Decreased Meiotic Intergenic Recombination and Increased Meiosis I Nondisjunction in exo1 Mutants of Saccharomyces cerevisiae

Corresponding author: Lorraine S. Symington, Department of Microbiology and Institute of Cancer Research, Columbia University, 701 W. 168th St., New York, NY 10032., lss5{at}columbia.edu (E-mail)

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Exonuclease I was originally identified as a 5' 3' deoxyribonuclease present in fractionated extracts of Schizosaccharomyces pombe and Saccharomyces cerevisiae. Genetic analysis of exo1 mutants of both yeasts revealed no major defect in meiosis, suggesting that exonuclease I is unlikely to be the primary activity that processes meiosis-specific double-strand breaks (DSBs). We report here that exo1 mutants of S. cerevisiae exhibit subtle but complex defects in meiosis. Diploids containing a homozygous deletion of EXO1 show decreased spore viability associated with an increase in meiosis I nondisjunction, while intergenic recombination is reduced about twofold. Exo1p functions in the same pathway as Msh5p for intergenic recombination. The length of heteroduplex tracts within the HIS4 gene is unaffected by the exo1 mutation. These results suggest that Exo1p is unlikely to play a major role in processing DSBs to form single-stranded tails at HIS4, but instead appears to promote crossing over to ensure disjunction of homologous chromosomes. In addition, our data indicate that exonuclease I may have a minor role in the correction of large DNA mismatches that occur in heteroduplex DNA during meiotic recombination at the HIS4 locus.

IN Saccharomyces cerevisiae, meiotic recombination is initiated by double-strand breaks (DSBs) that are subsequently processed to form 3' single-stranded tails (CAO et al. 1990 ; SUN et al. 1991 ). Spo11p is an atypical type II topoisomerase that is thought to catalyze DSB formation (KEENEY et al. 1997 ), but the identity of the nuclease(s) that processes the breaks to form single-stranded tails remains unknown. The processing reaction is thought to require the activity of a 5' 3' double-stranded exonuclease, a 5' 3' single-stranded exonuclease in conjunction with a DNA helicase, or a single-stranded endonuclease, again working with a DNA helicase. In some proposed models, the Mre11/Rad50/Xrs2 complex participates in the removal of Spo11p from DSB sites in meiosis and might also participate in the removal of the 5' strand (FURUSE et al. 1998 ; USUI et al. 1998 ; MOREAU et al. 1999 ). However, the in vitro exonuclease activities of yeast Mre11p and the human Mre11 complex are of the opposite polarity to that predicted for the processing reaction (FURUSE et al. 1998 ; TRUJILLO et al. 1998 ; USUI et al. 1998 ). Although the endonuclease activity of Mre11p might be sufficient to remove Spo11p and the 5' strand, another possibility is that the 5' strand is removed by the sequential action of Mre11p and a different nuclease.

The resulting 3' single-stranded tailed molecules participate in homologous pairing and strand invasion of a homologous duplex to form heteroduplex DNA. The length of heteroduplex DNA at the ARG4 locus is dictated by the extent of degradation of the 5' strand (SUN et al. 1991 ). DNA mismatches present in heteroduplex DNA are repaired by the mismatch correction system giving rise to gene conversion (6:2 or 2:6 segregation of markers) or restoration to the parental markers (4:4 segregation of markers). Failure to repair results in postmeiotic segregation of markers to yield 5:3 or 3:5 segregations, manifested as sectored spore colonies in S. cerevisiae. Although single base pair (except C/C) and small insertion/deletion mismatches are efficiently repaired by the mismatch repair system, palindromic insertions are refractory to repair (NAG et al. 1989 ). Hence, palindromic insertions can be used to monitor heteroduplex DNA formation without the complication of effects on meiotic recombination and sporulation caused by defects in the mismatch repair system. At the HIS4 locus in S. cerevisiae, heteroduplex formation in meiosis is initiated at the 5' end of the gene and often extends to the 3' end, a distance of 2.5 kb (PORTER et al. 1993 ).

