SUMMARY The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.

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...

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1͞Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1͞Rad10 and Msh2͞Msh3, but reveals a difference in their roles. When the f lanking homologous regions that anneal are 205 bp, the requirement for Msh2͞ Msh3 is as great as for Rad1͞Rad10; but when the annealing partners are 1,170 bp, Msh2͞Msh3 have little effect, while Rad1͞Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3 tail.In Saccharomyces cerevisiae, homologous recombination initiated by double-strand breaks (DSBs) can occur by at least two distinct pathways: gene conversion and single-strand annealing (SSA) (1-6). In both cases, the ends of the DSB are resected by a 5Ј to 3Ј exonuclease to produce long 3Ј ended single-strand tails (7,8). In gene conversion, these tails invade a homologous donor sequence and act as primers of new DNA synthesis. However, for this to occur, any nonhomologous bases at the 3Ј end must be removed, so that the primer end may basepair with the donor template. Similarly, in SSA, complementary strands of homologous regions flanking a DSB can anneal, producing an intermediate that has two nonhomologous 3Ј ended tails that must be removed before new DNA synthesis and ligation can occur. In both instances, removal of nonhomologous tails depends on the Rad1 and Rad10 proteins (9), which have been shown in vitro to cleave 3Ј ended nonhomologous tails and which carry out a related function in nucleotide excision repair (NER) (10-12). Other NER proteins are not required (13).In NER, Rad1͞Rad10 are presumably recruited to the site of DNA damage by other proteins of the NER repair complex. We were interested in whether other proteins were required to attract Rad1͞Rad10 to the sites of strand invasion or strand annealing. One set of proteins that recognize DNA distortions are the mismatch repair proteins, most notably Msh2, which has been shown to bind to mismatched bp (14), heteroduplex loops (14), and Holliday junctions (15). Msh2 has been shown to form heterodimers with Msh6 (16,17) and studies of the specificity of mismatch repair have led to the conclusion that Msh2͞Msh6 primarily recognize and correct single bp mismatches, while Msh2 and Msh3 act to correct heteroduplex DNA containing small loops formed by frameshift mutations (16, 18). All of these mismatch repair events ...

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.

The importance of safer approaches for gene therapy has been underscored by a series of severe adverse events (SAEs) observed in patients involved in clinical trials for Severe Combined Immune Deficiency Disease (SCID) and Chromic Granulomatous Disease (CGD). While a new generation of viral vectors is in the process of replacing the classical gamma-retrovirus–based approach, a number of strategies have emerged based on non-viral vectorization and/or targeted insertion aimed at achieving safer gene transfer. Currently, these methods display lower efficacies than viral transduction although many of them can yield more than 1% engineered cells in vitro. Nuclease-based approaches, wherein an endonuclease is used to trigger site-specific genome editing, can significantly increase the percentage of targeted cells. These methods therefore provide a real alternative to classical gene transfer as well as gene editing. However, the first endonuclease to be in clinic today is not used for gene transfer, but to inactivate a gene (CCR5) required for HIV infection. Here, we review these alternative approaches, with a special emphasis on meganucleases, a family of naturally occurring rare-cutting endonucleases, and speculate on their current and future potential.