Boyd, 1985; Sakaguchi, unpublished observations). On the basis of that homology each form is potentially involved in either nuclear (α, β) or mitochondrial (γ) DNA repair. The most complex and best- studied polymerase is the major synthetic enzyme polymerase a. That enzyme is composed of four different subunits with an aggregate molecular weight of 280 000 (Kaguni et al. 1983). Current efforts are being devoted to the development of an in vitro replication complex, which currently includes Drosophila DNA primase and ribonuclease H (DiFrancesco & Lehman, 1985). Analysis of that complex should ultimately contribute to an improved understanding of the synthetic aspects of repair and their impact on mutation and chromosome stability. Further characterization of the two smaller polymerases is expected to follow a similar pattern. The availability of these enzymes in pure form will greatly facilitate cloning of their structural genes, which will in turn permit a genetic analysis of enzyme function.

Topoisomerases, which are strongly implicated in DNA repair in other organisms, have also been extensively studied in Drosophila (Javaherian et al. 1982; Ackerman et al. 1985; Udvardy et al. 1985). In situ analysis of polytene chromosomes has revealed that topoisomerase II is a major chromosomal protein that is distributed evenly throughout the genome (Berrios et al. 1985). In contrast, topoisomerase I is associated primarily with the transcriptionally active regions of the chromosomes (Fleischmann et al. 1984). Identification of the corresponding genes through cloning is also expected to clarify the role of these enzymes in DNA repair.

The work reported from our laboratories is being supported by the Department of Energy (EV 70210) and the National Institutes of Health (GM 32040). We are grateful to Bruce Baker for permission to reproduce Fig. 2. The advice and participation of Paul Harris has been invaluable throughout this effort.

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