Exonuclease I was originally characterized because the biochemical activity suggested that it might play a role in processing DSBs in vivo (SZANKASI and SMITH 1992 ; HUANG and SYMINGTON 1993 ; FIORENTINI et al. 1997 ). The exo1 gene of Schizosaccharomyces pombe is transcriptionally induced during meiosis, suggesting a role in meiotic DNA metabolism (SZANKASI and SMITH 1995 ). The observations that exo1 mutants are radiation resistant, proficient at mating-type switching, and have wild-type levels of sporulation argue against the possibility that exonuclease I is the primary DSB-processing activity (SZANKASI and SMITH 1995 ; FIORENTINI et al. 1997 ). Instead, the elevated rates of spontaneous mutation observed in exo1 strains and the physical interaction between Exo1p and Msh2p are more consistent with a role for Exo1p in mismatch repair in mitotic cells. TSUBOUCHI and OGAWA 2000 isolated EXO1 as a high-copy suppressor of the methyl methanesulfonate sensitivity caused by mutations in MRE11, RAD50, and XRS2. They also show that EXO1 is induced in meiosis; processing of double-strand breaks is reduced in an exo1 dmc1 strain; the level of crossing over, but not the frequency of gene conversion, is reduced by the exo1 mutation; and Exo1p and Msh4p act in the same pathway for intergenic recombination. KHAZANEHDARI and BORTS 2000 also demonstrate that Exo1p and Msh4p act in the same pathway for crossing over but act in different pathways for spore viability. They show that the exo1 mutation confers a high rate of meiosis I nondisjunction and that the level of gene conversion in the mutant is reduced at some but not all alleles examined.

Below we report that Exo1p has several meiotic functions. Strains with the exo1 mutation have decreased spore viability and intergenic recombination and have elevated levels of chromosome nondisjunction. Genetic data argue that Exo1p acts in the same pathway as Msh5p to generate intergenic recombinants, but in overlapping pathways for spore viability. The extent of heteroduplex formation is not affected by the exo1 mutation, indicating that exonuclease I does not have an essential role in processing of double-strand breaks to generate long 3' single-stranded DNA molecules for strand invasion during meiotic recombination. In addition, the patterns of aberrant segregation observed in exo1 strains suggest that Exo1p functions in the repair of the DNA mismatches that lead to gene conversion events at some loci. These results are consistent with Exo1p having complex roles in both meiotic intergenic recombination and mismatch repair.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Media and genetic procedures:
With the exceptions noted below, standard protocols and media were used (SHERMAN et al. 1986 ). Sporulation plates contained 1% potassium acetate, 0.1% yeast extract, 0.05% glucose, 6 µg/ml adenine, and 2% agar. For studies on the frequencies of crossovers and aberrant segregation, diploids were sporulated at 18° and dissected onto plates containing rich growth medium. After colonies formed, they were replica plated to various omission media to score segregation of heterozygous markers. Postmeiotic segregation events at the HIS4 locus were detected as sectored His⁺/His^- colonies (DETLOFF et al. 1992 ). Nomenclature for aberrant segregation events follows the standard set for eight-spored fungi—for segregation of a heterozygous allele A/a, Mendelian segregation is designated 4A:4a, while a gene conversion event is 6A:2a or 2A:6a and single postmeiotic segregation events (one sectored colony) are 5A:3a and 3A:5a.

Studies on spore viability were done in two different genetic backgrounds. Diploids in the AS4/AS13 genetic background (described below) were sporulated at 18°. Diploids in the W303 background were sporulated at 30°. Following matings of the haploids to generate diploids, these strains were grown for either 15 or 60 generations prior to sporulation. The mating phenotype of germinated spores was determined by replica plating dissection plates to lawns of mating-type tester strains MCY14 and SJR13, followed by selection for diploids on minimal medium. Spores that failed to mate to the two tester strains were considered to be nonmaters due to missegregation of chromosome III.

