INTRODUCTION

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The processes by which damaged DNA is repaired and the mechanisms of genetic recombination are intimately related. Much of what we know about these events has come from studies of the yeast Saccharomyces cerevisiae, for which the development of new molecular biological and genetic approaches has made it possible to appreciate the many different pathways used by eukaryotic cells. The study of these processes in a simple, unicellular eucaryote has the obvious advantages of the ease of manipulation of DNA sequences (all of which are now precisely known) and the possibility of studying specific repair and recombination events induced synchronously in a large proportion of cells. Equally important is the growing conviction that the processes that one can study with relative ease in yeast are identical in most respects to the ways in which human cells repair DNA damage and generate genetic diversity. The expanding list of human genetic diseases associated with defects in DNA metabolism makes it especially important in understanding how these processes occur. Moreover, defining these mechanisms has taken on added importance in the quest to develop more efficient mechanisms of gene targeting and gene replacement in mammalian cells.

Recombination can be initiated by several types of DNA damage. Single-strand DNA (ssDNA) lesions may result during DNA replication or during repair, after UV irradiation or the alkylation or cross-linking of DNA bases, or from intermediates of type I topoisomerases. Double-strand breaks (DSBs) can appear as a consequence of ionizing radiation, by mechanical stress, by endonucleases, or by replication of a single-stranded nicked chromosome. The repair of DNA resulting from nucleotide excision repair, base excision repair, and other types of damage affecting one strand of the DNA duplex has been well reviewed elsewhere (37, 136, 390). This review will concentrate on the types of recombination created by DSBs. DSBs are the sole instigators of recombination in meiotic cells and are a major factor in recombination in mitotic cells, although the origin of spontaneous mitotic recombination remains unknown. In addition to its relevance as a fundamental biological process, DSB-mediated recombination is the basis of gene modification in yeast and in other eukaryotes.

We classify DSB repair events into two major categories. Homologous recombination events of several types are characterized by the need for the damaged DNA strands to base pair with a homologous partner, where the extent of interaction generally involves hundreds of nearly perfectly matched base pairs. In contrast, illegitimate or nonhomologous repair events can seemingly join ends of DNA with no complementary base pairs at the junction, although in general it turns out that most of these events make use of a very small number of base pairs (microhomology). In yeast, nonhomologous repair events generally occur at significantly lower frequencies than homologous events, so that one could argue that some of the distinctions between homologous and nonhomologous repair are artificial, especially since homologous recombination can occur with surprisingly short homologous regions, albeit at low frequency. However, these types of events are distinctly different, because they have different genetic requirements.

For a complete overview of recombination and DSB repair in yeast and other organisms, we also direct the reader to several other reviews that have recently appeared (220, 235, 347, 377, 413, 461, 462). Also, the present review deals essentially with the budding yeast S. cerevisiae, but there are more and more available data about recombination in the fission yeast Schizosaccharomyces pombe, an organism that seem to behave more like higher eukaryotes. Several recent reviews have appeared that will give the reader a good overview (133, 264, 359).

Although many of the fundamental ideas about recombination originated from studies of Drosophila, the analysis of fungi provided the opportunity to recover all four products of meiosis. This led to the discovery of non-Mendelian segregation of markers, both gene conversions and postmeiotic segregations, that provided the first insight into the molecular mechanisms of eukaryotic recombination. Although important observations were made with Neurospora crassa and Ascobolus immersus, it is Saccharomyces cerevisiae that has emerged as the model system of choice in studying both meiotic and mitotic recombination. The pioneering studies of Fogel and Mortimer with yeast (127-129, 193), as well as those of Rossignol et al. with Ascobolus (418) and Stadler with Neurospora (465), and insights from Hastings (176) and Whitehouse (549) established the basic framework by using naturally arising alleles in a variety of biosynthetic and pigment genes. However, it was the development of gene-targeting methods (183, 358, 421, 447) that allowed the creation of defined alterations of the genome and a refinement of these genetic approaches. The mechanism of gene targeting itself became the object of scrutiny, and much of our current thinking comes from the analysis of mitotic recombination of transformed DNA. More recently, two additional developments have provided new ways to investigate molecular events in greater detail. It is now possible to examine physical intermediates of recombination and thus to test the predictions of current recombination models. Moreover, in vitro biochemical studies of strand invasion, the central step of most recombination events, have provided direct tests of the role of proteins identified by genetic studies. The characterization of specific recombination proteins is discussed in a later section.

