INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Homologous recombination events can be either reciprocal or nonreciprocal in nature. Reciprocal recombination (crossing over) alters the linkage relationships of loci that flank the site of an exchange, whereas nonreciprocal recombination (gene conversion) is defined as the unidirectional transfer of information from one DNA molecule to another. All present models of recombination stipulate the formation of a heteroduplex recombination intermediate, which is formed by complementary base pairing of single strands derived from different duplexes. Correction of a mismatch in heteroduplex DNA can result in a gene conversion event, and such correction is effected by the same mismatch repair (MMR) machinery that corrects errors made during DNA replication. Cocorrection of a series of contiguous mismatches generates a conversion tract, the length of which is considered to be a minimal estimate of the extent of the heteroduplex intermediate formed. The point at which the recombining molecules exchange single strands to form heteroduplex DNA is referred to as a Holliday junction, and cleavage of this junction gives rise to crossovers and noncrossovers at roughly equivalent frequencies.

Homologous recombination usually involves allelic sequences on homologous chromosomes but also can occur between dispersed (ectopic) repeated sequences. Ectopic gene conversion provides a mechanism for homogenizing repeated sequences and hence is important in the concerted evolution of multigene families. In contrast to the relatively benign effects of ectopic gene conversion, ectopic crossing over results in various types of chromosome rearrangements which destabilize overall genome structure. Given the large amount of repetitive DNA present in the genomes of higher eukaryotes and the potentially deleterious nature of ectopic interactions, one would predict that allelic interactions should be highly favored over ectopic interactions. Two features of repeated sequences that might be important in limiting ectopic recombination are the length of the repeats and the degree of sequence identity between the repeats (38).

Studies with bacterial species (1, 22, 28, 29, 39, 46, 51, 57-59), Saccharomyces cerevisiae (4, 7, 12, 13, 20, 24, 30, 32, 33, 35, 36, 40, 43, 44), and higher eukaryotes (14, 15, 52, 56) have uniformly found that sequence divergence acts as a potent barrier to recombination. As first shown in conjugational crosses between Escherichia coli and Salmonella typhimurium (39), much of the recombination barrier associated with sequence divergence in prokaryotes derives from the action of the MMR machinery (1, 16, 22, 28, 29, 51, 57). The MMR system of E. coli (31) is used as a paradigm for eukaryotic MMR, and homologs of the bacterial MutS protein, which binds mismatched base pairs, and of the MutL protein, which interacts with MutS, have been identified in eukaryotes (11). In E. coli, elimination of either MutS or MutL results in a comparable increase in recombination between diverged sequences (1, 39). The antirecombination activity of MMR proteins also has been documented in yeast (7, 12, 13, 24, 32, 43, 44) and mammalian cells (9, 14), indicating that the barrier to recombination imposed by the MMR system is evolutionarily conserved. Interestingly, however, the antirecombination activities of yeast MutS and MutL homologs (Msh2p and Pms1p, respectively) are not equivalent. In yeast, elimination of Msh2p elevates recombination between diverged sequences to a greater extent than does elimination of Pms1p (references 12 and 44 and unpublished data), indicating that the antirecombination effect of Msh2p is not totally dependent on Pms1p. This observation is consistent with in vitro DNA binding studies demonstrating that the yeast MutL homologs increase the mismatch binding efficiency of the MutS homologs (19).

Systematic studies carried out with bacterial cells (28, 51) and yeast (13) have revealed a log-linear relationship between the rate of recombination and the degree of sequence divergence in both MMR-competent and MMR-defective cells. Such a relationship is predicted by models that assume that a minimal length of perfect homology (the "MEPS" [45]) is necessary to initiate recombination; the number of MEPS decreases exponentially with sequence divergence. In the yeast experiments, a single mismatch within a 350-bp substrate was sufficient to reduce recombination fourfold, and this inhibition was completely dependent on the MMR machinery (13). Additional mismatches had a cumulative negative effect on recombination; some of the inhibition was due to MMR-associated antirecombination activity, and some resulted from an MMR-independent process. The MMR-independent inhibition of recombination was attributed to an inability of the recombination machinery to recombine diverged sequences efficiently.