Strains:
Diploid strains used in the analysis of recombination were constructed by mating haploid strains derived from AS4 (MAT trp1-1 arg4-17 tyr7-1 ade6 ura3) or AS13 (MATa leu2-Bst ade6 ura3 rme1) (STAPLETON and PETES 1991 ) or from published derivatives of these strains. The haploid DTK299 was made from AS4 by one-step gene replacement using a PCR-generated DNA fragment designed to replace the EXO1 gene with the URA3 gene. The PCR fragment was generated by using the primers (5' AAAGGAGCTCGAAAAAACTGAAAGGCGTAGAAAGGAATGGGTATCCAAGGTgattgtactgagagtgcacc and 5' CCTCCGATATGAAACGTGCAGTACTTAACTTTTATTTACCTTTATAAACAAATTGGGgggctgtgcggtatttcacaccg) to amplify the URA3 gene from the plasmid pRS306 (SIKORSKI and HIETER 1989 ). Capital letters represent EXO1 sequences and are identical to those used previously to construct the exo1::HIS3 allele (FIORENTINI et al. 1997 ); the small letters represent sequences flanking URA3. Other strains with the exo1 mutation were constructed in the same way including: DTK300, derived from DNY24 [AS13 but his4-lopd, (NAG et al. 1989 )]; LSY612, derived from DNY16 [AS13 but his4-B2 (NAG et al. 1989 )]; LSY613, derived from DNY25 [AS13 but his4-lopc (NAG et al. 1989 )]; and LSY611, derived from PD98 [AS13 but his4-3133 (DETLOFF et al. 1992 )]. MSH5 was disrupted in DNY24, creating DTK492, using a PCR-generated DNA fragment from the primers (5' CAACTCATTCAAAATAACTTACTCATTCATATACTGCCACCAAATGGAATcgtacgctgcaggtcgac and 5' TTATTAACTTAAATATGTTACAGGTGGGCGTTTTTTTATTCTTTGATATAatgatgaattcgagctcg) to amplify the KanMX4 cassette from pFA6-kanMX4 (WACH et al. 1994 ); transformants were selected by resistance to geneticin. Capital letters in the primers represent MSH5 sequences, while small letters represent pFA6-kanMX4 sequences. DTK492 was crossed with AS4 and transformed by a one-step transformation using the msh5::URA3 plasmid pNH190-11 (HOLLINGSWORTH et al. 1995 ), selecting for Ura⁺ geneticin-resistant transformants to create the diploid DTK498. MSH5 was disrupted in a Ura^- derivative of DTK299 using pNH190-11, generating DTK502. EXO1 was disrupted in DTK492 using the exo1::URA3 primers described above, creating DTK504. Diploid strains were constructed by mating: DTK308 (DTK299 x DTK300), DTK332 (DTK299 x LSY612), DTK333 (DTK299 x LSY613), DTK335 (DTK299 x LSY611), and DTK505 (DTK502 x DTK504). Except for the exo1 and/or msh5 mutations, strains DTK332, DTK333, and DTK335 are isogenic with strains DNY16, DNY26, and PD98, while DTK308, DTK498, and DTK505 are isogenic with DNY27 (NAG et al. 1989 ; DETLOFF et al. 1992 ). The diploid strain DTK347 is also isogenic with the diploid DNY26 (NAG et al. 1989 ), except for a homozygous insertion of a 60-bp simple repetitive sequence (CCCGNN)₁₂ that replaces transcription factor binding sites located upstream of HIS4 (KIRKPATRICK et al. 1999 ).

