Repair of a double-strand break (DSB) in yeast can induce very frequent expansions and contractions in a tandem array of 375-bp repeats. These results strongly suggest that DSB repair can be a major source of amplification of tandemly repeated sequences. Most of the DSB repair events are not associated with crossover. Rearrangements appear in 50% of these repaired recipient molecules. In contrast, the donor template nearly always remains unchanged. Among the rare crossover events, similar rearrangements are found. These results cannot readily be explained by the gap repair model of Szostak et al. (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) but can be explained by synthesis-dependent strand annealing (SDSA) models that allow for crossover. Support for SDSA models is provided by a demonstration that a single DSB repair event can use two donor templates located on two different chromosomes.Tandem repeat instability is implicated in several human genetic diseases. The best-documented examples of deleterious rearrangements in tandem repeats are the massive amplifications of microsatellite DNA, known to be responsible for a dozen diseases, including fragile X syndrome and Huntington's disease (for reviews, see references 62 and 74). Rearrangements affecting minisatellites (repeats of 10 to 50 nucleotides) can be harmful, too (4). For example, expansions of a minisatellite are associated with epilepsy (30,31,73). During meiosis, minisatellites can display a very high rate of modification, including intra-allele duplications and deletions, and nonreciprocal interallelic transfer of information (2, 24). Recently, a human minisatellite was also found to display massive amplification (78). Rearrangements in tandem repeats are not specific to micro-and minisatellites. Expansions and contractions of larger tandem repeats have been observed in Drosophila melanogaster and yeast (48,49,69,75,76).While replication slippage can easily account for small changes in microsatellite copy number (63), the origin of massive amplifications remains a mystery. Since the predominant rearrangement events observed in minisatellites are nonreciprocal interallelic transfers of information, the meiotic instability affecting those sequences is thought to result from gene conversions rather than replication (24). Tandem repeat rearrangements observed in Drosophila are linked to P-M dysgenesis and have also been supposed to be the consequence of genetic recombination, because P-element excision is known to induce gene conversion (9,29,48,49,69).Gene conversions are most often explained by the doublestrand break (DSB) repair model, proposed by Resnick and Martin (54) and Szostak et al. (68) to account for recombination events in yeast and other fungi. Many of the features of this model, or of its revised version (67), have been experimentally verified in Saccharomyces cerevisiae. The initial observation that a DSB in the DNA double helix induced a gene conversion in mitotic cells (45) was corroborated by...

Break-induced replication (BIR) is a nonreciprocal recombinationdependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5 end of the LEU2 gene at one end was transformed into a strain in which the 5 end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2 ؉ . Ade1 ؉ Leu2 ؉ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ. D uring the past several years, some old ideas about how recombination occurs have received strong experimental support. Meselson and Weigle (1) first proposed that crossingover could be explained by a break-copy mechanism in which one end of a double-strand break (DSB) could invade an intact linear template molecule and initiate new DNA synthesis that could proceed to the end of the chromosome template. In essence, a recombination event led to the establishment of a unidirectional replication fork. Skalka (2) provided a more molecular view of this idea (a replicator's view of recombination, as she called it) to explain phage recombination. Mosig (3, 4) made a similar proposal to account for late DNA replication in phage T4. Formosa and Alberts (5) provided a key in vitro experimental demonstration for the formation of a replication fork by recombination. More recent studies by George and Kreuzer (6) of DSB-induced recombination, controlled by phage T4 genes in Escherichia coli have supported the idea that recombination leads to extensive replication. Similarly, recent experiments by Motamedi et al. (7) and by Kuzminov and Stahl (8) with phage have provided strong evidence that a major pathway to generate crossing-over involves extensive replication during break-copy recombination.These ideas were applied by Kogoma (9, 10) to explain origin-independent, recombination-dependent replication of the E. ...