The antirecombination activity of MMR proteins presumably derives from the recognition of mismatches in heteroduplex recombination intermediates, but how this impairs recombination is unclear. Because of the strand-nicking activity associated with the repair of replication errors in E. coli, a heteroduplex destruction model has been considered. According to this model, the attempted repair of multiple mismatches in close proximity would create convergent excision tracts and hence double-strand breaks, which would destroy the recombination intermediate (see reference 6). The efficiencies of transformation of highly mismatched heteroduplex molecules into MMR-competent versus MMR-defective E. coli are similar, however, and this finding is not consistent with the destruction of mismatched heteroduplex DNA (53). Also, there is no evidence of MMR-associated strand nicking in eukaryotes, and such nicking would be a prerequisite for heteroduplex destruction. A second model that has been proposed is the heteroduplex rejection model, which posits that MMR proteins modulate the formation of mismatch-containing heteroduplex DNA (3, 10, 57). One possibility is that MMR-associated helicase activity unwinds mismatch-containing heteroduplex DNA, thus resulting in rejection of the donor strand (10, 57). Alternatively, the MMR machinery might interact directly with the recombination machinery to cause either reverse branch migration or immediate resolution of a recombination intermediate when mismatched heteroduplex is detected (3). A specific prediction of heteroduplex rejection models is that the extent of heteroduplex DNA should be longer in MMR-defective cells than in wild-type cells. Consistent with such a model, Worth et al. (55) have demonstrated that both the rate and the extent of in vitro RecA-catalyzed heteroduplex formation were reduced in the presence of purified MutS and MutL proteins.

One approach to understanding the molecular basis of MMR-associated antirecombination activity is to ask whether recombination products in MMR-competent cells differ significantly from those in MMR-defective cells. Several parameters can be examined in experiments of this sort. One can, for example, examine the ratio of gene conversions to crossovers in order to determine if the MMR machinery biases the resolution of recombination intermediates in a mismatch-dependent manner (see, e.g., reference 5). Alternatively, comparison of gene conversion tracts in MMR-competent and MMR-defective cells will indicate whether the molecular nature of recombination intermediates is impacted by the MMR machinery. The endpoints of the conversion tracts can be examined, or the lengths of the tracts can be determined. A difference in the endpoints of conversion tracts between MMR-competent and MMR-defective cells would imply a role of the MMR machinery in determining preferential sites of recombination initiation and/or resolution; a difference in the lengths of conversion tracts would imply a role for the MMR machinery in monitoring the fidelity of base pairing during heteroduplex formation.

In the present study, we have used an intron-based inverted-repeat (IR) assay system to characterize mitotic gene conversion tracts in wild-type versus MMR-defective yeast strains. With the intron-based system, one selects specifically for events that reverse the orientation of the region between the recombination substrates; reorientation of this region (the invertible segment) can result from either intrachromatid crossover or sister chromatid conversion. Using 94%-identical substrates, we have found an MMR-dependent conversion gradient across the substrates, with an excess of conversion tract endpoints in identity intervals closest to the invertible segment in MMR-competent cells. Such clustering is most easily explained by assuming that recombination occurs predominantly through a sister chromatid conversion mechanism rather than via intrachromatid crossover. Conversion tract length calculations based on an unequal sister chromatid conversion model indicate that conversion tracts are significantly longer in MMR-defective cells than in wild-type cells. The relevance of the MMR-dependent shortening of conversion tracts to the antirecombination activity of MMR proteins is discussed.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Plasmid constructions. Plasmids with various chicken -tubulin isoform 2 cDNA sequences (c2 sequences) were constructed according to the general scheme outlined in Fig. 1 (see references 12 and 13 for more details). 5' and 3' cassettes were derived by replacing sequences in pSR266 (the basic HIS3::intron construct) with PCR-amplified c2 sequences. To construct plasmids pSR424, pSR584, and pSR610, a SmaI/SpeI restriction fragment containing the 5' cassette was inserted into a SpeI/NotI-digested plasmid containing the 3' cassette (the NotI site was filled in with the Klenow fragment of DNA polymerase). To construct plasmid pSR612, a SmaI/SpeI restriction fragment containing the 5' cassette was inserted into a NotI-digested plasmid containing the 3' cassette; the ends of both fragments were filled in with the Klenow fragment of DNA polymerase.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Construction of inverted-repeat substrates. The pGAL-HIS3::intron construct contained on plasmid pSR266 is shown at the top. Open boxes correspond to HIS3 sequences, solid boxes to artificial intron sequences, and shaded boxes to c2 sequences; boxes are not drawn to scale. The positions of the oligonucleotides used as PCR primers are indicated by arrows numbered as follows: 1, HIS3-702F; 2, HIS3-1751F; 3, HIS3-765R; 4, T3. The left recombinant c2 segment was amplified with primers 1 and 3; the right recombinant segment was amplified with primers 2 and 4. Only those restriction sites relevant to constructions are shown. RI, EcoRI; Sm, SmaI; Bam, BamHI; Spe, SpeI; Not, NotI; Bgl, BglII.