Since diploids derived from AS4 and AS13 exhibit lower spore viability than most other laboratory strains, we also used W303 derivatives to measure spore viability. Strains W303-1A (MATa leu2-3, 112 trp1-1 can1-100 ura3-1 ade2-1 his3-11, 15) and isogenic derivatives W303-1B (MAT), LSY496-10D (MATa exo1::HIS3), and LSY496-20A (MAT exo1::HIS3) were described previously (FIORENTINI et al. 1997 ). LSY625 (MATa exo1::URA3) was constructed from W303-1A as described above for the AS4 and AS13 exo1 derivatives. The wild-type diploid (W303) was constructed by crossing W303-1A with W303-1B while the exo1 diploid derivatives were constructed by crossing LSY496-10D with LSY496-20A (to generate LSY529a) and LSY496-20A with LSY625 (to generate LSY529b). The MSH5 gene was disrupted in the haploid W303-derived strains by the one-step replacement method using plasmid pNH190-11 (HOLLINGSWORTH et al. 1995 ). The resulting strains, LSY810 (MATa msh5::URA3), LSY811 (MAT msh5::URA3), LSY812 (MATa msh5::URA3 exo1::HIS3), and LSY813 (MAT msh5::URA3 exo1::HIS3), were crossed to make the diploids LSY899 (LSY810 x LSY811) and LSY900 (LSY812 x LSY813). The genotypes of the mating-type tester strains were: MATa suc2-437 lys2-802 (MCY14) and MAT lys2-802 (SJR13).

Statistical analysis:
Statistical comparisons were done using the Instat 1.12 program for Macintosh (GraphPad Software). Chi-square test or Fisher's exact variant were used for comparisons. Results were considered statistically significant if P < 0.05.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Reduced spore viability in diploids homozygous for the exo1 mutation:
The diploid strains W303 (EXO1/EXO1) and LSY529 (exo1/exo1) are isogenic except for the exo1 mutation. When these strains were sporulated after a limited number of generations (about 15) of vegetative growth following their construction, we found that the wild-type diploid had better spore viability than the homozygous exo1 diploid (94 vs. 87%; Table 1); by a chi-square test this difference is highly significant (P < 0.0001). Since strains with the exo1 mutation have elevated levels of spontaneous mutations (TISHKOFF et al. 1997 ), one interpretation of this result is that recessive lethal mutations accumulate in the diploid exo1 strain, leading to a loss of spore viability. By this interpretation, additional generations of vegetative growth of the diploid should lead to further reductions in spore viability. As shown in Table 1, although 60 generations of vegetative growth of LSY529 slightly reduced spore viability (from 87 to 79%), a reduction was also observed in the wild-type diploid (from 94 to 89%). We conclude, therefore, that the reduction in spore viability in the exo1 diploid is unlikely to reflect the accumulation of recessive lethal mutations.

Another potential cause of spore inviability is chromosome nondisjunction. A single nondisjunction event during meiosis I would be expected to result in two disomic spores and two spores missing one chromosome. Nondisjunction events involving two different chromosomes during meiosis I would be expected to result in either two double disome spores and two spores missing two chromosomes or four spores that are disomic for one chromosome and lack another. Thus, in strains with a moderate level of nondisjunction in meiosis I, one would expect to detect an elevation in classes of tetrads with two viable spores or with no viable spores. In contrast, classes of tetrads with three viable spores or one viable spore would not be substantially elevated as a consequence of meiosis I nondisjunction.

Two arguments are consistent with the conclusion that the exo1 mutations increase the rate of chromosome nondisjunction in meiosis I. First, when we compare the sum of the number of tetrads with two viable spores and no viable spores with the sum of the other classes of tetrads in exo1 and wild-type strains, we find a significant elevation in the exo1 strain (P < 0.0001). When we compare the sum of the number of tetrads with three viable spores and one viable spore with the sum of the other classes of tetrads, no significant elevation is observed in the exo1 strains (P = 0.6). The second argument is based on finding nonmating spores. If a nondisjunction event involves chromosome III (the location of the MAT locus), tetrads with two viable nonmating spores should be produced. We tested the mating phenotype of all of the spores derived from exo1 and wild-type strains shown in Table 1. As expected, all except six of the tetrads with four viable spores showed 4:4 segregation for MAT. The exceptions were gene conversion events observed in both strains. Of the tetrads with two viable spores, 11 of 93 from the LSY529 exo1 strains represented pairs of nonmating spores, indicative of chromosome III nondisjunction during meiosis I. No pairs of nonmating spores were observed for the wild-type strain. A second diploid version of LSY529 (LSY529b) was constructed in which one allele of EXO1 was disrupted with URA3 (exo1::URA3) and one allele with HIS3 (exo1::HIS3). As the EXO1 locus resides on chromosome XV, nondisjunction events involving chromosome XV can be detected by monitoring the segregation of these two alleles. From the 48 2:2 spores (Table 1), there were 2 with Ura⁺ His⁺ spores, indicative of nondisjunction of chromosome XV. This number might be an underestimate, as the Ura⁺ His⁺ spores grow very poorly, presumably due to the extra copy of chromosome XV.

