INTRODUCTION

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DNA mismatch repair has long been known to be involved in two important cellular processes: the repair of mismatches generated by misincorporation of nucleotides during DNA replication, and the processing of recombination intermediates, resulting in novel configurations of genetic markers (24, 37). More recently DNA mismatch repair has been found to be involved in three additional processes: regulation of recombination between divergent DNA sequences associated with genetic instability (40, 47); nucleotide excision repair (NER) involved in repair of physical and chemical damage to DNA (14, 22, 34); and the recognition of DNA damage and the initiation of responses to this damage, such as cell cycle arrest (1, 19).

The best-characterized mismatch repair pathway is the Escherichia coli MutHLS system (36, 37). The MutHLS system recognizes and repairs all single-base mismatches as well as insertions and deletions 3 bp in size. The efficiency of mismatch repair depends on the specific mispair formed; e.g., Pu · Pu mispairs are repaired at higher efficiencies than Py · Py mispairs. Repair of a given mispair generally depends on surrounding sequence context (21), but C · C mismatches appear not to be repaired in the sequence contexts that have been studied (36, 51). Repair proceeds through mismatch-dependent nicking of the unmethylated DNA strand opposite a GATC site containing a methylated adenine, degradation from the nick through the mismatch site, and then resynthesis of the excision tract (36); these excision repair tracts can span as many as several thousand nucleotides (53). The MutS protein recognizes and binds to DNA at the mismatch. MutL interacts with MutS bound to the mismatch and is required for optimal activity of MutH endonuclease which nicks the unmethylated strand.

Eukaryotes possess a mismatch repair system related to the bacterial MutHLS system (24, 37). Multiple homologs of MutS and MutL (but not MutH) have been identified in yeast and in mammals, and certain of these genes have been demonstrated to be required for mismatch repair in vivo and in vitro. Mutations in the gene encoding a human MutS homolog (hMSH2) are common in hereditary nonpolyposis colorectal cancer families (15, 31). This observation is consistent with the idea that defects in the mismatch repair system may lead to genomic instability which predisposes the affected individuals to certain types of cancers. DNA methylation does not appear to play a role in eukaryotic mismatch repair, and the in vivo mechanism by which eukaryotes distinguish the newly replicated DNA strand from the parental strand is currently unknown. In vitro, strand-specific repair is initiated from a nick in the DNA of one of the strands (37).

NER is the repair of damaged DNA involving excision of oligomers with lengths of 27 to 29 nucleotides in eukaryotes and 12 to 13 nucleotides in prokaryotes (44, 56). In eukaryotes, there appears to be some functional overlap between NER and DNA mismatch repair, and certain gene products are required for both processes. NER systems appear to be highly conserved through evolution from yeast to mammals. Genes required for NER in yeast were first identified as UV-sensitive rad mutations (16). In humans, genes required for NER have been identified as the seven repair-deficient complementation groups (A to G) of the disease xeroderma pigmentosum (XP) (44). XP is characterized by extreme UV sensitivity and a predisposition to certain types of cancer. In Drosophila, several genes required for DNA repair have been identified in screens for mutations which confer increased sensitivity to mutagens (8). One of these genes, mei-9, had previously been identified as a gene required for meiotic crossing over and normal meiotic chromosome disjunction (4). Mutant mei-9 females are able to generate heteroduplex DNA in recombination intermediates but are unable to repair mismatches within the heteroduplex regions and to resolve the recombination intermediates as reciprocal exchanges (11, 43). Drosophila mei-9 mutants have also been shown to be slow in clearing UV-induced pyrimidine dimers from genomic DNA, indicating that mei-9 is required for Drosophila NER in vivo (9). The mei-9 gene has been cloned from Drosophila and found to encode a homolog of the yeast NER protein Rad1 (46) and the human NER XP complementation group F protein (10, 48). We report here an in vitro system (51) using extracts of mitotic, postmitotic, or senescent Drosophila tissues, which efficiently catalyzes DNA mismatch repair. The specific activities of extracts derived from the entirely postmitotic cells of young and senescent adults were similar to those of extracts derived from rapidly dividing embryos. These results suggest that the mismatch repair system may function throughout the Drosophila life span. Specific activities of Drosophila extracts were considerably greater than those of HeLa cell extracts. Mismatch repair in Drosophila may be inducible, as specific activities are increased five- to sixfold by X-ray irradiation of flies. There are specific defects in repair in extracts generated from mei-9 mutant animals, consistent with previous reports that mei-9 is required in vivo for certain types of DNA mismatch repair (11, 43) and NER (9).