View larger version (66K): [in this window] [in a new window]   FIG. 2.   (A) Alignment of recombination substrates. Sequences of perfect identity are boxed and shaded. Although we refer to positions within the aligned sequences using the numbering system shown, it should be noted that the homology between c2 and c2-21mm does not actually begin until position 7. During the generation of the c2-21mm substrate, an unusually high density of PCR errors apparently was introduced at the 5' end of the product. The homology between c2 and c2-21mm ends at position 349, for a total of 343 bp of partially homologous (homeologous) sequence. There are 21 mismatches, 2 of which are contiguous, within the 343-bp region of homology. (B and C) Only nucleotides from position 301 are shown; restriction sites used in the restriction site polymorphism analyses are indicated above or below the alignments.

Yeast strain constructions. All strains used in this study were derived from isogenic strains SJR231 (MAT ade2-101_oc his3200 ura3-Nhe), GCY121 (MAT ade2-101_oc his3200 ura3-Nhe msh2 msh3::hisG), and GCY128 (MAT ade2-101_oc his3200 ura3-Nhe pms1) (see reference 12) by lithium acetate transformation (18). pSR424, pSR584, pSR610, or pSR612 was digested with StuI prior to transformation in order to target integration to the URA3 locus. Following selection of Ura⁺ transformants, single-copy integration of the relevant plasmid was confirmed by PCR or Southern blot analysis.

Generation of independent recombinants. One-milliliter cultures were grown nonselectively at 30°C for two days in YEP medium (1% yeast extract-2% Bacto Peptone) supplemented with 2% glycerol and 2% ethanol (YEPGE). Cells were harvested, washed once with H₂O, and resuspended in 200 µl of H₂O. An aliquot of 100 µl was plated on SGGEHis selective medium (synthetic complete medium supplemented with 2% glycerol, 2% galactose, and 2% ethanol but deficient in histidine) to select for His⁺ recombinants. Only one colony was taken from each culture in order to ensure that all recombinants analyzed were of independent origin.

Molecular analysis of recombinants. Genomic DNA was extracted by glass bead lysis (21) from each recombinant and used as a template for PCR amplification. The 5' and 3' recombination products were amplified by using primers homologous to sequences that flank the recombination substrates (see Fig. 1 for the positions of primers). The 5' product was amplified by using primers HIS3-702F (5'-GTTTCTGGACCATATG) and HIS3-765R (5'-GCACTCAACGATTAG), and the 3' recombination product was amplified by using primers HIS3-1751F (5'-GATGGCAAACATGTC) and T3 (5'-TGATGTCGGCGATATAGG). The PCR products were purified by using Qiaquick Spin Columns (Qiagen) and were used as templates for DNA sequencing. Oligonucleotides FAI (5'-ATGGACTAAAGGAGGCT) and T3 were used as sequencing primers for the 5' and 3' recombination products, respectively. All sequencing reactions were carried out with ABI Prizm Dye Terminator Cycle Sequencing Ready Reaction Kits and run on an ABI Prizm 377XL DNA Sequencer (Perkin-Elmer Applied Biosystems).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

