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1.
Nucleic Acids Res ; 50(2): 899-914, 2022 01 25.
Article in English | MEDLINE | ID: mdl-34967417

ABSTRACT

During meiosis, DNA double-strand breaks (DSBs) are formed at high frequency at special chromosomal sites, called DSB hotspots, to generate crossovers that aid proper chromosome segregation. Multiple chromosomal features affect hotspot formation. In the fission yeast S. pombe the linear element proteins Rec25, Rec27 and Mug20 are hotspot determinants - they bind hotspots with high specificity and are necessary for nearly all DSBs at hotspots. To assess whether they are also sufficient for hotspot determination, we localized each linear element protein to a novel chromosomal site (ade6 with lacO substitutions) by fusion to the Escherichia coli LacI repressor. The Mug20-LacI plus lacO combination, but not the two separate lac elements, produced a strong ade6 DSB hotspot, comparable to strong endogenous DSB hotspots. This hotspot had unexpectedly low ade6 recombinant frequency and negligible DSB hotspot competition, although like endogenous hotspots it manifested DSB interference. We infer that linear element proteins must be properly placed by endogenous functions to impose hotspot competition and proper partner choice for DSB repair. Our results support and expand our previously proposed DSB hotspot-clustering model for local control of meiotic recombination.


Subject(s)
Chromosomes, Fungal/metabolism , DNA, Fungal/metabolism , Escherichia coli/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces , DNA Breaks, Double-Stranded , DNA Repair , Homologous Recombination , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism
2.
Proc Natl Acad Sci U S A ; 115(40): E9333-E9342, 2018 10 02.
Article in English | MEDLINE | ID: mdl-30217891

ABSTRACT

Viable gamete formation requires segregation of homologous chromosomes connected, in most species, by cross-overs. DNA double-strand break (DSB) formation and the resulting cross-overs are regulated at multiple levels to prevent overabundance along chromosomes. Meiotic cells coordinate these events between distant sites, but the physical basis of long-distance chromosomal communication has been unknown. We show that DSB hotspots up to ∼200 kb (∼35 cM) apart form clusters via hotspot-binding proteins Rec25 and Rec27 in fission yeast. Clustering coincides with hotspot competition and interference over similar distances. Without Tel1 (an ATM tumor-suppressor homolog), DSB and crossover interference become negative, reflecting coordinated action along a chromosome. These results indicate that DSB hotspots within a limited chromosomal region and bound by their protein determinants form a clustered structure that, via Tel1, allows only one DSB per region. Such a "roulette" process within clusters explains the observed pattern of crossover interference in fission yeast. Key structural and regulatory components of clusters are phylogenetically conserved, suggesting conservation of this vital regulation. Based on these observations, we propose a model and discuss variations in which clustering and competition between DSB sites leads to DSB interference and in turn produces crossover interference.


Subject(s)
Chromosomes, Fungal/metabolism , DNA Breaks, Double-Stranded , Meiosis , Nuclear Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Chromosomes, Fungal/genetics , Nuclear Proteins/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics
3.
Methods Mol Biol ; 1471: 25-49, 2017.
Article in English | MEDLINE | ID: mdl-28349389

ABSTRACT

The fission yeast Schizosaccharomyces pombe is especially well suited for studying meiosis in molecular detail. Experiments with S. pombe strains that undergo a nearly synchronous meiosis-at variable temperatures-have elucidated the mechanisms of meiotic progression and the proteins that are involved. For example, studies focused on the initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs) have proven exceptionally informative. In meiosis, some regions of DNA have more frequent DSBs than the surrounding regions. These DSB hotspots can be visualized by Southern blot hybridization of restriction fragments ranging from kilobases (kb) to megabases (Mb) in size. More recently, the benefits of genome-wide analysis to map the distribution and frequency of meiotic DSBs have been attained, with resolution down to the nucleotide level. Infrequent, non-hotspot DSBs previously not detectable have been observed, creating a better understanding of how recombination is regulated. Additional genome-wide analyses have shown proteins that bind specifically to DSB hotspots, providing insight into how the DSB initiation complex functions. We describe here detailed methods for achieving these results.


