Intrachromosomal recombination between direct or indirect repeats. (A) Recombination between two direct repeats. Here, Leu⁺ cells can arise by a deletion or a pop-out event that removes all the intervening sequences (top) or by a simple gene conversion of one of the two repeats (bottom). Both kinds of event involve only one chromatid. (B) Another case of recombination between direct repeats. Leu⁺ cells can arise by simple gene conversion (top). However, because of the orientation of the mutations, deletion is unlikely to result in a functional LEU2 gene, but a LEU2 gene can result from a USCE. Crossing over between one repeat from one chromatid and a second repeat from the other chromatid will result in a triplication (bottom). (C) Another case of USCE. The proximal leu2 copy is deleted at the 5′ end, and the distal one is deleted at the 3′ end. USCE can reconstitute a LEU2 copy. (D) Selection of deletion events between truncated direct repeats. (E) Selection of recombination between indirect repeats. Deletions cannot occur. Obtaining Leu⁺ cells depends on gene conversion events not associated or associated with crossing over (top and bottom, respectively).

New restriction endonuclease fragments produced by reciprocal recombination. The appearance of these new restriction fragments can be monitored by Southern blotting at various time points of meiosis. Vertical lines stand for the restriction sites used in the diagnostic assay. P1 and P2, parental restriction fragments; R1 and R2, recombinant fragments resulting from crossing over.

DSB repair model of Szostak et al. (494). DSB formation is followed by 5′-to-3′ resection of the ends. The resulting 3′ ends are recombinogenic and can invade a homologous template, to initiate new DNA synthesis. Two HJs are formed and are resolved independently by cutting the crossed (open arrowhead) or noncrossed (closed arrowhead) strands, resulting in crossover or noncrossover products.

SDSA models. (A) Simple SDSA model. Both 3′ ends invade the template and initiate new DNA synthesis. (B) SDSA model with bubble migration. (C) SDSA model with crossing over. Following strand annealing of the second 3′ end with the displaced strand, a double HJ intermediate can occur. (D) Repair replication fork capture model. Strand invasion initiates both leading- and lagging-strand synthesis. Because of branch migration following DNA synthesis, this is an SDSA model.

Tripartite recombination. (A) A plasmid with a gapped copy of leu2 is gap repaired from two chromosomal templates. Genetic information must be recovered from two different chromosomes and assembled into the plasmid to create a complete LEU2 gene. (B) Two models to explain the production of a LEU2 gene. DNA synthesis can be initiated independently from both 3′ ends of the broken plasmid (top). Both newly synthesized strands are then unwound from the template and annealed. Alternatively, DNA synthesis can be initiated from one 3′ end, and template switching ensures the recovery of all the required LEU2 sequences (bottom). Then the newly synthesized strand anneals with the other 3′ end. In both cases, DNA synthesis is conservative: the newly synthesized DNA is unwound from its template.

BIR. (A) Three models to explain BIR. In model 1, the 3′ end of the DNA fragment (or broken chromosome) invades the template and initiates synthesis of one DNA strand by bubble migration. The complementary strand has to be synthesized later. In model 2, another (more likely) possibility is that the 3′ end initiates both leading- and lagging-strand synthesis, in a true replication fork. Here, DNA synthesis is semiconservative. The branched structure has then to be resolved by an endonuclease. In model 3, the initiation of a true replication fork is compatible with conservative DNA synthesis provided that branch migration follows the progression of the replication fork (bottom). Semiconservative replication is constrained to a small bubble. This hybrid model corresponds to the gene conversion model proposed in Fig. 8D. (B) One example of BIR. A DNA fragment with subtelomeric sequences, a centromere, and a terminal sequence homologous to a chromosomal region is transformed into yeast. The subtelomeric sequence can recombine with a chromosomal subtelomeric region to result in a true telomere (ellipse). This step is not shown. The other end of the DNA fragment can acquire all the sequences distal to the chromosomal homologous region, up to the telomere.