The intron-based IR recombination system. The relevant features of the intron-based IR recombination system are diagrammed in Fig. 1 (for a more complete description of the system, see references 12 and 13). A galactose-inducible HIS3 gene containing an artificial intron (HIS3::intron) was the starting point for all constructs. 5' or 3' recombination cassettes were constructed by replacing sequences downstream of the 5' intron splice consensus element or upstream of the intron TACTAAC element, respectively, with a fragment derived from c2 cDNA. 5' cassettes contained the 5' end of HIS3, the 5' portion of the intron, and a c2 recombination substrate, whereas 3' cassettes were composed of a different c2 recombination substrate, the 3' portion of the intron, and the 3' end of HIS3. The 5' and 3' cassettes were combined in reverse orientation relative to each other, resulting in the 3' portion of HIS3::intron being flanked by c2 IRs. Following integration of the IR construct at the URA3 locus in an appropriate his3 strain, cells are phenotypically His. Recombination between the c2 repeats can reverse the orientation of the intervening region (referred to below as the invertible segment), thus placing the 5' and 3' parts of the HIS3 gene in the same orientation and reconstituting a functional intron. Such recombinants are phenotypically His⁺ and can be identified on an appropriate selective medium.

Gene conversion tracts in an MMR-competent strain. The initial c2-derived substrates used to examine gene conversion tracts were 350 bp and 94% identical (c2 and c2-21mm). c2-21mm was generated from a c2 plasmid template by low-fidelity PCR. The c2-c2-21mm pair of substrates was chosen for sequencing studies because the potential mismatches are more randomly distributed than those in naturally diverged sequences. As illustrated in Fig. 2A, the mismatches divide the substrates into 21 intervals of perfect identity ranging in size from 2 to 37 bp. The c2 substrates were oriented so that their 3' ends were proximal and their 5' ends were distal to the intervening invertible segment (orientation 1 in Fig. 3). Following the isolation of a His⁺ recombinant, each recombinant c2 segment was amplified by PCR using flanking primers (see Fig. 1), and the PCR products were sequenced individually. A given mismatch was considered to have undergone a gene conversion event if the same nucleotide was present in both recombinant c2 segments; if the recombinant c2 segments still differed at the position of the original mismatch, then the site was considered not to have undergone gene conversion. A gene conversion tract encompasses a series of contiguous mismatches.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Orientation of c2 substrates relative to the HIS3::intron invertible segment. Arrows indicate the 5'-to-3' (nucleotides 1 to 350, respectively, as in Fig. 2) orientations of the c2 sequences.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Conversion tract endpoint distributions. The percentage of endpoints expected in each interval was subtracted from the observed percentage, and the residual value was plotted. Positive or negative percentages indicate excesses or deficits of endpoints, respectively. WT, wild type.

Analysis of conversion tract endpoints in interval 318 to 350 by using a restriction site polymorphism. A restriction site polymorphism just upstream of interval 318 to 350 was used to more accurately quantify the number of conversion tracts that end in this interval. A mismatch at position 309 establishes a TaiI site (5'-ACGT-3') in the c2 substrate but a HinP1I site (5'-GCGC-3') in the c2-21 mm substrate. Conversion tracts that either start or end in interval 318 to 350 are assumed to include the mismatch at position 309, and thus both recombination products should have either a TaiI site or a HinP1I site. The observation that only 1 of the 62 recombinants sequenced had an endpoint in interval 309 to 318 validates the use of the polymorphism at position 309 as a marker for assigning endpoints to interval 318 to 350. Forty-seven additional His⁺ recombinants were isolated and were analyzed for the presence or absence of the restriction site polymorphism. Eighteen (38%) of the recombinants converted the mismatch at position 309. This value agrees well with the DNA sequencing results, where 34% (21 of 62 total recombinants sequenced) of the recombinants had an endpoint(s) in this interval. It should be noted that the restriction site polymorphism studies indicate whether a given recombinant has a conversion tract endpoint in interval 318 to 350 but provide no information as to whether the underlying event is simple or complex. Because of this uncertainty, the percentage of recombinants with an endpoint(s) in interval 318 to 350, instead of the percentage of total endpoints in this interval, was calculated for all constructs.