Subject(s)
DNA Breaks, Double-Stranded , Meiosis , Oligonucleotide Array Sequence Analysis/methods , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Chromatin Immunoprecipitation/methods , Genome, Fungal , Genome-Wide Association Study , In Situ Hybridization/methods , Schizosaccharomyces pombe Proteins/genetics
4.
PLoS Genet ; 12(6): e1006102, 2016 06.
Article in English | MEDLINE | ID: mdl-27304859

ABSTRACT

To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species.


Subject(s)
Chromosome Segregation/genetics , DNA Helicases/metabolism , DNA Repair/genetics , Rad51 Recombinase/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , DNA Breaks, Double-Stranded , DNA, Cruciform/genetics , DNA, Fungal/metabolism , Endodeoxyribonucleases/genetics , Gene Deletion , Gene Library , Holliday Junction Resolvases/metabolism , Meiosis/genetics
5.
PLoS Genet ; 11(5): e1005225, 2015 May.
Article in English | MEDLINE | ID: mdl-25993311

ABSTRACT

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.


Subject(s)
Casein Kinase I/metabolism , Chromosome Segregation , Protein Kinases/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Casein Kinase I/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Breaks, Double-Stranded , DNA, Fungal/genetics , Homologous Recombination , Meiosis , Mutation , Phosphorylation , Promoter Regions, Genetic , Protein Kinases/genetics , Schizosaccharomyces pombe Proteins/genetics , Synaptonemal Complex/metabolism , Cohesins
6.
Nat Protoc ; 9(1): 223-31, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24385151

ABSTRACT

Synchronous cultures are often indispensable for studying meiosis. Here we present an optimized protocol for induction of synchronous meiosis in the fission yeast Schizosaccharomyces pombe. Chemical inactivation of an ATP analog-sensitive form of the Pat1 kinase (pat1-as2) by adding the ATP analog 1-NM-PP1 in G1-arrested cells allows the induction of synchronous meiosis at optimal temperature (25°C). Importantly, this protocol eliminates detrimental effects of elevated temperature (34°C), which is required to inactivate the commonly used temperature-sensitive Pat1 kinase mutant (pat1-114). The addition of the mat-Pc gene to a mat1-M strain further improves chromosome segregation and spore viability. Thus, our protocol offers highly synchronous meiosis at optimal temperature, with most characteristics similar to those of wild-type meiosis. The synchronization protocol can be completed in 5 d (not including strain production, which may take as long as 2 or 3 months).


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Cell Culture Techniques , Meiosis/drug effects , Protein Serine-Threonine Kinases/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/cytology , Adenosine Triphosphate/pharmacology , Protein Serine-Threonine Kinases/genetics , Schizosaccharomyces pombe Proteins/genetics , Temperature
7.
Nucleic Acids Res ; 42(1): 359-69, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24089141

ABSTRACT

Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.


Subject(s)
DNA, Fungal/metabolism , Meiosis/genetics , Recombination, Genetic , Schizosaccharomyces/genetics , Temperature , DNA Breaks, Double-Stranded , DNA Repair , DNA, Cruciform/metabolism , Mutation , Protein Serine-Threonine Kinases/genetics , Schizosaccharomyces pombe Proteins/genetics
8.
Nucleic Acids Res ; 40(19): 9633-46, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22855558

ABSTRACT

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81-Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81-Eme1 is unclear. In cells lacking Nse5-Nse6 of the Smc5-Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5-Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5-Smc6 complex in HJ resolution via Mus81-Eme1.


Subject(s)
Chromosomal Proteins, Non-Histone/physiology , Meiosis/genetics , Recombinational DNA Repair , Schizosaccharomyces pombe Proteins/physiology , Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/genetics , Crossing Over, Genetic , DNA Breaks, Double-Stranded , Escherichia coli Proteins/metabolism , Gene Deletion , Holliday Junction Resolvases/metabolism , Mitosis/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics
9.
Cell Cycle ; 11(8): 1626-33, 2012 Apr 15.
Article in English | MEDLINE | ID: mdl-22487684

ABSTRACT

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2-3 in these mutants than in starvation-induced pat1(+) meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.