	MATERIALS AND METHODS

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Drosophila strains and culture. All Drosophila strains are as previously described (32). mei-9^AT2 and mei-9^a Drosophila stocks were obtained from R. Scott Hawley, University of California, Davis, Calif. Oregon-R strain flies were grown on cornmeal-molasses-agar medium (2). Embryos were collected on molasses-agar plates coated with yeast paste. Flies were cultured and aged as previously described (55). To obtain flies of defined ages, flies were cultured at 25°C until 0 to 2 days posteclosion, and then the male flies were maintained at 29°C, at 50 flies per vial. Aging flies were transferred to fresh vials every 4 days to prevent growth of bacteria or fungus in the cultures. "Young" refers to flies 4 to 5 days posteclosion, and "old" refers to flies 35 days posteclosion. At 35 days posteclosion at 29°C, only ~15% of the cohort is still surviving, and thus these flies are highly senescent.

X-ray treatment. Young male flies were separated from female flies and distributed at 50 flies per vial. These flies were X-ray irradiated for 4 h at 320 rads/min. The flies were allowed to recover for 48 h before preparation of extracts. The mock-irradiated control flies were handled and cultured identically to the irradiated flies.

E. coli strains. E. coli NR9099, NR9162, and CSH50 and bacteriophage M13mp2 have been previously described (7, 26, 27, 29, 41). Mutant M13mp2 derivatives, previously described (26-28), were obtained from T. A. Kunkel, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, N.C.

Preparation of single-stranded DNA. To prepare phage stocks, mutant phage were grown for 5 to 6 h by adding 50 µl of plaque suspension to 5 ml of an early-log-phase culture of CSH50 cells in LB medium at 37°C. Cultures were then transferred to sterile microcentrifuge tubes and centrifuged at 10,000 rpm for 5 min at room temperature. Supernatants were aliquoted to sterile tubes; these stocks can be stored indefinitely at 4 or 20°C without loss of infectivity. One-half milliliter of the appropriate phage stock was added to an early-log-phase culture of CSH50 cells (2.5 ml) and allowed to stand for 5 min at room temperature. These infected cells were diluted into 500 ml of fresh LB medium and incubated overnight at 37°C with constant vigorous shaking. The phage were precipitated from 500 ml of the supernatant by adding 0.25 volume of NaCl and 20% PEG-8000 and stirring for 1 h in the cold room, followed by centrifugation at 11,800 rpm for 30 min at 4°C. The virus pellet was resuspended by adding the cationic detergent cetyltrimethylammonium bromide (24 ml/pellet obtained from 250 ml of the supernatant) and vortexing vigorously. The suspension was centrifuged at 14,000 rpm for 15 min, and the pellet was resuspended in 7 ml of 1.2 M NaCl/250 ml of supernatant. DNA was precipitated by adding 17.5 ml of ethanol and incubating the sample at 20°C overnight. The DNA was pelleted by centrifuging at 14,000 rpm for 15 min at 4°C. The pellet was washed with 70% ethanol, spun for 5 min, dried, and resuspended in 2 ml of Tris-EDTA (pH 8.0), and the final DNA concentration was determined.

Preparation and purification of RF DNA. Replicative-form (RF) DNA was prepared and purified by using the Wizard Maxipreps DNA purification system.