Division of interval 318 to 350. A divided construct (Fig. 3) was created in order to determine whether the length of interval 318 to 350 is important for its hot spot activity. The divided construct was derived by introducing a T-to-A transversion at position 334 in c2-21mm, which creates an additional mismatch between the recombination substrates and splits interval 318 to 350 into two smaller intervals of equal length (see Fig. 2B). Conversion of the TaiI/HinP1I restriction site polymorphism at position 309 was used to monitor tracts that ended in the 318-to-350 region of the divided construct. Sixty recombinants were examined, and 40% (24 of 60) had an endpoint in interval 318 to 350. This percentage is similar to that obtained with the undivided orientation 1 construct (34 and 38% by DNA sequence and restriction site polymorphism analysis, respectively) and is different from the expected value (P < 0.01 by the ² test). Based on the results obtained with the divided construct, we conclude that the excess of endpoints in interval 318 to 350 in the undivided orientation 1 construct is not due to the length of uninterrupted identity in this interval. In addition to dividing interval 318 to 350, the T334A mutation in the c2-21mm sequences created a DdeI site (5'-CTNAG-3'). This new polymorphism was used to further refine the positions of endpoints within the two halves of interval 318 to 350. Sixteen of the 24 recombinants had an endpoint in interval 318 to 334, 7 had an endpoint in interval 334 to 350, and 1 had an endpoint in each interval.

Extension of the 3' ends of the substrates. An extended construct (Fig. 3) was created in order to test the hypothesis that interval 318 to 350 is a hot spot because it is at the extreme end of the substrate and hence borders a region of complete nonhomology. The original 350-bp substrates were extended at the 3' end by adding two additional intervals of perfect identity (see Fig. 2C). The first additional interval (interval 347 to 368) in the extended construct was 20 bp, and the second (interval 368 to 375) was 6 bp. A restriction site polymorphism was created by mutations at positions 346 and 347 (C346A and C347T), thus creating an EcoRV site (5'-GATATC-3') in the extended c2-21mm sequences while maintaining a BamHI site (5'-GGATCC-3') in the extended c2 sequences. The two introduced mismatches separate the previous 318 to 350 interval (318 to 346 in the extended construct) from the newly added intervals 347 to 368 and 368 to 375. By monitoring the conversion of the mismatches at positions 309 and 346/347, we were able to determine how many conversion tracts ended either in interval 318 to 346 or in interval 347 to 375. Among 49 recombinants analyzed, 17 (35%) had an endpoint in interval 318 to 346. This percentage is very similar to the proportion of orientation 1 recombinants with endpoints in interval 318 to 350 and is significantly different from the expected value (P = 0.01 by the ² test). Interestingly, the two new intervals in the extended construct also contained a disproportional number of endpoints. Because the new intervals contain 7.6% (26 of 344) of the nucleotide identity in the extended substrates, approximately 15% of recombinants would be expected to contain an endpoint in the extended region. Thirteen of the 49 recombinants (27%), however, had an endpoint in the newly added intervals, which is significantly more than expected (P < 0.05 by the ² test). Based on the results with the extended substrates, it appears that an interval needs only to be near, rather than at, the 3' end of the c2 substrate in order to contain an excess of conversion tract endpoints. This result is consistent with the general observation (Fig. 4A) that there is an excess of endpoints in the 3' halves of the substrates and a deficit of endpoints in the 5' halves. This "gradient" either could be related to the lower density of mismatches in the 3' halves of the substrates or could reflect the proximity of the 3' ends of the substrates to the invertible segment. These two possibilities were distinguished by reversing the orientations of the c2 substrates relative to the invertible HIS3::intron sequences.