Subject(s)
Adenosine Triphosphate/pharmacology , Protein Serine-Threonine Kinases/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Temperature , Adenosine Triphosphate/analogs & derivatives , Alleles , Chromosome Segregation/drug effects , Meiosis/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Schizosaccharomyces/cytology , Schizosaccharomyces pombe Proteins/antagonists & inhibitors , Schizosaccharomyces pombe Proteins/genetics , Spores, Fungal/drug effects
10.
Trends Genet ; 27(10): 411-21, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21782271

ABSTRACT

The unique segregation of homologs, rather than sister chromatids, at the first meiotic division requires the formation of crossovers (COs) between homologs by meiotic recombination in most species. Crossovers do not form at random along chromosomes. Rather, their formation is carefully controlled, both at the stage of formation of DNA double-strand breaks (DSBs) that can initiate COs and during the repair of these DSBs. Here, we review control of DSB formation and two recently recognized controls of DSB repair: CO homeostasis and CO invariance. Crossover homeostasis maintains a constant number of COs per cell when the total number of DSBs in a cell is experimentally or stochastically reduced. Crossover invariance maintains a constant CO density (COs per kb of DNA) across much of the genome despite strong DSB hotspots in some intervals. These recently uncovered phenomena show that CO control is even more complex than previously suspected.


Subject(s)
Chromatids/genetics , Crossing Over, Genetic , Meiosis , Recombination, Genetic , Synaptonemal Complex/genetics , Animals , Caenorhabditis elegans , Chromatids/metabolism , DNA Breaks, Double-Stranded , DNA Repair , Humans , Mice , Saccharomyces cerevisiae , Schizosaccharomyces , Synaptonemal Complex/metabolism
11.
Cell ; 142(2): 243-55, 2010 Jul 23.
Article in English | MEDLINE | ID: mdl-20655467

ABSTRACT

Crossovers between meiotic homologs are crucial for their proper segregation, and crossover number and position are carefully controlled. Crossover homeostasis in budding yeast maintains crossovers at the expense of noncrossovers when double-strand DNA break (DSB) frequency is reduced. The mechanism of maintaining constant crossover levels in other species has been unknown. Here we investigate in fission yeast a different aspect of crossover control--the near invariance of crossover frequency per kb of DNA despite large variations in DSB intensity across the genome. Crossover invariance involves the choice of sister chromatid versus homolog for DSB repair. At strong DSB hotspots, intersister repair outnumbers interhomolog repair approximately 3:1, but our genetic and physical data indicate the converse in DSB-cold regions. This unanticipated mechanism of crossover control may operate in many species and explain, for example, the large excess of DSBs over crossovers and the repair of DSBs on unpaired chromosomes in diverse species.


Subject(s)
Crossing Over, Genetic , DNA Repair , Meiosis , Schizosaccharomyces/metabolism , DNA Breaks, Double-Stranded , DNA, Cruciform , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/metabolism
12.
Methods Mol Biol ; 557: 235-52, 2009.
Article in English | MEDLINE | ID: mdl-19799186

ABSTRACT

The fission yeast Schizosaccharomyces pombe has many biological characteristics that make it an ideal model organism for the study of meiosis. A nearly synchronous meiosis is one of the most important. Under certain environmental and genetic conditions, large cultures of S. pombe can be induced to undergo meiosis in a timely and predictable manner that allows for changes in the DNA to be observed and analyzed by gel electrophoresis. Initiation of meiotic recombination via programmed DNA double-strand breaks, the formation of joint molecule recombination intermediates, and the resolution of these intermediates into crossover DNA products can all be seen with consistent timing during the progression of a synchronous meiotic induction. The timing of recombination events, the genetic requirements for the formation and disappearance of recombination intermediates, and the analysis of the DNA structures of those intermediates allow a comparison of meiotic recombination in fission yeast with that in the only other species similarly studied, the budding yeast Saccharomyces cerevisiae.


Subject(s)
DNA, Fungal/analysis , Meiosis/genetics , Recombination, Genetic/genetics , Schizosaccharomyces/genetics , Cell Culture Techniques/methods , DNA, Fungal/chemistry , DNA, Fungal/isolation & purification , Electrophoresis, Agar Gel/methods , Models, Biological , Schizosaccharomyces/chemistry , Schizosaccharomyces/growth & development
13.
PLoS Genet ; 4(11): e1000267, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19023408