Construction of heteroduplexes containing mispairs. Heteroduplex DNA was generated as described previously (50). Briefly, the appropriate mutant RF DNA was digested to completion with restriction endonuclease AvaII, which incises once in M13mp2 at position 264 (where position +1 is the first transcribed base of lacZ). This genome-length linear (minus-strand) DNA was annealed to mutant, single-stranded, circular, viral (plus-strand) DNA, to generate a completely double-stranded but nicked heteroduplex molecule. This nicked, circular DNA species was purified by 0.8% agarose gel electrophoresis. The nicked heteroduplex migrated as a distinct band, well separated from other DNA species on the gel (data not shown). The nicked heteroduplex was then isolated by electroelution, ethanol precipitated, and resuspended in 10 mM Tris-HCl (pH 8.0)-1 mM EDTA.

Preparation of competent cells, transfection, and plating. Preparation of competent cells, transfection, and plating were done essentially as described previously (7, 42). NR9162 cells which are deficient in mismatch repair system were derived from MC1061 and yield a very high efficiency for transfection, ~10⁵ plaques/ng of DNA. Plating efficiency was not affected by pretreatment of the DNA with HeLa cell or Drosophila embryo extracts (data not shown). Transfection was accomplished with a Bio-Rad Gene Pulser set at 1.8 kV, 400 , and 25 µF. Typical time constant is 9.3 ms. Immediately following electroporation, 1 ml of SOC medium (42) (at room temperature) was added to the cells, which were kept at room temperature. An appropriate amount of the electroporated cells was used to yield 200 to 500 plaques per plate for this assay. The total number of plaques was determined by counting all plaques for which the color phenotype was obvious. Tiny plaques, plaques at the edge of the plate, or those on regions of the plate smeared by a drop of water were not counted.

Preparation of extracts. Extracts from Drosophila embryos and adults were prepared essentially as previously described (35) except that both of the centrifugation steps were performed at 10,000 × g instead of 100,000 × g. Briefly, adults of Drosophila melanogaster Oregon-R were cultured in population cages, and the embryos were collected for 0 to 18 h on apple juice-agar plates coated with a thin layer of live yeast paste. The embryos were washed extensively in distilled H₂O and then dechorionated by immersion in 2.25% sodium hypochlorite (bleach) for 90 s. The embryos were then rinsed extensively with 0.7% NaCl-0.04% Triton X-100 solution and finally with distilled H₂O. The dechorionated embryos were filtered through Miracloth (Calbiochem), blotted dry from below, resuspended in 25 to 50 ml of homogenization buffer (20 mM HEPES-NaOH [pH 7.5], 5 mM magnesium acetate, 50 mM potassium acetate, 1.0 mM EGTA, 0.5 mM EDTA, 0.1% [vol/vol] Triton X-100, 10% [vol/vol] glycerol, 10 mM Na₂S₂O₅, 1.0 mM dithiothreitol, 5 µg of leupeptin per ml, 5 µg of pepstatin A per ml), and incubated on ice for 10 min. The embryo slurry was refiltered, blotted dry and weighed. The embryos were then homogenized (1 ml of buffer and 0.04 ml of 100 mM phenylmethylsulfonyl fluoride/g of embryos) in a Dounce homogenizer with a tight-fitting (A) pestle. The volume of the homogenate was measured, and one-ninth volume of 5 M NaCl was added to produce a final concentration of 0.5 M NaCl. This mixture was then homogenized with a B pestle and incubated for 30 min on ice with occasional stirring with a sterile plastic pipette. The homogenate was then centrifuged at 10,000 × g for 1 h at 4°C. The supernatant was collected, carefully avoiding the loose chromatin pellet. The supernatant was recentrifuged at 10,000 × g for 1 h at 4°C, and the supernatant was collected. The crude extract was frozen in liquid nitrogen as drops and stored at 80°C. Protein estimation was done by Bradford's method.

