Genetic evidence that the meiotic recombination hotspot at the HIS4 locus of Saccharomyces cerevisiae does not represent a site for a symmetrically processed double-strand break (original) (raw)
Related papers
Genetics, 1996
Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SP013 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad+ strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad' and in rad5OA cells that cannot initiate normal meiotic DSBs. We find that the RtlDSO gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad5OA cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad5OA diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination.
A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces cerevisiae
Genetics, 1996
We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARC4 locus. The “canonical” DSBR model, in which only 5′ ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a “trans” configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Mo...
Genetics, 1996
Meiotic recombination in Saccharomyces cerevisiae is initiated by double- strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad+ strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13: : HO induced gene conversions both in Rad+ and in rad50 delta cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50 delta cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome ...
Genome-Wide Redistribution of Meiotic Double-Strand Breaks in Saccharomyces cerevisiae
Molecular and Cellular Biology, 2007
Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.
Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae
The EMBO Journal
We present a scheme for locating double-strand breaks (DSBs) in meiotic chromosomes of Saccharomyces cerevisiae, based on the separation of large DNA molecules by pulsed field gel electrophoresis. Using a radSOS mutant, in which DSBs are not processed, we show that DSBs are widely induced in S.cerevisiae chromosomes during meiosis. Some of the DSBs accumulate at certain preferred sites. We present general profiles of DSBs in chromosomes III, V, VI and VII. A map of the 12 preferred sites on chromosome III is presented. At least some of these sites correlate with known 'hot spots' for meiotic recombination. The data are discussed in view of current models of meiotic recombination and chromosome segregation.
1996
Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SP013 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad+ strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad' and in rad5OA cells that cannot initiate normal meiotic DSBs. We find that the RtlDSO gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad5OA cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad5OA diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination.
PLoS ONE, 2013
Recombination during meiosis in the form of crossover events promotes the segregation of homologous chromosomes by providing the only physical linkage between these chromosomes. Recombination occurs not only between allelic sites but also between non-allelic (ectopic) sites. Ectopic recombination is often suppressed to prevent non-productive linkages. In this study, we examined the effects of various mutations in genes involved in meiotic recombination on ectopic recombination during meiosis. RAD24, a DNA damage checkpoint clamp-loader gene, suppressed ectopic recombination in wild type in the same pathway as RAD51. In the absence of RAD24, a meiosis-specific recA homolog, DMC1, suppressed the recombination. Homology search and strand exchange in ectopic recombination occurred when either the RAD51 or the DMC1 recA homolog was absent, but was promoted by RAD52. Unexpectedly, the zip1 mutant, which is defective in chromosome synapsis, showed a decrease, rather than an increase, in ectopic recombination. Our results provide evidence for two types of ectopic recombination: one that occurs in wild-type cells and a second that occurs predominantly when the checkpoint pathway is inactivated.
Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae
Microbiology and molecular biology reviews : MMBR, 1999
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.
Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae
Genetics, 2002
In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-O...