Chromosome nondisjunction during meiosis II would be expected to produce tetrads with three viable spores and one inviable spore. None of the tetrads with three viable spores contained a nonmating spore, indicating no detectable meiosis II nondisjunction in the wild-type or exo1 strains. In addition, as described above, the fraction of tetrads with three viable spores was not significantly affected by the exo1 mutation. In summary, we conclude that the spore inviability in exo1 strains reflects an increase in chromosome nondisjunction during meiosis I.

A number of other yeast mutants have been identified that decreased spore viability associated with reduced intergenic recombination including msh4, msh5, mlh1, mlh3, zip1, zip2, and tam1 (SYM et al. 1993 ; ROSS-MACDONALD and ROEDER 1994 ; HOLLINGSWORTH et al. 1995 ; CHUA and ROEDER 1997 , CHUA and ROEDER 1998 ; WANG et al. 1999 ). The MSH4 and MSH5 genes are meiosis specific and encode homologues of bacterial MutS proteins (ROSS-MACDONALD and ROEDER 1994 ; HOLLINGSWORTH et al. 1995 ). Unlike other MutS homologues, msh4 and msh5 mutants show no apparent defect in mismatch correction during mitosis or meiosis. Instead, the proteins are proposed to form a heterodimer that binds to Holliday junctions and promotes resolution of recombination intermediates to form crossover products (POCHART et al. 1997 ). The meiotic phenotype of exo1 mutants resembles msh4 and msh5 mutants, except that the defect in spore viability in exo1 strains is less severe. mlh1 mutants also show a decrease in intergenic recombination and spore viability and epistasis studies indicate that MLH1 functions in the same pathway as MSH4 and MSH5 (HUNTER and BORTS 1997 ). To determine whether EXO1 functions in the same pathway as MSH5, double mutants were generated and examined for spore viability. In the W303 genetic background, a strain homozygous for the msh5 mutation (LSY899) had substantially reduced spore viability (55% spore viability from 230 tetrads dissected). In the diploid strain LSY900, homozygous for both msh5 and exo1, the spore viability was not significantly different from the msh5 homozygote (53% from 637 tetrads dissected). A strain homozygous for exo1 and msh4 mutations showed similar spore viability (47% from 200 tetrads dissected).

We also examined the effects of the exo1 and msh5 mutations on spore viability in a different genetic background [crosses of AS4- and AS13-derived haploid strains (STAPLETON and PETES 1991 )]. In the EXO1/EXO1 diploid strain DTK347, we observed 85% spore viability. In the exo1/exo1 strains DTK308, DTK332, DTK333, and DTK335 (differing in alleles at the HIS4 locus), the average spore viability was 78%, a significant (P < 0.0001) reduction from that observed for the wild-type strain. An msh5/msh5 strain (DTK498) had a spore viability of 80%, also a significant reduction (P = 0.0001) compared to wild type. The average spore viability in a strain homozygous for deletions in both exo1 and msh5 (DTK505) was 67%, a significant reduction compared to wild-type, msh5, and exo1 strains (P < 0.0001), but above the level expected for independent pathways (80% x 78% = 62.4%). Thus, in two different genetic backgrounds, spore viability is reduced by mutations in EXO1 and MSH5, and analysis of spore viability in the double mutant indicates that Exo1p and Msh5p function in the same or overlapping pathways for chromosome disjunction. This contrasts with the results obtained with exo1 msh4 homozygous diploids in two other strain backgrounds, in which synergistic decreases in spore viability were observed (KHAZANEHDARI and BORTS 2000 ; TSUBOUCHI and OGAWA 2000 ).