Models for gene targeting (ends-out recombination). (A) A common view is that ends-out recombination is a double-crossover event. (B) Gene targeting could occur by the assimilation of one strand of the transformed DNA, which will thereafter convert the recipient by mismatch repair. (C) A third model, suggested by the work of Morrow et al. (326), envisions that the 3′ ends initiate BIR, resulting in a new chromosome that will replace or recombine with the original one.

Formation and correction of hDNA. (A) In the model of Holliday (185), hDNA would be corrected after HJ resolution. Conversion would be the consequence of a correction in favor of the invading strand. Correction in favor of the recipient strand would yield a restoration event. (B) Some evidence suggest that hDNA formed during strand invasion is corrected immediately. In the Szostak et al. (494) model, conversion would result from correction in favor of the recipient strand (see Fig. 17A and B), which is the opposite of Holliday’s proposition. (C) hDNA can also result from annealing in SDSA models. Then it is impossible to define an invading and a recipient strand, and correction may be unbiased.

Detection of restoration events in yeast. This experiment, developed for yeast by Kirkpatrick and Petes (230), is derived form an analogous experiment with Ascobolus by Hastings (174). A well corrected allele (y) is located between two high-PMS alleles (x and z). If the three alleles are found in the same heteroduplex, correction of the central allele might occur, even if the two flanking high-PMS alleles are not corrected. Correction in favor of the resident strand will result in gene conversion of y, while PMS will be observed for x and z. If correction occurs in favor of the invading strand (restoration event), there will be a 4:4 Mendelian segregation for y, even though simultaneous PMS will be observed for x and z.

End joining of HO endonuclease-cleaved DNA. The 4-bp complementary ends can be ligated, which is an efficient process, or can be joined by NHEJ mechanisms that are inefficient. If the bases marked “f” pair, fill-in synthesis will generate a 3-bp insertion. If the bases marked “d” pair and the short tails are clipped off, a 3-bp deletion can result. Other, much larger deletions can occur in the same fashion, using bases exposed by the 5′-to-3′ resection of the DSB ends.

Genetic assays for recombination. (A) Selection of heteroallelic recombination. Here, a functional LEU2 gene results from a conversion event not associated with crossing over. (B) Gene conversion associated with crossing over. (C) The LEU2 recombinant gene results from a reciprocal crossover event without any detectable gene conversion. (D) Assay for loss of heterozygosity. This results in Leu⁻ cells. The event described here corresponds to a gene conversion with crossing over. (E) Loss of heterozygosity can also occur by reciprocal exchange between the centromere and the marker during the G₂ stage, followed by segregation in the next cell division.

Methods to create new alleles. (A) Gene disruption by recombination with a plasmid containing a leu2 copy deleted at both the 5′ and 3′ ends. This results in a duplication, where both copies are mutated. This duplication can be obtained by selecting with the URA3 marker. (B) The pop-in/pop-out method. This two-step method requires first the integration of a plasmid with a mutated copy of leu2 (pop-in, selected for with the URA3 marker) and then the excision of the plasmid (pop-out, selected for as loss of URA3) leaves only one copy of leu2, which can be the original one or the mutated one (the case shown here). (C) One-step gene replacement. Some of the Ura⁺ transformants have also integrated the mutation in leu2 and become Leu⁻. (D) One-step method of gene knockout. Most of the central part of the gene is replaced by the selectable URA3 marker.

Mating-type switching in yeast. The MAT locus, which determines the a or α mating type, switches by gene conversion, using one of two silent cassettes, HMRa and HMLα, located on the same chromosome. The gene conversion event is initiated by the HO endonuclease, which creates a DSB at the border of the varying region (called Ya or Yα, according to the genotype). MAT, HMR, and HML share homology on both sides of the Y regions (W, X, and Z regions). Both strands of DNA are shown in the middle diagram. A PCR assay has been used to detect DNA synthesis during MAT switching. Using oligonucleotides P1 and P2, one cannot obtain any PCR products in MATa cells, but a PCR product appears when the cell switches to MATα, as soon as DNA synthesis initiated from the Z region of MAT proceeds to copy Yα from MATα.

