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.

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 ∼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.

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 ϩ 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. I N most eukaryotes homologous recombination ocobserved more recently in a second organism, the distantly related fission yeast Schizosaccharomyces pombe (Cercurs at high levels during meiosis to aid the proper segregation of homologs at the first meiotic division vantes et al. 2000). There are two lines of indirect evidence for meiotic ds breaks in other organisms. First, and to increase genetic diversity among gametes, the products of meiosis. The two meiotic cell divisions revarious eukaryotes encode homologs of the Spo11 protein, which is essential for meiotic DNA ds break formaduce the diploid number of chromosomes in the precursor cells to the haploid number in the gametes. The tion in S. cerevisiae (Keeney et al. 1997). Where tested in other organisms, these proteins are essential for meiotic general mechanism of reductional segregation of homologs is highly conserved: in most eukaryotes recombinarecombination or viable gamete formation (Keeney 2001), and in S. pombe the homolog, called Rec12, is tion between homologs provides a physical connection between them that imparts tension when the homologs also essential for meiotic DNA ds break formation (Cervantes et al. 2000). Second, in mice a modified histone, are properly arranged to segregate to opposite poles of the cell (Nicklas 1997). In the absence of recombina-␥-H2AX, thought to associate with chromatin specifically near ds breaks, appears as foci on meiotic chromotion, homologs frequently missegregate, resulting in aneuploid gametes; the subsequent zygotic progeny are somes at the time expected for recombination; these foci are Spo11 dependent (Rogakou et al. 1998; Mahafrequently sick or dead, underscoring the importance of un...

SUMMARY 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 non-crossovers 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 vs. homolog for DSB repair. At strong DSB hotspots, intersister repair outnumbers interhomolog repair ~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.

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 Ͼ25 kb away. Possible mechanisms for generating crossovers in such break-free intervals are discussed. I N eukaryotic cells, DNA double-strand breaks (DSBs) ferred sites at widely separated physical distances ‫-05ف(‬ 100 kb on average). In contrast, crossovers are observed are a dangerous form of DNA damage and yet are also crucial for the process of meiosis. During vegetative at a relatively constant frequency per unit of physical distance (Young et al. 2002); i.e., crossovers are not growth, DSBs are a threat because, left unrepaired, they cause chromosomal fragmentation. In addition, repair consistently more frequent in intervals where strong breaks are observed than in apparently break-free interof DSBs can promote genomic instability, including DNA rearrangements resulting from homologous revals. It was proposed that the DSB and crossover patterns could be reconciled if meiotic DSBs are able to generate combination. In contrast, the deliberate generation of DSBs appears to be a conserved feature of meiosis precrossovers over large distances (tens of kilobases) around their positions (Young et al. 2002). cisely because of their ability to promote homologous recombination and generate crossovers. Crossovers, in Two factors control how the frequency of DSBs relates to the frequency of crossovers. First, DSBs can be return, are critical for correct meiotic chromosome segregation and the promotion of genetic diversity among paired using either sister chromatids or homologous chromosomes (homologs), but genetically observable the products of meiosis. The importance of DSB formation in generating meiotic crossovers has been well escrossovers can arise only from interhomolog events. The relative frequency of intersister and interhomolog tablished in Saccharomyces cerevisiae and Schizosaccharomyces pombe and in...