ABSTRACT

The fission yeast Schizosaccharomyces pombe Rec12 protein, the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and becoming covalently linked to the DNA ends of the break. This protein-DNA linkage has previously been detected only in mutants such as rad50S in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms. Here, we show that we can detect Rec12-DNA linkages in Sc. pombe rad50(+) cells, which are proficient for DSB repair. In contrast to the results from Sa. cerevisiae, genome-wide microarray analysis of Rec12-DNA reveals indistinguishable meiotic DSB distributions in rad50(+) and rad50S strains of Sc. pombe. These results confirm our earlier findings describing the occurrence of widely spaced DSBs primarily in large intergenic regions of DNA and demonstrate the relevance and usefulness of fission yeast studies employing rad50S. We propose that the differential behavior of rad50S strains reflects a major difference in DSB regulation between the two species--specifically, the requirement for the Rad50-containing complex for DSB formation in budding yeast but not in fission yeast. Use of rad50S and related mutations may be a useful method for DSB analysis in other species.


Subject(s)
DNA Breaks, Double-Stranded , DNA, Fungal/chemistry , Meiosis , Recombination, Genetic/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , DNA, Fungal/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics
14.
Genetics ; 179(3): 1157-67, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18562672

ABSTRACT

RecQ helicases are found in organisms as diverse as bacteria, fungi, and mammals. These proteins promote genome stability, and mutations affecting human RecQ proteins underlie premature aging and cancer predisposition syndromes, including Bloom syndrome, caused by mutations affecting the BLM protein. In this study we show that mutants lacking the Rqh1 protein of the fission yeast Schizosaccharomyces pombe, a RecQ and BLM homolog, have substantially reduced meiotic recombination, both gene conversions and crossovers. The relative proportion of gene conversions having associated crossovers is unchanged from that in wild type. In rqh1 mutants, meiotic DNA double-strand breaks are formed and disappear with wild-type frequency and kinetics, and spore viability is only moderately reduced. Genetic analyses and the wild-type frequency of both intersister and interhomolog joint molecules argue against these phenotypes being explained by an increase in intersister recombination at the expense of interhomolog recombination. We suggest that Rqh1 extends hybrid DNA and biases the recombination outcome toward crossing over. Our results contrast dramatically with those from the budding yeast ortholog, Sgs1, which has a meiotic antirecombination function that suppresses recombination events involving more than two DNA duplexes. These observations underscore the multiple recombination functions of RecQ homologs and emphasize that even conserved proteins can be adapted to play different roles in different organisms.


Subject(s)
DNA Helicases/chemistry , DNA Helicases/metabolism , Meiosis , Recombination, Genetic , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/metabolism , Sequence Homology, Amino Acid , Alleles , Crossing Over, Genetic , DNA Breaks, Double-Stranded , DNA Repair , Gene Conversion , Gene Duplication , Mutation/genetics , Protein Structure, Tertiary , RecQ Helicases , Schizosaccharomyces pombe Proteins/chemistry , Spores, Fungal/cytology
15.
PLoS Genet ; 3(8): e141, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17722984

ABSTRACT

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) made by Spo11 (Rec12 in fission yeast), which becomes covalently linked to the DSB ends. Like recombination events, DSBs occur at hotspots in the genome, but the genetic factors responsible for most hotspots have remained elusive. Here we describe in fission yeast the genome-wide distribution of meiosis-specific Rec12-DNA linkages, which closely parallel DSBs measured by conventional Southern blot hybridization. Prominent DSB hotspots are located approximately 65 kb apart, separated by intervals with little or no detectable breakage. Most hotspots lie within exceptionally large intergenic regions. Thus, the chromosomal architecture responsible for hotspots in fission yeast is markedly different from that of budding yeast, in which DSB hotspots are much more closely spaced and, in many regions of the genome, occur at each promoter. Our analysis in fission yeast reveals a clearly identifiable chromosomal feature that can predict the majority of recombination hotspots across a whole genome and provides a basis for searching for the chromosomal features that dictate hotspots of meiotic recombination in other organisms, including humans.