Various studies suggest that eukaryotic chromosomes may occupy distinct territories within the nucleus and that chromosomes are tethered to a nuclear matrix. These constraints might limit interchromosomal interactions. We have used a molecular genetic test to investigate whether the chromosomes of Saccharomyces cerevisiae exhibit such territoriality. A chromosomal double-strand break (DSB) can be efficiently repaired by recombination between f lanking homologous repeated sequences. We have constructed a strain in which DSBs are delivered simultaneously to both chromosome III and chromosome V by induction of the HO endonuclease. The arrangement of partially duplicated HIS4 and URA3 sequences around each HO recognition site allows the repair of the two DSBs in two alternative ways: (i) the creation of two intrachromosomal deletions or (ii) the formation of a pair of reciprocal translocations. We show that reciprocal translocations are formed approximately as often as the pair of intrachromosomal deletions. Similar results were obtained when one of the target regions was moved from chromosome V to any of three different locations on chromosome XI. These results argue that the broken ends of mitotic chromosomes are free to search the entire genome for appropriate partners; thus, mitotic chromosomes are not functionally confined to isolated domains of the nucleus, at least when chromosomes are broken.Mitotic recombination in Saccharomyces cerevisiae can be initiated by site-specific double-strand breaks (DSBs) created by the HO endonuclease (1, 2). One efficient mechanism of DNA repair, termed single-strand annealing (SSA), occurs intrachromosomally and results in deletions between homologous DNA sequences flanking the DSB (3). This process involves extensive 5Ј to 3Ј degradation of DNA from the site of a DSB until flanking regions of homology are exposed and the complementary sequences on either side of break can anneal. SSA occurs efficiently even when the flanking homologous regions are separated by as much as 15 kb or when the extent of shared homology of the flanking regions is only a few hundred bp (4, 5). It is important to note that SSA is not a minor pathway in yeast, used only when cells cannot repair a break by gene conversion. When a DSB is created in a region that can be repaired either by intrachromosomal gene conversion or by SSA, more than two-thirds of the events occur by SSA (5). Very similar results are observed when a different site-specific endonuclease, I-SceI, is expressed in S. cerevisiae (6).But is SSA inherently an intrachromosomal pathway? Given a choice, would the two ends of one DSB reanneal more readily than the ends of two independent DSBs on different chromosomes? There is abundant evidence from many organisms that chromosomes exist in association with a nuclear matrix that may significantly restrict the ability of different chromosomes or chromosome regions to interact with each other (7-9). These studies suggest that chromosomal DNA is arranged in loops of 50-100 kb. Other st...

To study targeted recombination, a single linear 2-kb fragment of LEU2 DNA was liberated from a chromosomal site within the nucleus of Saccharomyces cerevisiae, by expression of the site-specific HO endonuclease. Gene targeting was scored by gene conversion of a chromosomal leu2 mutant allele by the liberated LEU2 fragment. This occurred at a frequency of only 2 ؋ 10 ؊4 , despite the fact that nearly all cells successfully repaired, by single-strand annealing, the chromosome break created by liberating the fragment. The frequency of Leu ؉ recombinants was 6-to 25-fold higher in pms1 strains lacking mismatch repair. In 70% of these cases, the colony was sectored for Leu ؉ ͞Leu ؊ . Similar results were obtained when a 4.1-kb fragment containing adjacent LEU2 and ADE1 genes was liberated, to convert adjacent leu2 and ade1 mutations on the chromosome. These results suggest that a linear fragment is not assimilated into the recipient chromosome by two crossovers each close to the end of the fragment; rather, heteroduplex DNA between the fragment and the chromosome is apparently formed over the entire region, by the assimilation of one of the two strands of the linear duplex DNA. Moreover, the recovery of Leu ؉ transformants is frequently defeated by the cell's mismatch repair machinery; more than 85% of mismatches in heteroduplex DNA are corrected in favor of the resident, unbroken (mutant) strand.Despite the fact that Saccharomyces is celebrated for its ability to carry out homologous recombination, the frequency of gene replacement by transformation of a linear exogenous DNA is surprisingly low (1-3). This could be due to several factors. For example, ''naked'' DNA, not yet associated with histones or other nuclear proteins, might be rapidly degraded before it has a chance to undergo recombination. Alternatively, the replacement of homologous DNA sequences may be an inherently inefficient process. Previously we showed that the frequency of gene replacement can be increased by providing additional copies of the target sequence (3), a result that suggests that the search for homology is a rate-limiting step in the process. In these and previous experiments it was also not known if there was a special subpopulation of cells that were especially adept at this process, either because they were more proficient in recombination or because they were able to take up more copies of the transforming DNA.To avoid some of the uncertainties inherent in transformation, we have devised a way to liberate a single linearized fragment of DNA from a chromosome within the nucleus and to examine its capacity to be ''captured'' by homologous recombination. The chromosomal region illustrated in Fig. 1A contains a LEU2 gene flanked by HO endonuclease recognition sites. When a galactose-inducible HO endonuclease is expressed, cleavage occurs at these sites, liberating a 2.0-kb LEU2 fragment and leaving a broken chromosome. The chromosomal break itself can be very efficiently repaired by the process of single-strand annealing (4-6), ...