Mismatch repair reaction. The mismatch repair reaction was as previously described (51). The standard mismatch repair reaction mixture (25 µl) contained 30 mM HEPES (pH 7.8), 7 mM MgCl₂, 4 mM ATP, 200 µM each CTP, GTP, and UTP, 100 µM each dATP, dGTP, dTTP, and dCTP, 40 mM creatine phosphate, 100 µg of creatine phosphokinase per ml, 15 mM sodium phosphate (pH 7.5), 5 ng of purified heteroduplex DNA, and the extract. After incubation at 37°C for the desired time, the reaction was terminated by the addition of 25 µl of stop mix (2% sodium dodecyl sulfate, 50 mM EDTA-Na₂ [pH 8.0], 2 mg of proteinase K per ml) and further incubated for 30 min. To this, 30 µl of tRNA (800 µg of tRNA per ml-4.6 M ammonium acetate) was added, and the sample was precipitated with 80 µl of isopropanol, extracted twice with 80 µl of phenol-chloroform-isoamyl alcohol, reprecipitated, and finally resuspended in 40 µl of distilled water. Transfection of E. coli NR9162 with this DNA was carried out by using a Bio-Rad Gene Pulser electroporation system. Repair efficiency is expressed in percent as 100 × (1  ratio of percentages of mixed bursts obtained from extract-treated and untreated samples) (51).

	RESULTS

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The mismatch repair assay. The mismatch repair assay is based on the analysis of plaque color phenotypes resulting from the transfection of a mutS E. coli strain with purified M13mp2 heteroduplex DNAs that have been treated with various extracts (45, 51). The heteroduplexes contain base mispairs in the E. coli lacZ gene; this gene can carry out  complementation, yielding blue phage plaques on host indicator plates (51). The composition of the mispair is such that expression of one strand yields a light or dark blue plaque phenotype whereas the other strand contains a stop codon and leads to a white plaque. Upon transfection, any unrepaired molecules are potentially capable of forming mixed-color plaques at a certain frequency, as well as pure-color plaques. Pure-color bursts are obtained even without extract treatment, and this is characteristic of M13 phage replication in E. coli (51). When the template is first treated with extract and repair occurs, the ratios change such that mixed bursts decrease, and one or both pure bursts will increase depending on the extent of repair of either strand (51).

Mismatch repair requires a nick and is directed toward the nicked strand. The methylation state of adenine in d(GATC) sequences serves as the signal for strand discrimination of mismatch correction by the E. coli methyl-directed MutHLS pathway; a nick is made by MutH endonuclease, in the presence of MutL, at the d(GATC) site in the unmethylated strand (25, 33, 36). However, a persistent nick or strand break can bypass the requirements for both d(GATC) sequences and mutH for the E. coli mismatch repair system in vitro (30, 54). Transfection experiments suggest that a nick may suffice to determine strand specificity of mismatch repair in mammalian cells (17). Nuclear extracts derived from Drosophila K_c and HeLa cell lines have been found to correct single-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific nick located 808 bp from the mismatch (20). To determine whether a strand-specific and site-specific nick is required for efficient repair of G · G and T · G heteroduplexes in the Drosophila embryo extract, the relative frequencies of repair of the nicked and unnicked strands were assayed.

Repair of mispairs G · A, C · A, A · A, C · T, and T · T is not specific for the nicked strand. When the G · A and C · A mispairs were assayed for repair of the nicked and nonnicked DNA strands, repair was found to be specific for the nonnicked (plus) strand (Table 2). A likely explanation for this result is that the G · A and C · A mispairs were being repaired by the activity of an A-glycosylase. Consistent with this idea, repair of the A · A mispair was found to occur on both strands (Table 2). Repair of the A · G mispair was found to be specific for the nicked strand; however, since the nicked strand also contains the A of the A · G mispair, this might or might not represent an A-glycosylase activity. Similarly, repair of mispairs C · T and T · T was not specific for the nicked strand. This repair may be the result of a T-glycosylase activity. A T-glycosylase has recently been purified from HeLa cell extracts (38).