Physical characterization of the double-HJ intermediate predicted by Szostak et al. (494). (A) Two-dimensional gel electrophoresis of DNA from meiotic cells at the pachytene stage. Restriction enzyme-digested DNA samples are run first slowly on a low-concentration (0.4%) agarose gel. The migration lane is then cut and inserted across a high-concentration (0.8 to 1.0%) agarose gel, for a second, quick migration. Various molecules or events can be identified by Southern blotting, such as parental molecules (Mom and Dad), recombinant molecules (Rec), DSBs, and JMs. There are three different JMs, corresponding to the Mom-Mom, Dad-Dad, and Mom-Dad (the bigger signal in the middle). D, dimension. This figure has been adapted from Fig. 1 of reference 439. (B) JMs are double Holliday junctions. The JMs corresponding to interchromosome recombination (Mom-Dad) can be extracted from the gel. These molecules are resolved into parental and recombinant molecules (437) by the RuvC resolvase, showing that they include HJs. The JMs can also be run on a denaturing electrophoresis gel, and the size and specific hybridization pattern of the strands will indicate if they are recombinant or nonrecombinant strands. The theoretical outcome expected for simple HJs is two parental strands and two recombinant strands (left). This was not observed (437, 438). Instead, only parental strands were observed, the expected outcome for double HJs (right).

Some data in favor of SDSA models for meiotic recombination. Recombination can occur between two alleles differing by several mutations (A to C). These mutations can be on the same side of a meiotic DSB, as y and z, or on two different sides, as x and y or x and z. If these mutations result in high-PMS alleles, the revised DSB repair model of Szostak et al. (479, 494) predicts that y and z can be found in the same heteroduplex (D) and can be observed as two simultaneous PMS with the y and z alleles from one parent found in the same sector (F). In contrast, x will form a heteroduplex DNA on a different chromatid from y and z. In an SDSA model, y and z can also be found in the same heteroduplex (E). However, x can be found in another heteroduplex but on the same chromatid (E), resulting in simultaneous PMS with y and z, but now the x allele from one parent is found in the sector containing the y and z alleles from the other parent (G). This configuration, which is not predicted by the Szostak et al. model, has been observed by Porter et al. (384) and Gilbertson and Stahl (148).

DSB-induced expansions and contractions of a tandem repeat. (A) To test directly if a DSB can induce rearrangements in tandem repeats in yeast, Pâques et al. (368) tested HO-induced gene conversions with a homologous donor sequence containing an intervening interval including 8 repeats of 375 bp. Perfect copying of the template should introduce the whole intervening repeated locus into the repaired molecule. However, only about half of the repaired chromosomes acquire an unmodified repeated array. The others have a variable number of repeats ranging from 1 to 13 copies. These frequent rearrangements are restricted to the repaired recipient molecule, with the donor template remaining unmodified. Similar result have been obtained with an artificial or real yeast 36-bp minisatellite locus (365) and with a microsatellite CTG locus (402). (B) The rearrangements may result from replication slippage occurring during the semiconservative kind of DNA synthesis predicted by Szostak et al. (494); however, the rearrangement would be expected to be found in the donor template as well as in the recipient. The clustering of the rearrangements in the recipient molecule can be better explained by an SDSA model. (C) In this SDSA model, both 3′ ends initiate DNA synthesis. The newly synthesized strands are then unwound from their template and annealed. Because of the redundant structure, there are many possibilities of annealing, resulting in expansions or contractions. (D) Another possibility is that the kind of DNA synthesis associated with SDSA (bubble migration for example) would easily generate slippage-like events. If DNA synthesis stops before the new strands overlap with the other 3′ end, reinvasion has to occur. Because of the redundant structure, there are many possibilities of reinvasion, which would be responsible for the expansions and contractions that are always found on the recipient molecule because the newly synthesized sequences return to the repaired molecule. Resolution by annealing can occur, but HJs also can be formed and lead to crossovers, as proposed in Fig. 8C. This last feature has the advantage of explaining why the infrequent crossover events found in this experiment were associated with tandem repeat rearrangements as often as were the noncrossover events.