Recombination can be assessed genetically, for example by measuring gene conversion between heteroalleles of an easily scored nutritional marker (129, 448) (Fig. 1A and B). As initially defined in meiosis, a gene conversion is a nonreciprocal transfer of genetic information from one homologous chromosome to another. Although one might imagine that heteroallelic recombination could occur by a precise reciprocal exchange of DNA in the interval between the two alleles (Fig. 1C), studies of the fate of the alleles in diploids where a prototrophic cell has arisen show that more than 90% of the events are actually gene conversions, in which only one of the two participating alleles is unchanged (165, 268, 329). The literature is unfortunately replete with false distinctions between "gene conversions" (by which the authors mean gene conversions not associated with an exchange of flanking markers) and "reciprocal recombination" (by which the authors generally mean gene conversions associated with crossing over). Interchromosomal recombination can also be assessed by the loss of heterozygosity of nutritional markers. This can occur by gene conversion between two alleles of a scored marker (Fig. 1D) or by a reciprocal exchange anywhere between the marker and the centromere during the G₂ stage of the cell cycle, followed by chromosome segregation (Fig. 1E).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   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.

One can also assay specifically for crossovers. The simplest system involves a pair of alleles, distal to which are other markers that can be used to measure crossing over (Fig. 1E). Prototrophic recombinants can then be assessed for the arrangement of these flanking markers. If crossover occurs in G₂, thendepending on the segregation of chromosomeshalf of the prototrophic diploids should be homozygous for one or the other distal marker (Fig. 1E). If recombination occurs in G₁, crossing over will be genetically silent, although the two heterozygous distal markers will have exchanged positions. It is also possible to detect such events if there are closely enough linked polymorphisms that can be analyzed on genomic blots or by inducing a chromosome loss event (to see which markers become coordinately lost) or sporulating the diploids and determining the genetic linkage of these markers to other markers on the chromosome.

Genetic exchange between (identical) sister chromatids cannot usually be detected; thus, to detect crossovers between sister chromatids, one must examine events involving tandem repeats that give rise to unequal sister chromatid exchange (USCE). The first such assays monitored the fate of an inserted gene into the repeated ribosomal DNA (rDNA) array (375, 495). One can score the appearance of sectored colonies, where one half is derived from a cell that lost the inserted marker. In some of these cases, the opposite sector has two copies of the marker and thus most probably arose from USCE. It should be noted, however, that in many instances only one copy of the marker is found in the opposite sector, which can be explained by intrachromosomal recombination or by gene conversion between misaligned rDNA repeats (144).

A second approach to look for USCE events is to examine prototrophic recombinants between tandem repeats of heteroalleles. Especially when the orientation of alleles is such that a simple "pop-out" event will not produce a prototrophic recombinant, a significant fraction of prototrophs prove to be triplications (Fig. 2B) resulting from USCE (199, 236). If the markers are arranged as shown in Fig. 2A, prototrophs containing a single copy of the repeat could be generated by USCE but could also arise from intrachromatid recombination. Another way to study USCE is to use partially overlapping truncated genes (119, 120). Here, two overlapping parts of a gene are inserted in an orientation that will not permit intrachromatid crossing over to produce a heritable recombined, complete gene. In contrast, USCE will yield a full-length gene (Fig. 2C).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   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).

Intrachromatid recombination can also be examined by using direct or inverted repeats, either at a chromosomal location or on a plasmid. Direct-repeat assays are commonly used. In one such assay, two copies of a gene, one truncated at the 3' end and the other truncated at the 5' end but with a homologous region, will recombine to restore gene function (452) (Fig. 2D). Alternatively, the deletion can also be scored by the loss of a marker in the interval between direct repeats (239, 503, 504) (Fig. 2D). Inverted repeats can also be used, either when recombination between heteroalleles will yield a functional gene (Fig. 2E) or when recombination will cause an inversion of the region in between the repeats to produce a complete gene (551) (Fig. 2E). However, recent results suggest that some apparently intrachromatid events might result from interchromatid gene conversion (71). In addition, recombination can be studied between sequences on two plasmids or between a plasmid and a chromosome.

