A DNA double chain break stimulates triparental recombination in Saccharomyces cerevisiae.

Abstract

Mitotic recombination between his3 heteroalleles on heterologous chromosomes is stimulated by a DNA double chain break delivered in vivo at a site 8.6 kilobase pairs distant from one his3 allele and unlinked to the other. The induced recombination at his3 is accompanied by gap repair at the break site using the uncut homolog as a template. The DNA between the break site and his3 is not deleted in most ofthe His+ recombinants.In Saccharomyces cerevisiae a DNA double chain break is repaired by homologous recombination (1-4). It has been imagined that the ends, after some exonucleolytic degradation, invade the intact homolog, the 3'-OH ends prime synthesis that repairs the gap, and the resulting dimer is resolved, sometimes producing recombinant products (3). In this double chain break repair (DCBR) model the site of the double chain break is coincident with the site of recombination. In phage A Red recombination, a double chain end is thought to be resected on one chain to promote a long region of heteroduplex DNA (5-8). By contrast, in the major Escherichia coli recombination pathway the RecBCD enzyme apparently enters the DNA helix at the site of a double chain break, travels along the molecule, encounters a specific DNA sequence termed Chi, and stimulates recombination in the neighborhood of Chi (9, 10). Thus, in the RecBCD pathway the site ofthe exchange is arbitrarily distant from the site of the double chain break. In fungi no evidence exists for a mechanism analogous to that in the RecBCD pathway; however, there is evidence of spatially discrete but statistically correlated multiple events of recombination (11)(12)(13)(14), some of which are difficult to reconcile with the current models of recombination (11,13,14).In this communication we demonstrate that in vegetative cells of S. cerevisiae a double chain break-induced repair event is accompanied by ectopic recombination 8.6 kilobase pairs (kb) away. These coincident recombination events, involving three chromosomes, yield products that are not altered between the two sites of recombination. MATERIALS AND METHODSStrains and Methods. Yeast strains are listed in Table 1 and their construction is described in Fig. 1. Media and biochemical methods are as described in Ray et al. (17) and in ref. 19. YEPL is YEP broth with 3 M lactate (pH 6.5). The E. coli strain was DH1 (15).Induction of HO. A single colony of the appropriate strain growing on SD-ura was suspended in YEPL broth and incubated with shaking for 8 hr. Aliquots were plated on YEP-gal, and the plates were incubated for 18 hr. Cells were washed off the plates, washed and diluted in water, and RESULTSDouble Chain Break in cis to his3-D Stimulates Ectopic Recombination. A difference between the current view of DCBR recombination in yeast (3) and that for RecBCDcatalyzed recombination in E. coli (1, 9, 10) is that in the latter process there is no degradation of DNA between the site of double chain break and the site of initiation ofrecombination. In yeast, can recombination initiated by a doub...

A unique pathway of double-strand break repair operates in tandemly repeated genes.

“…The RecBCD complex is thought to enter a DNA duplex at a DSB, travel along the duplex until encountering a specific sequence (Chi site) and then initiate recombination near that site (55). DSB-induced stimulation of recombination at a site distant from the DSB has been observed in yeast cells (35). Rejoining of duplexes by this mechanism would always result in the loss of units and would usually result in the loss of URA3, consistent with the data.…”

“…Strand recision following DSB formation may be an important step in DSB-induced recombination in S. cerevisiae (1,6,28,29,35,36,39,53,54,56). Exonucleolytic digestion in a 5'-to-3' direction has been demonstrated following HO cleavage at MAT (59).…”

“…Simple gap repair is a predominant mechanism of DSB repair in repeated sequences in Rad+ cells (28,36). DSB repair frequently involves the formation of extensive regions of heteroduplex DNA in sequences flanking the break (16,28,29,35,36). The frequency with which a marker near a DSB undergoes gene conversion, whether by gap expansion or heteroduplex formation followed by mismatch repair (see reference 54), is directly correlated to the distance of the marker from the DSB (28,29,36).…”

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…Physical monitoring of recombination at MAT has yielded much of what we know about DSB-induced mitotic recombination (reviewed in Haber 1995Haber , 2006Pâques and Haber 1999;Krogh and Symington 2004;Hicks et al 2011). Some related studies have been done by inserting small HO endonuclease recognition sites at other locations and from the induction of other site-specific endonucleases, most notably I-SceI (Rudin and Haber 1988;Nickoloff et al 1989;Ray et al 1989;Plessis et al 1992;McGill et al 1993;Liefshitz et al 1995;Weng et al 1996;Inbar and Kupiec 1999;Wilson 2002;Storici et al 2003;Lydeard et al 2007Lydeard et al , 2010Jain et al 2009;Marrero and Symington 2010). Additional information about DSB repair has been gleaned from the analysis of DSB-induced recombination in meiotic cells (reviewed in Kleckner 1996;Borner et al 2004;Keeney and Neale 2006;Longhese et al 2009).…”

A Two-Pathway Analysis of Meiotic Crossing Over and Gene Conversion in Saccharomyces cerevisiae

“…Such tetrads Step 6: The 39-whisker arising by branch migration on the resulting noncrossover product may promote crossing over with the blue chromatid or a third chromatid. might result from a pairing-pathway noncrossover being accompanied by a not-so-incidental exchange (Ray et al 1989) provoked by the 39-end of a protruding SS-DNA whisker (Hotchkiss 1971) (Figure A1). …”