Meiotic DNA Breaks at the S. pombe Recombination Hot Spot M26
Molecular Cell, 2002
they seem to occur preferentially in 1100 Fairview Avenue North, A1-162 regions of higher than average GϩC content (Gerton et Seattle, Washington 98109 al., 2000). In contrast, the ade6-M26 hot spot of the distantly related yeast Schizosaccharomyces pombe does re-Summary quire a discrete sequence, 5Ј-ATGACGT-3Ј, for activity (Schuchert et al., 1991). The M26 hot spot was discov-The ade6-M26 allele of Schizosaccharomyces pombe ered originally as a unique allele of the ade6 gene, ade6creates a well-defined meiotic recombination hot spot M26 (Gutz, 1971), but the heptamer sequence has since that requires a specific sequence, 5-ATGACGT-3, been shown to be an active hot spot at multiple positions and the Atf1•Pcr1 transcription factor for activity. We within ade6 and also in the distantly located ura4 gene find that M26 stimulates the formation of meiosis-spe-(Fox et al., 1997 and Figure 1). The M26 sequence serves cific double-strand DNA breaks at multiple sites suras a binding site for the Atf1•Pcr1 transcription factor, rounding M26. Like hot spot activity, breakage requires an essential component for hot spot activity (Kon et the M26 heptamer, Pcr1, and the general recombinaal., 1997). Mutation of any one base in the heptamer tion factor Rec12. When the M26 heptamer is moved abolishes both its hot spot activity and its ability to bind to new positions within ade6, new break sites are ob-Atf1•Pcr1 in vitro (Schuchert et al., 1991; Wahls and served spanning 2-5.0ف kb around the moved hep-Smith, 1994). However, M26 is not the only sequence to tamer. Break frequency is strongly correlated with which Atf1•Pcr1 can bind: at least two other sequences recombination frequency for these alleles. The occlosely related to M26, TGACGTA and TGACGTC, bind currence of breaks at M26 suggests mechanistic to Atf1•Pcr1 and are also meiotic hot spots (Fox et al., similarities to hot spots in the distantly related yeast 2000). Saccharomyces cerevisiae. Like S. cerevisiae, meiosis-induced DSBs occur in S. pombe, the only other organism in which such breaks Introduction have been reported (Cervantes et al., 2000; Murakami and Nurse, 2001). Meiotic recombination and break for-During meiosis, four haploid cells (gametes or spores) mation are intimately associated in S. pombe since both are generated from a single diploid cell. One of the hallrequire the action of at least eight meiotic rec gene marks of meiosis is a greatly elevated level of recombiproducts. Therefore, it is quite likely that these breaks nation between homologous chromosomes prior to the initiate recombination. If the M26 hot spot operates by first meiotic division, typically 100-to 1000-fold higher a mechanism similar to that of hot spots of S. cerevisiae, than mitotic rates (reviewed in Esposito and Wagstaff, then it should also be the site of a DNA break. However, 1981; Fox and Smith, 1998). This recombination serves repeated searches in this laboratory and others failed at least two important functions. First, it forms attachto show either double-or single-strand breaks in the ments (chiasmata) between homologs, which facilitate vicinity of M26 (Ponticelli et al., 1988; Szankasi et al., proper segregation of chromosomes at the first meiotic 1988; Bä hler et al., 1991), raising the possibility that M26 division. Second, it shuffles alleles between homolomay operate by a fundamentally different mechanism gous chromosomes, thus increasing the genetic diverfrom hot spots of S. cerevisiae. sity on which natural selection can act. Characterization of DNA break sites in S. cerevisiae Recombination does not occur evenly throughout the has been greatly aided by separation-of-function mutagenome of many organisms, but at lower than average tions in the RAD50 gene, known as rad50S mutations frequency in some regions (cold spots) and higher than (Cao et al., 1990; Sun et al., 1991; de Massy et al., 1995; average in others (hot spots). It was first shown that Liu et al., 1995; Xu and Petes, 1996). These mutant alleles double-strand DNA breaks (DSBs) are associated with allow breaks to be formed but not resected or repaired a hot spot of recombination at the ARG4 locus of the during meiosis. The absence of resection results in the budding yeast Saccharomyces cerevisiae (Sun et al., formation of discrete broken fragments rather than a 1989). Nicolas et al. (1989) reported that markers near heterogeneous mixture of partially resected fragments, the break site convert more frequently than markers far while the failure to repair results in broken fragments from the break site, supporting the proposal that meiotic accumulating to higher levels during the course of meiorecombination is initiated at double-strand breaks (Ressis. Thus, in a rad50S background, DNA break sites are nick, 1976; Szostak et al., 1983). Other genetically demore easily detected than in wild-type. fined hot spots in S. cerevisiae have subsequently been A rad50S allele has also been recently created in shown to be sites of DSBs, and these breaks may initiate S. pombe. This allele creates a lysine-to-isoleucine substitution at position 81 of the protein, similar to one of the commonly used rad50S alleles in S. cerevisiae (Farah