Specific activity of mismatch repair in Drosophila extracts is maintained throughout development and aging. To compare mismatch repair activities during different stages of Drosophila development, it was necessary to measure the specific activities of extracts from embryos and from young and old adult flies. We also compared the specific activities of Drosophila extracts with those of HeLa cell extracts. The specific activities for correction of the mispairs G · G and C · C were determined by assaying repair in the linear range both for protein amount and time of incubation (Fig. 1). Conditions were identified for each type of extract (HeLa, Drosophila embryo, and Drosophila adult) for which repair activity was approximately linear with regard to protein concentration and time of reaction.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Mismatch repair efficiencies of HeLa cell and Drosophila extracts. The percent repair efficiency was calculated as a function of amount of extract protein for the various types of extracts. Reaction mixtures were incubated for 15 min for HeLa cell extracts and for 2 min for Drosophila extracts. Results are expressed as total repair efficiencies and are based on counts of several hundred to several thousand plaques per data point. A small amount of C · C repair was observed in HeLa cell extracts, but it was not strand specific. The data are plotted as means ± standard errors. Open circles, repair of G · G; closed circles, repair of C · C.

Mismatch repair activities in adults are induced by X-ray irradiation. It was of interest to determine whether the mismatch repair activity detected in the Drosophila extracts was an inducible system. To test for inducibility, extracts were prepared from adult flies which had been subjected to 76,800 rads of X-ray irradiation and from mock-irradiated control flies. The specific activity of the extracts for repair of G · G mismatch was found to be five- to sixfold induced by X-ray irradiation, indicating that the mismatch repair system detected in the extracts is inducible in adult flies (Table 3).

Relative efficiency of repair of different mismatches. To determine the relative efficiency of repair of different mismatches in Drosophila extracts, a variety of base mispairs were assayed for specific activity of repair in two independent embryo extracts (Table 4). A · A, T · G, and G · G were found to be the most efficiently repaired, C · C, T · T, and C · T were intermediate, and C · A, G · A, and A · G were the least efficiently repaired. In E. coli, the MutHLS system can also repair 1- to 3-bp loops but does not efficiently repair a 5-bp loop. The Drosophila embryo extract was found to efficiently repair both 1- and 5-bp loops; however, the repair was not nick dependent (Table 4).

Extracts of mei-9 mutant animals exhibit defects in nick-dependent DNA mismatch repair. The Drosophila mei-9 gene is homologous to yeast rad1 and is required for meiotic recombination and certain types of mismatch repair and NER in vivo in Drosophila. To determine whether mei-9 gene function is required for mismatch repair in the in vitro assay, extracts were prepared from animals homozygous for either of two strong mei-9 alleles, as well as from heterozygous controls. Both the mei-9^AT2 and mei-9^a mutant alleles were found to significantly reduce the repair of G · G and T · G mispairs. Relative to extracts of heterozygous flies, repair of G · G and T · G mispairs was reduced 5.7- and 9-fold, respectively, in mei-9^AT2 mutant extracts and reduced 12.5- and 10-fold, respectively, in mei-9^a mutant extracts. The decrease in repair relative to wild-type flies may be even greater, as even the heterozygous fly extracts appear to be slightly reduced in activity relative to wild-type extracts. (Fig. 2). The mei-9 mutations did not significantly affect the repair of the A · G or G · A mispairs (Fig. 2) or the other mispairs such as C · C, where repair was non-nick dependent (data not shown). Thus, mei-9 mutations specifically reduce nick-dependent DNA mismatch repair in vitro.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Repair of G · G, T · G, A · G, and G · A mispairs by Drosophila wild-type and mei-9 mutant extracts. Extracts were prepared from Oregon-R strain (wild-type) young adults and from flies heterozygous (het) and homozygous (hom) for mei-9 alleles mei-9AT2 and mei-9a, as indicated. Results are expressed as specific activity (percent repair efficiency/minute/microgram of protein) and are based on counts of several hundred to several thousand plaques per assay. Reaction mixtures contained 0.5 µg of protein and were incubated for 2 min at 37°C, which is within the approximately linear range of the assay (Fig. 1C). The data are plotted as means ± standard errors of two to four independent measurements. All mispairs were efficiently repaired by a mixture of wild-type and mei-9 mutant extracts (data not shown).

