HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways. In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions. Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product. Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb. Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded. Gap repair processes are independent of distance but are reduced in rad52 mutants and in Gl-arrested cells.In both mitotic and meiotic cells, double-strand breaks (DSBs) can initiate homologous recombination events. In the yeast Saccharomyces cerevisiae, this process has been characterized by analyzing the outcome of repair of DSBs induced by X rays, transformation of linear substrates into cells, and introduction of site-specific lesions in both mitotic and meiotic cells (1,18,36,37,44,54 1A) proposes that a DSB or gap is repaired by a gene conversion event involving the concerted participation of both broken ends, which invade and copy DNA from an intact, homologous donor copy. More recent considerations have prompted a modification of this model to include the creation of extensive single-stranded regions by 5'-to-3' exonucleolytic digestion of the DSB (55,63). This model predicts that gene conversion can occur both with and without an accompanying crossing over and that these two products occur with equal frequencies (Fig. 1A).Similar recombination events have been described for mammalian cells. Linearized DNA molecules can be used to effect site-directed gene replacement (13,27,50) and gap repair of transformed DNA (2). Recently, a system that carries out gap repair in vitro has been developed; however, some of the products obtained had not been predicted by a simple gap repair model (15). A number of studies of recombination in mammalian cells suggest that there is an alternative pathway of recombination, termed single-strand annealing (SSA, Fig. 1B) (3,6,(21)(22)(23)61). In this pathway (Fig. 1B), deletions are formed between sequences in direct orientation. A DSB produced between the directly repeated sequences is attacked by a 5'-to-3' exonuclease whose degradation produces complementary single strands which then anneal. This mechanism also accounts for products resembling gene conversions that have arisen by the interaction of several DNA molecules (22 (29)(30)(31).With yeast, the molecular events associated with recombinational repair of DSBs can be observed in vivo by physically monitoring DNA after the synchronous induction of a DSB. With mitotic cells, it has been possible to carry out a detailed analysis of DSB repair by using two highly efficient site-specific endonucleases: the HO endonuclease, whic...

Novel recombinational repair of a site-specific double-strand break (DSB) in a yeast chromosome was investigated. When the recognition site for the HO endonuclease enzyme is embedded in nonyeast sequences and placed between two regions of homology, expression of HO endonuclease stimulates recombination between the homologous flanking regions to yield a deletion, the apparent product of an intrachromosomal exchange between direct repeats. This deletion-repair event is very efficient, thus preventing essentially all the potential lethality due to the persistence of a DSB. Interestingly, unlike previous studies involving spontaneous recombination between chromosomal repeats, the recombination events stimulated by HO-induced DSBs are accompanied by loss of the sequences separating the homologous regions greater than 99.5% of the time. Repair is dependent on the RAD52 gene. The deletion-repair event provides an in vivo assay for the sensitivity of any particular recognition site to HO cleavage. By taking advantage of a galactose-inducible HO gene, it has been possible to follow the kinetics of this event at the DNA level and to search for intermediates in this reaction. Deletion-repair requires approximately 45 min and is inhibited when cycloheximide is added after HO endonuclease cleavage.The repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae seems to occur predominantly by recombination between the broken ends of a DNA molecule and intact, homologous sequences. When chromosomal DSBs are induced by gamma-irradiation, efficient repair depends on the presence of either an unbroken homologous chromosome in a diploid or an intact sister chromatid in a haploid cell after DNA replication (36,37). Similarly, the repair of spontaneous breaks at or near the MAT locus in heterothallic (ho) strains frequently occurs by recombination between the fragment of broken chromosome containing the centromere and the intact homologous chromosome (27); in some cases, complete loss of the broken chromosome is observed. Additionally, the repair of dicentric chromosomes, broken during mitosis, was also often observed to occur by recombination between one end of the DSB and a homologous segment elsewhere on the remaining monocentric broken chromosome (11). Orr-Weaver et al. (33) (9,19,29). In a haploid homothallic strain in which both HML and HMR have been deleted, an HO-induced DSB at MAT has been shown to be lethal, presumably because no homologous sequences are available from which to repair the break (18). In diploids lacking the donor loci, and in which only one of the two MAT genes can be cleaved by HO endonuclease, the broken chromosome is repaired by gene conversion from the intact MAT locus. Unlike a normal mating-type switch, repair associated with crossing-over and/or homozygosis of the distal arm of chromosome III occurred in over 25% of the cases (17).Although most DSB repair in yeast cells proceeds by homologous recombination, a low frequency of apparently nonhomologous repair events has been described. Small del...

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We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.

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