Reorientation of the substrates relative to the invertible segment. The orientation of each of the c2 substrates was reversed relative to the HIS3::intron sequences, thus placing the original 3' substrate ends (with an excess of endpoints) distal to the invertible segment and the original 5' ends (with a deficit of endpoints) adjacent to the invertible segment. Substrates in the reverse orientation will be referred to as being in orientation 2 (see Fig. 3). As described above, the proportion of recombinants with a conversion tract endpoint in interval 318 to 350 (note that the same nucleotide receives the same coordinate for orientations 1 and 2) was analyzed by using the TaiI/HinP1I restriction site polymorphism. Only 23% (11 of 48; P > 0.5 by the ² test for this number of endpoints compared to that expected) of the orientation 2 recombinants had an endpoint in interval 318 to 350, compared to 34 to 40% of recombinants with an endpoint in this interval with the orientation 1 construct, the divided construct, and the extended construct. This result suggested that altering the orientation of the recombination substrates might have reversed the gradient of conversion tract endpoints evident with the orientation 1 substrates. To examine this further, the first 26 recombinants derived by using the orientation 2 substrates were subjected to DNA sequence analysis in order to determine the precise positions of all conversion tract endpoints. Twenty-two recombinants had continuous conversion tracts, two had complex conversion tracts, and two had no detectable conversion tract. This distribution of recombinant classes was the same as that obtained with the orientation 1 substrates (50 continuous tracts, 6 complex tracts, and 6 simple crossovers; P > 0.90 by the ² contingency test). The distribution of conversion tract endpoints for the orientation 2 substrates is given in Table 1 and is compared graphically to the expected distribution in Fig. 4B. Whereas recombinants derived from the orientation 1 c2 substrates had an excess of endpoints near the 3' ends of the substrates (Fig. 4A), the orientation 2 substrates yielded recombinants with a clear excess of endpoints in the 5' halves of the c2 sequences. These data suggest that the distribution of endpoints is determined primarily by the relative proximity of an interval to the invertible HIS3::intron segment located between the substrates rather than by interval size or sequence. In other words, intervals close to the invertible segment are more likely to contain a conversion tract endpoint than are intervals that are farther away from the invertible segment.

Conversion tract endpoints in MMR-defective cells. The yeast MMR system exerts a strong antirecombination effect on the 94%-identical substrates used in the conversion tract analysis above (13). Therefore, we examined gene conversion tracts in cells defective in mismatch binding activity (an msh2 msh3 mutant) in order to ascertain whether the MMR system impacts the nature of recombination intermediates. Because there is no gene conversion via MMR of heteroduplex DNA in MMR-defective cells, any heteroduplex formed during recombination will be segregated at the next round of DNA replication. Replication of the unrepaired donor DNA thus will produce the equivalent of a gene conversion tract.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study, an intron-based recombination assay system has been used to generate recombinants between a pair of 94%-identical substrates oriented as IRs (see Fig. 1). The IR substrates flank the 3' end of an intron-containing HIS3 gene, which is in reverse orientation relative to the 5' end of the gene. Reorientation of the 3' HIS3::intron segment via recombination involving the flanking IRs reconstitutes a functional HIS3 gene, and such events can be identified as His⁺ colonies on histidine-deficient medium.

Although reorientation or flipping of a segment of DNA is generally considered to result from intrachromatid crossing over between flanking IRs (Fig. 5A), a sister chromatid gene conversion event also can flip the region between IRs (42). In sister chromatid gene conversion (Fig. 5B), one of the chromatids loops around and pairs with the sister; the substrate upstream of the invertible segment on one chromatid pairs with the downstream substrate on the sister chromatid, and the substrate downstream of the invertible segment likewise pairs with the upstream substrate on the sister. The invertible segment is thus flanked by sister-sister pairings, each of which involves diverged rather than identical substrates. Gene conversion events that initiate in the paired sequences on one side of the invertible segment, extend through the invertible segment, and terminate in the paired sequences on the other side will flip the invertible segment. It should be noted that neither intrachromatid gene conversion nor sister chromatid crossing over will yield His⁺ recombinants. Intrachromatid gene conversion does not reorient the invertible segment, so recombinants remain His; sister chromatid crossing over gives rise to acentric and dicentric recombinant chromosomes, resulting in inviable progeny.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Reversal of the orientation of a segment of DNA between IRs by either intrachromatid cross over or sister chromatid gene conversion. The open and shaded boxes correspond to the IR substrates that flank the invertible HIS3::intron segment, which is represented by a loop with an arrowhead indicating the orientation. Small open circles represent centromeres.