	DISCUSSION

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We have characterized an in vitro system for DNA mismatch repair that uses extracts of Drosophila embryos and adults. Repair was nick dependent and specific for the nicked DNA strand of the heteroduplex for mispairs T · G and G · G. In contrast, repair of A · A, C · A, G · A, C · T, T · T, and C · C was not nick dependent and may be catalyzed by glycosylases. A-glycosylase activity has been observed in other systems, for example, the MutY activity of E. coli (3). A T-glycosylase has previously been purified from HeLa cell extracts (38). The range of relative activities for the repair of the different mispairs was similar to that previously reported for extracts of mammalian cells and Drosophila tissue culture cells. Strikingly, the Drosophila embryo and adult extracts have >100-fold-higher specific activity for nick-dependent repair than HeLa tissue culture cell extracts.

The developing Drosophila embryo exhibits the most rapid S phase known for a multicellular eukaryote, and it is therefore not surprising that these cells would express high levels of the factors required for postreplicative DNA mismatch repair. In E. coli, cessation of cell division by entry into stationary phase causes a down-regulation of DNA mismatch repair activities: a 10-fold decrease in mutS activity and a 3-fold decrease in mutH activity (13). In contrast, extracts of the entirely postmitotic cells of adult Drosophila had a specific activity for mismatch repair equal to that of the rapidly dividing Drosophila embryo. Moreover, equally high specific activity was obtained from extracts of highly senescent Drosophila adults. This finding indicates that the relevant activities are continuously expressed up to the end of the Drosophila life span. The continued high level expression of these DNA mismatch repair activities suggests that they may be required to maintain DNA sequence integrity throughout the adult Drosophila life span.

DNA mismatch repair activity in the postmitotic cells of adult Drosophila may be inducible, in that the specific activity of extracts was increased five- to sixfold by pretreatment of the adults with X rays. X rays produce hydroxyl radicals, which in turn cause double-strand breaks in DNA (5). In Drosophila, repair of double-strand breaks created by transposable element excision appears to involve exonucleolytic digestion of the free DNA ends and then resynthesis of the double-strand DNA gap, using homologous sequences as the template (12). X-ray-induced double-strand breaks may be repaired by a similar pathway, and we hypothesize that mismatch repair activities may be induced to correct any misincorporated nucleotides in the newly synthesized DNA. Consistent with this idea, double-strand break repair in yeast is associated with high rates of DNA synthesis errors (49). Alternatively, X-ray-generated oxygen radicals may directly damage certain DNA bases, and activities involved in both NER and mismatch repair may be induced to repair this damage.

Requirement for mei-9 activity. Mutations in the Drosophila mei-9 gene were first identified in a screen for mutations causing increased levels of meiotic nondisjunction (4). The meiotic nondisjunction results from a reduced level of meiotic crossing over (reciprocal exchange) to <10% of wild-type levels. While reciprocal exchange is greatly reduced by mei-9 mutations, meiotic gene conversion is nearly normal. In addition, mei-9 females exhibit high levels of postmeiotic segregation, where progeny can inherit a single maternal chromosome yet be mosaic for both maternal alleles of a particular gene on that chromosome (11, 43). These data have been interpreted as indicating that mei-9 females are able to generate heteroduplex DNA in recombination intermediates but are unable to repair mismatches within the heteroduplex regions and to resolve the recombination intermediates as reciprocal exchanges (46).