Reduced spore viability associated with elevated levels of meiosis I chromosome nondisjunction is often a consequence of decreased meiotic crossing over (ROEDER 1997 ). Consequently, as described below, we examined the effects of the exo1 mutation on the frequency of reciprocal and nonreciprocal meiotic recombination.

Reduced meiotic crossing over in strains homozygous for the exo1 mutation:
We examined meiotic recombination in four pairs of diploid wild type and exo1 mutant strains (Fig 1). Each diploid was constructed from AS4- and AS13-derived haploid strains and was heterozygous for mutations at the HIS4 locus (located on chromosome III), the linked (centromere-proximal) LEU2 locus, and the centromere-linked TRP1 locus. We monitored crossovers between HIS4 and LEU2 and between LEU2 and CEN3, as well as the frequency of aberrant segregation at the HIS4 locus. As will be discussed in more detail below, the four pairs of strains differed in the nature and/or position of the heterozygous his4 mutations.

View larger version (22K): In this window In a new window Download PPT slide   Figure 1. Location of markers within the his4 gene. Haploid strains LSY611, LSY613, DTK300, and LSY612 were made by one-step gene replacement of strains PD98, DNY25, DNY24, and DNY16, respectively, using a PCR-generated DNA fragment designed to disrupt the EXO1 gene. The vertical arrow indicates the position of the recombination initiation site upstream of the HIS4 gene; the horizontal arrows indicate the direction of transcription. The lollipops indicate the position of palindromic insertions within his4. The inverted triangle represents the nonpalindromic insertion within his4. Each haploid was crossed to the His+ strain DTK299 to create the diploids DTK308, DTK332, DTK333, and DTK335. The corresponding diploid strain number is given in parentheses.

As shown in Table 2, for all four pairs of strains the map distances between HIS4 and LEU2 and between LEU2 and the centromere were reduced by the exo1 mutation. This reduction was statistically significant (P < 0.03) for both genetic intervals in every strain pair, except for the HIS4-LEU2 interval in strains PD99 and DTK335 (P = 0.2). We conclude, therefore, that the exo1 mutation reduces but does not eliminate crossing over. It is likely that this reduction is responsible for the elevation in chromosome nondisjunction and the reduced spore viability observed in the exo1 strains.

The relationship between EXO1-dependent crossing over and the previously defined pathway for crossing over that involves MLH1, MLH3, MSH4, and MSH5 (described above) was determined by examining the level of intergenic recombination in msh5 and msh5 exo1 strains (Table 2). Deletion of MSH5 reduced the map distance between HIS4 and LEU2 to the same degree as a deletion of EXO1 [from 39 cM to 24 (msh5) and 22 cM (exo1), respectively]. The HIS4–LEU2 map distance in the double mutant (19 cM) was not statistically different than the distances in the single mutants, indicating that the two gene products function in the same pathway for intergenic recombination.

Effect of the exo1 mutation on aberrant segregation at the HIS4 and ARG4 loci:
Three of the pairs of AS4 x AS13 strains used in this study are heterozygous for palindromic insertions; one member of each pair was wild type and one member was homozygous for exo1. The location of these insertions relative to the HIS4 initiating codon are: -50 (his4-B2), +467 (his4-lopc), and +2327 (his4-3133) (Fig 1). The fourth pair of strains (DNY27 and DTK308) was heterozygous for a nonpalindromic insertion at the same position in HIS4 as the his4-lopc insertion. Tetrads from each strain were dissected and the level of aberrant segregation at the HIS4 locus was monitored (Table 3). All of the strains are also heterozygous for a mutation in ARG4; the level of aberrant segregation of this marker was also determined.