Distributions of conversion tract endpoints in a wild-type strain. DNA sequence analysis of both products derived from individual recombination events allowed for the determination of conversion tract endpoints. In the discussion that follows, it is assumed that conversion tracts are an accurate representation of the extent of heteroduplex DNA present in recombination intermediates. We recognize the formal possibility, however, that conversion tract endpoints may not necessarily correspond to positions where recombination events begin and end. At least in MMR-competent cells, endpoints could correspond to repair borders. We also note that the repair of heteroduplex DNA in mitosis is not likely to be 100% efficient (41), so some of the conversion tracts in MMR-competent cells may arise via replicative resolution of heteroduplex intermediates, which is the likely mechanism for generating conversion tracts in MMR-defective cells. Regardless of the mechanism for generating a conversion tract, however, this mechanism presumably operates after the completion of heteroduplex DNA formation and thus should not affect the extent of heteroduplex formed. In determining conversion tract parameters, only the no-conversion and continuous, asymmetric conversion tract classes, which together accounted for 90% of His⁺ recombinants, were considered. The relative rarity of symmetric or discontinuous conversion tracts is in general agreement with mitotic data obtained in previous studies (2, 49).

Distributions of conversion tract endpoints in MMR-defective strains. Conversion tract endpoints were determined in MMR-defective strains in order to ascertain whether endpoint distributions are influenced by the yeast MMR machinery. The clear endpoint clustering evident with the c2 substrates in either orientation 1 (Fig. 4A) or orientation 2 (Fig. 4B) was abolished in an msh2 msh3 background (Fig. 4C and D). Similarly, in a pms1 mutant (Fig. 4E) there was no indication of the prominent endpoint clustering that was evident in wild-type cells. The data presented in Fig. 4 thus demonstrate that the biased endpoint distributions evident in wild-type cells are dependent on a functional MMR system. As will be elaborated further below, we speculate that the alteration of conversion tract endpoints by MMR proteins is related to the documented antirecombination activity of these proteins.

Intrachromatid crossover versus sister chromatid conversion. As shown in Fig. 6, estimates of conversion tract lengths are very different for intrachromatid crossovers and sister chromatid conversion events that have the same endpoints. In principle, an intrachromatid crossover can be distinguished from sister chromatid conversion if the recombination intermediate can be captured and analyzed physically or if the timing of the recombination event in the cell cycle (G₁ versus G₂) can be determined. Given the low frequency with which our diverged substrates recombine, however, neither approach is feasible at present. As argued below, however, we believe that the majority of the His⁺ recombinants detected by our assay system occur via sister chromatid conversion rather than via intrachromatid crossover.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Estimation of conversion tract lengths. Open and shaded boxes correspond to IR substrates with four hypothetical mismatches (a through d), which divide the 350-bp substrates into five 70-bp intervals of perfect identity (1 through 5). As shown, intrachromatid and sister chromatid events that have identical endpoints (one endpoint in interval 2 and the other in interval 4) correspond to conversion tracts of different lengths: 140 and 350 bp for intrachromatid crossover and sister chromatid conversion events, respectively. If the products are viewed linearly along one chromosome, however, they appear identical.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   Replication of an asymmetric heteroduplex intermediate formed during intrachromatid crossing over in a MMR-defective strain. Both strands of the DNA duplexes (1 and 2, 1 and 1', or 2 and 2') are shown, with arrows indicating the 3' ends. Thin and thick vertical lines correspond to the inverted substrates that flank the invertible HIS3::intron segment (represented by a loop with the orientation indicated by an arrowhead). Dashed horizontal lines (a and b) indicate conversion tract endpoints. Resolution of the asymmetric heteroduplex intermediate yields two His+ products, of which one has a conversion tract (right) and the other appears as a simple crossover (left).