Subject(s)
DNA Breaks, Double-Stranded , DNA, Intergenic/physiology , Meiosis/genetics , Schizosaccharomyces/genetics , Blotting, Southern , Chromosome Mapping , Chromosomes, Fungal , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/physiology
16.
Cell ; 127(6): 1167-78, 2006 Dec 15.
Article in English | MEDLINE | ID: mdl-17174892

ABSTRACT

Crossing-over between homologous chromosomes facilitates their accurate segregation at the first division of meiosis. Current models for crossing-over invoke an intermediate in which homologs are connected by two crossed-strand structures called Holliday junctions. Such double Holliday junctions are a prominent intermediate in Saccharomyces cerevisiae meiosis, where they form preferentially between homologs rather than between sister chromatids. In sharp contrast, we find that single Holliday junctions are the predominant intermediate in Schizosaccharomyces pombe meiosis. Furthermore, these single Holliday junctions arise preferentially between sister chromatids rather than between homologs. We show that Mus81 is required for Holliday junction resolution, providing further in vivo evidence that the structure-specific endonuclease Mus81-Eme1 is a Holliday junction resolvase. To reconcile these observations, we present a unifying recombination model applicable for both meiosis and mitosis in which single Holliday junctions arise from single- or double-strand breaks, lesions postulated by previous models to initiate recombination.


Subject(s)
Crossing Over, Genetic , DNA, Cruciform/genetics , Meiosis , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Chromosomes, Fungal/genetics , Chromosomes, Fungal/physiology , DNA, Fungal/genetics , DNA-Binding Proteins/physiology , Endonucleases/physiology , Mutation , Saccharomyces cerevisiae Proteins/physiology , Schizosaccharomyces pombe Proteins/physiology , Sister Chromatid Exchange/physiology
17.
Genetics ; 169(2): 595-605, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15545638

ABSTRACT

In Schizosaccharomyces pombe, meiosis-specific DNA breaks that initiate recombination are observed at prominent but widely separated sites. We investigated the relationship between breakage and recombination at one of these sites, the mbs1 locus on chromosome I. Breaks corresponding to 10% of chromatids were mapped to four clusters spread over a 2.1-kb region. Gene conversion of markers within the clusters occurred in 11% of tetrads (3% of meiotic chromatids), making mbs1 a conversion hotspot when compared to other fission yeast markers. Approximately 80% of these conversions were associated with crossing over of flanking markers, suggesting a strong bias in meiotic break repair toward the generation of crossovers. This bias was observed in conversion events at three other loci, ade6, ade7, and ura1. A total of 50-80% of all crossovers seen in a 90-kb region flanking mbs1 occurred in a 4.8-kb interval containing the break sites. Thus, mbs1 is also a hotspot of crossing over, with breakage at mbs1 generating most of the crossovers in the 90-kb interval. Neither Rec12 (Spo11 ortholog) nor I-SceI-induced breakage at mbs1 was significantly associated with crossing over in an apparently break-free interval >25 kb away. Possible mechanisms for generating crossovers in such break-free intervals are discussed.


Subject(s)
Chromosome Breakage , Crossing Over, Genetic , DNA, Fungal , Gene Conversion , Meiosis , Schizosaccharomyces/genetics , Chromatids , Chromosomes, Fungal , Genes, Fungal , Genetic Markers , Models, Genetic , Restriction Mapping
18.
Genetics ; 167(2): 593-605, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15238514

ABSTRACT

During meiosis DNA double-strand breaks initiate recombination in the distantly related budding and fission yeasts and perhaps in most eukaryotes. Repair of broken meiotic DNA is essential for formation of viable gametes. We report here distinct but overlapping sets of proteins in these yeasts required for formation and repair of double-strand breaks. Meiotic DNA breakage in Schizosaccharomyces pombe did not require Rad50 or Rad32, although the homologs Rad50 and Mre11 are required in Saccharomyces cerevisiae; these proteins are required for meiotic DNA break repair in both yeasts. DNA breakage required the S. pombe midmeiosis transcription factor Mei4, but the structurally unrelated midmeiosis transcription factor Ndt80 is not required for breakage in S. cerevisiae. Rhp51, Swi5, and Rad22 + Rti1 were required for full levels of DNA repair in S. pombe, as are the related S. cerevisiae proteins Rad51, Sae3, and Rad52. Dmc1 was not required for repair in S. pombe, but its homolog Dmc1 is required in the well-studied strain SK1 of S. cerevisiae. Additional proteins required in one yeast have no obvious homologs in the other yeast. The occurrence of conserved and nonconserved proteins indicates potential diversity in the mechanism of meiotic recombination and divergence of the machinery during the evolution of eukaryotes.


Subject(s)
Conserved Sequence , DNA Repair/genetics , DNA, Fungal/genetics , Fungal Proteins/genetics , Yeasts/genetics , DNA Damage , Meiosis/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics
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