These approaches take advantage of the ease with which DNA sequences can be inserted into yeast by the introduction of a linearized fragment of transforming DNA. Two general methods are used to create new alleles on a chromosome. In the first (357, 358), a circular plasmid containing an in vitro-modified gene and a selectable marker such as URA3 is targeted by a DSB created by a restriction endonuclease within a region of homology (Fig. 3A and B). The integration creates a tandem duplication of nonidentical sequences. The process can effectively be reversed by screening for the loss of URA3, either randomly (447) or, more conveniently, by selecting such events on medium containing 5-fluoroorotic acid, whose presence is lethal to cells with a functional URA3 gene (43). Some of the pop-out events that eliminate the URA3 gene and other plasmid sequences will leave behind the modified DNA sequences in place of the original sequences (Fig. 3B). In this way it is possible to introduce known alleles into a specific strain, so that all derivatives are isogenic. Other genes, such as LYS2, can be selected for positively and negatively (69), but it is essentially the URA3 marker which has been used for this kind of procedure. Alternatively, one can introduce modified sequences in one step by transformation with a linearized fragment containing a selectable marker (421). This kind of recombination event is often called "ends-out" recombination, since the DSB ends point to opposite directions (Fig. 3C and D). Ends-out recombination is often used to disrupt or delete genes (Fig. 3D) or to insert other, adjacent unselected sequences into a novel location (Fig. 3C). Ends-out transformation with linearized, modified DNA is the basis of most knockout strategies in all organisms, about which more is discussed later.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   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.

A powerful tool in studying mechanisms of recombination is the physical analysis of DNA to detect recombination in the absence of any easily scored genetic marker. For example, in a diploid in which the two homologous chromosomes have polymorphic restriction sites flanking a region of interest, it is possible to identify cases in which crossing over has occurred by the appearance of novel restriction fragments generated by reciprocal exchange (Fig. 4). This method also permits an analysis of the kinetics of recombination by isolating samples at intervals after the initiation of a recombination event (49, 50).

View larger version (5K): [in this window] [in a new window]   FIG. 4.   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.

An advantage of the physical assay is that it can give information not only about the products of recombination but also about intermediate steps. Knowledge of the structure of the recombinant molecule can help in constructing models, but these models will become tangible only when their individual steps can be physically monitored.

One may also examine the extent to which recombination can occur even under conditions where cells are unable to complete recombination or even to continue growing. For example, one could ask if recombination can be completed when cells are arrested at different stages of the cell cycle or after the elevation of the cells to the restrictive temperature of a conditional-lethal mutation. Thus, it is possible to carry out what we have termed in vivo biochemistry, i.e., to infer the biochemical roles of specific enzymes by determining which steps in recombination are affected by the inactivation of that enzyme and where the mutants become blocked (160).

Very recently, physical analysis of DNA has been dramatically applied to examine the position of meiotic crossovers along the entire genome in a single experiment. High-density oligonucleotide arrays, capable of detecting more than 3,700 allelic differences between two divergent yeast strains, were hybridized with the DNA from each of the four segregants of a meiotic tetrad, allowing Winzeler et al. (553) to map the position of every crossing over and many gene conversions not accompanied by reciprocal exchange.

Synchronous induction of DSBs. Analysis of the kinetics of recombination, to discover the time of appearance of intermediates and products, is dependent upon the ability to initiate recombination synchronously in a large population of cells. This occurs naturally in meiotic cells, where DSBs arise at specific hot spots in a few percent of all chromatids. In mitotic cells, synchronous initiation of recombination can be accomplished by the induction of a site-specific endonuclease. Two such systems have been developed in yeast. The HO endonuclease recognizes a degenerate target of 22 bp (345) and normally cleaves only one site in the entire yeast genome: the mating-type (MAT) locus. Constructs in which the HO gene is fused to a galactose-inducible promoter have made it possible to express HO simply by adding galactose to cells grown on lactate, glycerol, or raffinose (203), three carbon sources that do not repress the galactose-inducible promoter. A second endonuclease is I-SceI, normally encoded and expressed only in yeast mitochondria to facilitate the movement of a mobile intron,  (LSU.I) (82, 101, 102, 290). A synthetic version of this gene, replacing codons whose usage is different in mitochondria and the cytoplasm, was constructed, again under the control of a galactose-inducible promoter (381). A 45- to 90-min induction of either HO or I-SceI leads to the cleavage of a significant fraction (30% for I-SceI, 100% for HO) of target sites. HO endonuclease is also turned over rapidly, so that no activity remains 30 min after the end of the induction period (547). Once a DSB has been created, intermediate steps in recombination, along with the appearance of final products, can be identified.