Lengths of conversion tracts in wild-type cells. We believe that our data are most consistent with the sister chromatid conversion model, which involves coconversion of sequences flanking the invertible HIS3::intron segment. The extent of a conversion tract thus becomes the sum of two conversion tracts, one on either side of the invertible segment. Furthermore, those events with no conversion tract would not have two endpoints in the same interval but rather would have an endpoint in each of the relevant identical intervals that flank the invertible segment. For each recombinant examined, DNA sequencing data were used to calculate minimal, maximal, and average tract lengths. Table 2 presents the mathematical mean values for the minimal, maximal, and average tract lengths, as well as the mean number of mismatches included in conversion tracts. With the c2 substrates in orientation 1, the mean of the average conversion tract lengths was 275 bp in wild-type cells, and tracts included an average of 14.4 mismatches. Very similar results were obtained with the orientation 2 c2 substrates, where the mean of the average conversion tract lengths was 230 bp and tracts included an average of 14.2 mismatches. The mean of the average tract lengths (as well as the means of the minimal and maximal lengths) was slightly larger with the orientation 1 substrates than with the orientation 2 substrates, although the mean number of mismatches converted was the same. This likely reflects the fact that the orientation 1 substrates had the end with the lower mismatch density (the 3' end) closest to the invertible segment (whose conversion was selected for), whereas the orientation 2 substrates had the end with the higher mismatch density closest to the invertible segment. The relevant parameter for regulating heteroduplex length in MMR-competent cells thus appears to be the number of mismatches traversed. An examination of the distribution of conversion tract endpoints for the orientation 1 and orientation 2 substrates (Fig. 4A and B, respectively) is consistent with this interpretation. As noted previously, there is an excess of endpoints proximal to the invertible segment for both substrate orientations. If 14 total mismatches are included in heteroduplex intermediates, then conversion tracts should include an average of 7 mismatches on each side of the invertible segment and the transition from an excess to a deficit of endpoints should occur approximately 7 mismatches from the invertible segment. As predicted, the position of the shift from an excess of endpoints to a deficit of endpoints occurs seven and eight mismatches away from the invertible segment for the orientation 1 and orientation 2 substrates, respectively.

Lengths of conversion tracts in MMR-defective cells. Gene conversion tracts presumably arise via repair of heteroduplex DNA and replicative segregation of heteroduplex DNA, respectively, in wild-type and MMR-defective cells. Although they are generated in mechanistically distinct manners, we suggest that conversion tracts generated in the presence or absence of MMR should nevertheless be an accurate reflection of the underlying heteroduplex recombination intermediate. Measuring gene conversion tracts may not be the ideal way to obtain estimates of heteroduplex DNA in mitotically dividing cells, but it is presently the only way to do so.

Relation of conversion tract lengths and endpoints to the antirecombination activity of MMR proteins. If one assumes that gene conversion tracts are an accurate representation of the extent of heteroduplex DNA formed in the presence of MMR proteins, then our data indicate that MMR proteins regulate the formation of heteroduplex DNA during mitotic recombination. Specifically, heteroduplex tracts are shorter and cover fewer mismatches in wild-type cells than in MMR-defective cells (Table 2). The tract shortening can account for the biased distribution of conversion tract endpoints observed in wild-type cells, where endpoints were clustered at the substrate end closest to the invertible segment (Fig. 4). To our knowledge, no other model could account for such an MMR-dependent clustering of conversion tract endpoints. The idea that MMR proteins might regulate heteroduplex formation when mismatches are present is not a new one (see references 10 and 37), but exactly how this might occur is not clear. MMR proteins could monitor the fidelity of the initial strand exchange reaction, they could regulate the extension of heteroduplex DNA, or they could bias how recombination intermediates are resolved. It also is possible that these proteins could act at more than one step. Recent data from mammalian cells suggest that the initiation, but not the extension, of heteroduplex formation is blocked by mismatches, although the involvement of the MMR machinery was not examined (56). Based on studies of meiotic gene conversion polarity gradients, Alani et al. (3) suggested that MMR proteins specifically block the extension of symmetric heteroduplex intermediates and should, therefore, bias resolution in favor of noncrossovers (see also reference 23). The relevance of the meiotic studies to the data reported here, however, is unclear, since our assay involves recombination between highly mismatched substrates in mitosis.