SSA. SSA can occur when a DSB appears between (or within) two direct repeats. Resection of the DSB ends produces two complementary single strands that are annealed. After excision of the nonhomologous 3′ ends and new DNA synthesis, ligation restores two continuous strands.

hDNA formation and correction analyzed by meiotic tetrad analysis. (A) Tetrad analysis. Sporulation of a heterozygous diploid most of the time results in two spores with one allele and two spores with the other one. This is the normal Mendelian segregation (4:4). However, other patterns are also observed. Non-Mendelian segregations include conversions and PMS. In PMS, the two alleles will segregate only after the first cell division following meiosis, resulting in a sectored colony. Various patterns of conversions and PMS are shown here. Gene conversion are often referred to as 2:6 or 6:2 segregations, since PMS events are referred to as 3:5, 5:3, or aberrant 4:4 (to differentiate it from normal Mendelian 4:4). This nomenclature is derived from the one used with filamentous fungi, where meiosis is followed by an additional cell division, resulting in eight spores instead of four. Note that in yeast, the aberrant 4:4 pattern is very rarely observed. (B) Molecular interpretation of non-Mendelian segregation. Strand invasion results in an asymmetric hDNA (step 1). Asymetric hDNA is found on only one chromatid. The displaced strand is used as template for DNA synthesis and yields a homoduplex (step 2). If hDNA is not corrected, asymetric hDNA results in 5:3 or 3:5 segregation. If corrected, it results in 6:2 or 2:6 segregation, depending whether correction occurs in favor of the recipient strand or in favor of the invading strand. In addition to the initial asymmetric hDNA, symmetric hDNA could result from branch migration (step 3). Symmetric hDNA is found over the same portion of two different chromatids. If not corrected, it will result in aberrant 4:4 segregation (step 4). Such events are rare in yeast but are common in Ascobolus.

Correction of hDNA resulting from strand invasion. (A) In the Szostak et al. (479, 494) model, correction in favor of the recipient strand leads to a gene conversion. (B) In the Szostak et al. (479, 494) model, correction in favor of the invading strand leads to a restoration at the B locus. Therefore, if one looks only at the B locus, nothing is detectable (normal Mendelian segregation). However, b is now associated with A and C, and B is associated with a and c (apparent double crossover). (C) In an SDSA model, correction in favor of the recipient strand results in a gene conversion. (D) In an SDSA model, correction in favor of the invading strand leads to a second round of mismatch repair that is initiated when the two strands of the template are reassociated. The final outcome can be gene conversion (genetic definition) or no detectable event (even no apparent double crossover). This outcome will be found with the SDSA models described in Fig. 8B, to D. In the SDSA model described in Fig. 8A, where a second strand is synthesized on the template, an additional mismatch will appear when the two newly synthesized strands are annealed (not shown). Four outcomes are then possible (no apparent event, apparent double crossover, gene conversion of B, gene conversion of b).

Three hypotheses to explain the gradient of meiotic gene conversion. (A) Restoration-conversion hypothesis. Next to the DSB, the mismatch is corrected preferentially in favor of the recipient molecule, resulting in a gene conversion. Further away, it will be corrected increasingly randomly, with the nick losing its ability to target the invading strand for the mismatch repair proteins. (B) Heteroduplex rejection model. Mismatches cause branch migration or unwinding of the heteroduplex, mediated by the mismatch repair proteins. Afterwards, the remaining hDNA is always corrected in favor of the recipient molecule. (C) Formation of a conversion gradient in an SDSA model. Both strands of the hDNA are processed by the mismatch repair machinery, which excises ssDNA tracts from each nick to the next mismatch. As a consequence, mismatches next to the DSB region result in conversion events whereas mismatches distal to the break more often result in restoration.