In meiosis, gene conversions are accompanied by higher levels of crossing over than in mitotic cells. To determine whether the special properties of meiotic recombination can be attributed to the way in which Spo11p creates double-strand breaks (DSBs) at special hot spots in Saccharomyces cerevisiae, we expressed the site-specific HO endonuclease in meiotic cells. We could therefore compare HO-induced recombination in a well-defined region both in mitosis and meiosis, as well as compare Spo11p-and HO-induced meiotic events. HO-induced gene conversions in meiosis were accompanied by crossovers at the same high level (52%) as Spo11p-induced events. Moreover, HO-induced crossovers were reduced 3-fold by a msh4⌬ mutation that similarly affects Spo11p-promoted events. In a spo11⌬ diploid, where the only DSB is made by HO, crossing over was significantly higher (27%) than in mitotic cells (<7%). This single meiotic DSB failed to induce the formation of a synaptonemal complex. We also show that HO-induced gene conversion tract lengths are shorter in meiotic than in mitotic cells. We conclude that a hallmark of meiotic recombination, the production of crossovers, is independent of the nature of Spo11p-generated DSBs at special hotspots, but some functions of Spo11p are required in trans to achieve maximum crossing over. M eiotic recombination in Saccharomyces cerevisiae differs from mitotic recombination in several respects. Beyond the fact that meiotic gene conversions occur 100 to 10,000 times more often than equivalent spontaneous mitotic events, the individual events themselves have different outcomes. Most notably, meiotic gene conversions have a higher association with crossing over than is seen in mitotic cells (reviewed in refs. 1 and 2). In addition to generating genetic diversity, crossing over is critical in ensuring accurate chromosome segregation during meiosis (3, 4).The origins of most spontaneous gene conversion events in mitosis are unknown, but they are certainly different from the way in which double strand breaks (DSBs) are induced by the meiosis-specific Spo11 protein (5, 6). DSBs created by Spo11p are generated at many nearby sites in meiotic hotspots (7,8). These hotspots represent a very special chromosomal context influenced by perhaps 10 other genes, the deletion of which abolishes or severely reduces DSB formation (reviewed in refs. 1, 3, 4, 9, and 10). Deletion of genes encoding Mre11p, Rad50p, and Xrs2p causes changes in the chromatin structure of hotspots (11). Similarly, the absence of protein components such as Hop1p that comprise the axial elements lying between sister chromatids also reduces DSB formation (12).Other mutations reduce the proportion of meiotic gene conversions accompanied by meiotic crossovers without affecting DSB formation. The zip1⌬ or zip2⌬ mutations, which eliminate formation of the central element of the synaptonemal complex (SC), have this effect (13,14), providing support for the general view that crossover regulation depends on this structure (3, 4). Deletion of ...