Physical monitoring of HO-induced mitotic gene conversion: the example of MAT switching. A paradigm for mitotic recombination is HO endonuclease-induced recombination, and more specifically, MAT switching. During switching (Fig. 5), the Ya- or Y-specific sequences at MAT that specify the mating type are replaced by sequences copied from two unexpressed donor sequences, HML and HMRa (reviewed in references 161, 163, 164, and 471). The initiating event is a DSB catalyzed by the HO endonuclease at the Y/Z junction of the recipient MAT locus.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Mating-type switching in yeast. The MAT locus, which determines the a or  mating type, switches by gene conversion, using one of two silent cassettes, HMRa and HML, 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 Y, 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 MAT, as soon as DNA synthesis initiated from the Z region of MAT proceeds to copy Y from MAT.

Relationship between gene conversion and crossovers. Gene conversion is defined as a nonreciprocal transfer of genetic information from one molecule to its homologue. Usually this occurs between two alleles of a gene (Fig. 1 and 2); however, gene conversions can embrace many contiguous genes, including the entire distal part of a chromosome arm. Gene conversions were initially defined in meiosis, where one could observe non-Mendelian segregation of alleles. The pioneering work of Mortimer and Fogel (329) established several key characteristics of gene conversions, including the idea that gene conversions exhibited polarity, whereby the probability that a nearby marker would be coconverted along with a specified gene-converted marker decreased with the distance between the markers. In meiosis, gene conversion tracts are on average 1 to 2 kb (46, 95, 293, 348, 478). In mitosis, some gene conversions cover very short distances (216, 305, 342) while others extend for hundreds of kilobases (see below).

(i) DSB repair model of Szostak et al. Because gene conversions are strongly associated with crossovers, molecular models were designed to account for this fact, culminating in the DSB repair model first suggested by Resnick and Martin (401) and later elaborated by Szostak and coworkers (479, 494) (Fig. 6). These models were based on earlier conceptions by Holliday (185) and by Meselson and Radding (312).

View larger version (17K): [in this window] [in a new window]   FIG. 6.   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.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   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).

(ii) Synthesis-dependent strand annealing. Because many mitotic gene conversions were infrequently associated with crossing over, a second family of gene conversion models emerged, beginning with those of Nasmyth (339) and Thaler and Stahl (499) and further elaborated by both Hastings (175) and McGill et al. (305). Similar alternative models appeared to explain results in other organism such as Drosophila (111, 150, 340), mammals (30), E. coli (250, 331), and Ustilago (121). The name we use for these kinds of mechanism, synthesis-dependent strand annealing (SDSA), was coined by Nassif et al. (340). The basic feature of these models is that the newly synthesized DNA strands are displaced from the template and returned to the broken molecule, allowing the two newly synthesized strands to anneal to each other. This could occur either because there are topoisomerases or helicases that actively dismantle the replication structure (305, 500) (Fig. 8A) or because the replication "bubble" remains small, with the newly synthesized strand being continuously unwound from its template (the bubble migration model) (130) (Fig. 8B). In both cases, DNA synthesis is conservative (all the newly synthesized sequences are on the same molecule) instead of semiconservative as in the Szostak et al. model. SDSA models were first designed to explain a lack of crossovers, but they received experimental backing from other observations that could best be explained by such a mechanism.

View larger version (26K): [in this window] [in a new window]   FIG. 8.   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.

View larger version (16K): [in this window] [in a new window]   FIG. 9.   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).

View larger version (10K): [in this window] [in a new window]   FIG. 10.   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.

View larger version (16K): [in this window] [in a new window]   FIG. 11.   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.