The level of aberrant segregation for strains containing a palindromic insertion at HIS4 is only slightly affected by the exo1 mutation (Table 3); the effects on aberrant segregation are considerably smaller than on crossing over. The exo1 mutation has its strongest effect on the level of aberrant segregation for the strain with the nonpalindromic insertion (compare DNY27 and DTK308); an interpretation of this result will be given below. The exo1 mutation does not significantly reduce the frequency of aberrant segregation of the palindromic insertion near the 3' end of HIS4 (comparison of PD98 and DTK335). Since most of the heteroduplexes located at the 3' end of HIS4 reflect processing of a DSB located at the 5' end of HIS4, this result indicates that Exo1p is not required for producing the single-stranded DNA "tail" required for heteroduplex formation.

The effect of deletion of exo1 on aberrant segregation of a heterozgyous allele at the ARG4 locus contrasts with the effect on aberrant segregation at HIS4. In the wild-type strain DNY27, 44 out of 359 tetrads exhibited aberrant segregation at ARG4 (24 6:2 events and 20 2:6 events). In the exo1 strains (DTK308, DTK332, DTK333, and DTK335) there were 37 tetrads of a total of 870 that exhibited aberrant segregation of the ARG4 marker (23 6:2 events and 14 2:6 events). This decrease in the level of aberrant segregation, from 12% in the wild type to 4% in the exo1 mutant strains, is highly significant (P < 0.0001). All events in both wild-type and exo1 strains were gene conversions; no postmeiotic segregation (PMS) tetrads were detected. Removal of Exo1p affects the observed level of aberrant segregation differently at HIS4 and ARG4. At HIS4, the data indicate that the level of aberrant segregation is not significantly affected by loss of Exo1p, whereas the level of aberrant segregation observed at the ARG4 locus is reduced. Locus-specific effects resulting from the loss of Exo1p have been reported previously (KHAZANEHDARI and BORTS 2000 ). This study on the effects of a deletion of EXO1 on the aberrant segregation rate of a number of two- or four-base insertions into HIS4 and LEU2 demonstrated that the HIS4 alleles, but not the LEU2 alleles, showed reduced aberrant segregation when EXO1 was deleted (KHAZANEHDARI and BORTS 2000 ).

Effect of the exo1 mutation on patterns of aberrant segregation at the HIS4 locus:
In strains heterozygous for a single marker, most tetrads segregate 4:4 (nomenclature used for eight-spored fungi). As discussed in the Introduction, the most common aberrant segregation types are of two classes, gene conversion events (6:2 or 2:6 segregations) or postmeiotic segregation events (5:3 or 3:5 segregation). PMS tetrads reflect asymmetric heteroduplex formation between wild-type and mutant alleles and failure to correct the resulting DNA mismatch. The efficiency of repair of a mismatch is reflected by the fraction of the aberrant segregation events that are PMS. In S. cerevisiae, single base mismatches (excluding C/C) and mismatches resulting from nonpalindromic insertions are efficiently repaired (low level of PMS compared to gene conversion), whereas C/C mismatches and mismatches resulting from small palindromic insertions are inefficiently repaired (PETES et al. 1991 ).