(iii) Synthesis-dependent strand annealing with crossing over. Gene conversion events not associated with crossing over can easily be explained by either the Szostak et al. model or the SDSA model. However, most formulations of SDSA do not allow for crossing over accompanying DSB repair; if this were the case, one might predict that outcomes characteristic of SDSA would not be found for gene conversions accompanied by crossing over. However, a version of SDSA that includes the possibility of crossing over has been suggested by Ferguson and Holloman (121). In this model, strand invasion is initiated by one end of the DSB and would proceed to copy across the template until one of two events occur. First, the newly synthesized strand may simply anneal with the second end, yielding a gene conversion without crossing over (Fig. 8B). Alternatively, the displaced D-loop created by the first strand may anneal with the second end, producing a single HJ that can be resolved with or without crossing over. We have subsequently proposed a similar model (368), wherein a double HJ would be formed instead of a single HJ (Fig. 8C). This idea of stabilizing the D-loop by annealing to the second end of the DSB was a feature of the mechanism proposed by Szostak et al., but there is a significant difference that might be used experimentally to distinguish between them: in the original DSB repair model the two HJs are found on either side of the DSB (494), while in our SDSA version, the two HJs are both on one side of the DSB (368). Crossovers could occur on either side of the DSB, depending on which end initiates DNA synthesis. We recognize that different positions of a double HJ could also result from branch migration of the HJs (437).

(iv) Repair replication fork capture. In both the Szostak et al. DSB repair model and the standard version of SDSA, there seems to be a need only for leading-strand polymerization, primed by the two 3' ends of the invading DNA strands. However, it would be possible for the invasion of one 3'-ended single strand to establish a modified replication fork, similar but not identical to the leading- and lagging-strand process of origin-dependent DNA replication (Fig. 8D).

The first observation of such events was made by Esposito (112), who found gene conversion tracts that apparently extended from the TRP5 locus to the ADE5 locus on chromosome VII. Since the entire yeast genome has now been sequenced, we now know that ADE5 and TRP5 are 400 kb apart. Coconversion of the TRP5 and LEU1 loci, 25 kb apart on chromosome VII, was also observed at a frequency 1,200-fold higher than if those events were independent (152), with coconversion also of intervening markers (153). Such high levels of coconversions could be explained in two ways. First, the conversion tracts might be very large. Second, gene conversion could occur in a subset of cells that are especially prone to recombination, and these cells would convert any locus at a very high rate. This second hypothesis was ruled out by Golin and Tampe (154), who showed that only genetically linked loci were converted at high frequencies. These authors also showed that the coconversion frequency decreased with the distance between two loci, down to a certain distance (35 kb), where it no longer depended on distance. Thus, they defined two processes of coconversion, a distance-dependent one and (for very long distances) a distance-independent one. However, the authors did not propose any fundamentally different recombination model to explain the two kinds of events, perhaps because, at that time, recombination events occurring in yeast were explained in term of the Meselson and Radding or Szostak et al. models, involving the formation of heteroduplexes of extensive lengths.

Similar asymmetrical inheritance of distal markers was seen when recombination was initiated by the HOT1 sequence (528). Coconversions involving up to 70 kb accounted for 90% of the conversion events at one locus. Actually, the same study also showed that the rate of coconversion was 60% even for spontaneous recombination (not induced by HOT1). Although these events are gene conversion events (i.e., asymmetrical inheritance), Voelkel-Meiman and Roeder (528, 529) invoked a replicative model of DSB repair, analogous to the recombination-induced replication of phage T4 (130, 330) or of the E. coli chromosome (242). It is this kind of model that has since been favored to explain very long tracts of gene conversion. Three different versions are shown in Fig. 12A.

View larger version (12K): [in this window] [in a new window]   FIG. 12.   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.

DSB repair leading to long tracts of gene conversion has also been observed by Malkova et al. (291), who used MATa/MAT-inc diploids to study the repair of an HO-induced DSB. Nearly all of the repair of MATa occurs by recombination with the MAT-inc (noncleavable) homologous chromosome, since the cut chromosome lacks the donors HML and HMR. In a wild-type strain, nearly all the repair events are short-patch gene conversions, most of the time without crossovers. In rad52 diploids, the broken chromosome is almost always lost, but in rad51 diploids, the repair efficiency is 45% of the wild-type level. The repair of the DSB in rad51 diploids does not occur by classical gene conversion, however. All of the cells in which repair had occurred had become homozygous for MAT-inc and for all markers distal to MAT. This RAD52-dependent, RAD51-independent repair process was termed break-induced replication (BIR).