In the AS4 x AS13 genetic background, the HIS4 locus has a very high level of aberrant segregation (NAG et al. 1989 ). Most aberrant segregation events at the HIS4 locus reflect an initiating double-strand DNA break near the 5' end of the gene (FAN et al. 1995 ), followed by heteroduplex formation that often extends from the break to the 3' end of HIS4 (PORTER et al. 1993 ). One argument supporting the conclusion that heteroduplex formation usually involves the entire HIS4 coding region is that the level of aberrant segregation for markers leading to inefficiently repaired DNA mismatches at the 5' and 3' ends of the HIS4 gene is similar (DETLOFF et al. 1992 ). For most of the exo1 strains, there was a significant increase in the number of PMS events at the HIS4 locus compared with the wild-type diploids, confirming the role of Exo1p in mismatch repair (Table 3). Both the his4-B2 and his4-lopc strains showed a significant (P = 0.04 and 0.03, respectively) increase in the number of PMS tetrads when EXO1 was deleted. The greatest increase (P = 0.007) in such tetrads was seen in the strain DTK308 containing the well-repaired his4-lopd allele, which generates a 26-base loop when contained in a heteroduplex tract. We previously demonstrated (KIRKPATRICK and PETES 1997 ) that correction of this loop was also dependent on the products of the RAD1 and MSH2 genes, although deletion of either of these two genes has a greater effect on correction of his4-lopd mismatches than a deletion of EXO1. In all pairs of strains, the exo1 member of the pair had a reduced frequency of gene conversion relative to the wild-type member (Table 3). For example, in strains heterozygous for the nonpalindromic his4-lopd insertion, the gene conversion frequency was reduced from 26 to 14% by the exo1 mutation, and the PMS frequency was elevated from 4.3 to 7%. These results are the first demonstration of a role for Exo1p in DNA mismatch repair during meiosis; other studies did not show an increase in the frequency of PMS of heteroalleles when EXO1 was deleted (KHAZANEHDARI and BORTS 2000 ; TSUBOUCHI and OGAWA 2000 ). Also, in our study no increase in PMS was detected at the ARG4 locus, as described above. The alleles that show no increase in PMS are those that will generate a small mismatch (one to four bases) when present in heteroduplex, while the alleles that have a significant increase in PMS are those that will generate a large mismatch (26 to 36 bases) in heteroduplex DNA. We propose that Exo1p may have a role specifically in the repair of large mismatches.

Conclusions:
In conclusion, we find that Exo1p promotes meiotic crossing over, ensuring proper disjunction of chromosomes during meiosis I. This function involves a previously identified pathway involving the MSH4, MSH5, MLH1, and MLH3 gene products (ROSS-MACDONALD and ROEDER 1994 ; HOLLINGSWORTH et al. 1995 ; HUNTER and BORTS 1997 ; WANG et al. 1999 ), as the level of crossovers in strains lacking both Exo1p and Msh5p is higher than expected if the two proteins work in distinct pathways. Loss of Exo1p also leads to a decrease in spore viability. This decrease is not due to the accumulation of mutations during mitotic growth and is partially overlapping with the decrease in spore viability seen in strains lacking Msh5p. Furthermore, the length of the heteroduplex tract at the HIS4 locus does not appear to be reduced in exo1 mutant strains, indicating that the Exo1p deoxyribonuclease does not participate in generating long 3' single-stranded tailed molecules following double-strand break generation during meiotic recombination at HIS4. It is possible that the nuclease function of Exo1p acts postsynaptically to direct resolution of recombination intermediates instead of the predicted presynaptic function. Alternatively, the EXO1 protein could participate in generating a small amount of single-stranded DNA, after which another protein or protein complex continues the process of generating the long 3' single-stranded tailed molecules present at HIS4. In addition, we find that the exo1 mutation affects the frequency of aberrant segregation in a manner that suggests that Exo1p has a minor role in the repair of DNA mismatches at HIS4. Exo1p is involved in the repair of a 26-bp loop mismatch in heteroduplex DNA, although to a lesser extent than several other proteins (Rad1p and Msh2p) known to act on DNA loops of this size (KIRKPATRICK and PETES 1997 ). Finally, there are locus-specific effects to the loss of Exo1p, as the level of aberrant segregation at ARG4 is significantly lowered by loss of Exo1p, unlike the situation at HIS4. Also in contrast to the data from the HIS4 locus, repair of mismatches at ARG4 is not affected by the exo1 mutation, indicating that at ARG4 initiation, rather than repair, is primarily affected by the exo1 mutation.

	FOOTNOTES

¹ Present address: Department of Genetics, Cell Biology, and Development, University of Minnesota, 250 Biological Sciences Bldg., 1445 Gortner Ave., St. Paul, MN 55108.

	ACKNOWLEDGMENTS

This work was supported by Public Health Service grants from the National Institutes of Health GM41784 (L.S.S.), 2 T32 CA09503 (J.R.F.), and GM24110 (T.D.P.). D.T.K. is a Special Fellow of the Leukemia and Lymphoma Society.

Manuscript received July 23, 1999; Accepted for publication September 6, 2000.

	LITERATURE CITED

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