Another recent study supports the idea that one-ended strand invasion events do indeed result in extensive DNA synthesis involving whole chromosome arms. Morrow et al. (326) investigated the process of chromosome fragmentation developed by Vollrath et al. (530). Vollrath et al. transformed a linear fragment of DNA into yeast, with one end including Y' subtelomeric sequences and therefore able to generate a new telomere by recombination with Y' sequences on another chromosome end. The other end is homologous to a yeast chromosomal sequence far from a telomere. They recovered recombined chromosomes, including the transformed DNA linear fragment, with a new telomere on its Y' side and, on the other side, all the sequences distal to the yeast homologous sequence (Fig. 12B). An obvious explanation was that this chromosome arose by a reciprocal exchange between the yeast gene present in the linear fragment and the chromosome. However, Morrow et al. showed that this was unlikely to be the case, because this new recombined chromosome was often found in addition to and not instead of the intact homologous yeast chromosome (Fig. 12B). Therefore, this kind of event had to involve extensive new DNA synthesis initiated from the non-Y' end of the transformed fragment, adding a whole chromosome arm to this end. Here, also, the authors propose that a true replication fork would be initiated, leading to semiconservative replication, as during recombination-induced replication in E. coli or phage T4 (242, 330).

One theoretical feature of BIR is that after strand invasion of one 3' end, there is no possible stabilization of the displaced strand by annealing with the second DSB end. Therefore, one has to envision two possibilities: either DNA synthesis occurs by bubble migration (130) (Fig. 12A, scheme 1), or BIR involves a true replication fork (Fig. 12A, scheme 2). However, with a bubble migration model, the synthesis of a complementary strand would be a secondary event. In contrast, recombination-induced replication forks have a rather well-characterized precedent in E. coli (242) and in bacteriophage T4 replication (330), a good reason to prefer this kind of model. We note that there is a satisfying unity of mechanism between BIR and the replication fork capture model described above. The third model (Fig. 12A, scheme 3) is directly derived from this fork capture model, with the progression of the replication fork closely followed by branch migration, resulting in conservative DNA synthesis. Once BIR starts, it can proceed to the chromosome end or be converted into gap repair if the second end of the DSB becomes involved.

BIR may also be a biologically very important repair pathway for the repair of chromosome ends. A chromosome that has lost a telomere has a single DSB end, and no second end can participate in a gene conversion repair event. One-ended events have been proposed for a long time to explain recombination at telomeres in wild-type cells (104, 534). Using HO-induced chromosome breaks in a diploid in which only one end of the broken chromosome has significant homology to its homologue, Bosco and Haber (51) found that repair was highly efficient; close to 70% of the broken chromosomes were repaired by apparently copying the 25 kb distal to the DSB from the homologous chromosome.

BIR accounts for the recombination-dependent maintenance of telomeres in cells in which telomerase, the enzyme that normally adds short TG_1-3 sequences at the end of yeast chromosomes, is deleted. Although most of these telomerase-deficient cells die, a small proportion survive by apparently frequent recombination that regenerates and disperses sufficient TG_1-3 at every chromosome end to keep cells alive (286). In addition, there are frequent rearrangements of subtelomeric sequences, including the proliferation to many ends of a subtelomeric Y' element found normally at some chromosome ends. This whole process depends on the RAD52 gene and is affected by other recombination genes (260). It is not known if the cells that survive in this way have undergone some change that distinguishes them from the vast majority of cells that die without telomerase. Perhaps they have become hyperrecombination mutants, but genetic analysis has failed to reveal a single mutation to account for their survival (285).

A similar phenomenon has been observed in S. pombe. Cells with the trt1⁺ gene, encoding the catalytic unit of the telomerase, deleted can survive by two different processes: circularization of the chromosomes or apparent elongation of chromosomal ends without telomeres by a recombinational pathway, which might be BIR (337). The recombination process in trt1 mutant cells is enhanced by a mutation in the taz1⁺ gene, which encodes a telomeric DNA binding protein (337). This protein probably prevents the chromosome ends from entering the recombination process which is the fate of regular DNA ends.