Budding yeast Pch2, a widely conserved meiotic protein, is involved in the initiation of meiotic recombination

Budding yeast Pch2 protein is a widely conserved meiosis-specific protein whose role is implicated in the control of formation and displacement of meiotic crossover events. In contrast to previous studies where the function of Pch2 was implicated in the steps after meiotic double-strand breaks (DSBs) are formed, we present evidence that Pch2 is involved in meiotic DSB formation, the initiation step of meiotic recombination. The reduction of DSB formation caused by the pch2 mutation is most prominent in the sae2 mutant background, whereas the impact remains mild in the rad51 dmc1 double mutant background. The DSB reduction is further pronounced when pch2 is combined with a hypomorphic allele of SPO11. Interestingly, the level of DSB reduction is highly variable between chromosomes, with minimal impact on small chromosomes VI and III. We propose a model in which Pch2 ensures efficient formation of meiotic DSBs which is necessary for igniting the subsequent meiotic checkpoint responses that lead to proper differentiation of meiotic recombinants.     

Branching out: meiotic recombination and its regulation

Homologous recombination is a dynamic process by which DNA sequences and strands are exchanged. In meiosis, the reciprocal DNA recombination events called crossovers are central to the generation of genetic diversity in gametes and are required for homolog segregation in most organisms. Recent studies have shed light on how meiotic crossovers and other recombination products form, how their position and number are regulated and how the DNA molecules undergoing recombination are chosen. These studies indicate that the long-dominant, unifying model of recombination proposed by Szostak et al. applies, with modification, only to a subset of recombination events. Instead, crossover formation and its control involve multiple pathways, with considerable variation among model organisms. These observations force us to 'branch out' in our thinking about meiotic recombination.     

New and old ways to control meiotic recombination

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.     

Both conserved and non-conserved regions of Spo11 are essential for meiotic recombination initiation in yeast

DNA double-strand breaks (DSBs) are the initiators of most meiotic recombination events. In Saccharomyces cerevisiae, at least ten genes are necessary for meiotic DSB formation. However, the molecular roles of these proteins are not clearly understood. The meiosis-specific Spo11 protein, which shows sequence similarity with a subunit of an archaeal topoisomerase, is believed to catalyze the meiotic DSB formation. Spo11 is also required for induction of meiotic DSBs at long inverted repeats and at large trinucleotide repeat tracts. Here we report the isolation and characterization of temperature-sensitive spo11-mutant alleles to better understand how Spo11 functions, and how meiotic DSBs are generated at various recombination hotspots. Analysis of mutation sites of isolated spo11-mutant alleles indicated that both N-terminal and C-terminal non-conserved residues of Spo11 are essential for the protein’s function, possibly for interaction with other meiotic DSB enzymes. Several of the mutation sites within the conserved region are predicted to lie on the surface of the protein, suggesting that this region is required for activation of the meiotic initiation complex via protein-protein interaction. In addition to the conditional mutants, we isolated partially recombination-defective mutants; analysis of one of these mutants indicated that Ski8, as observed previously, interacts with Spo11 via the latter’s C-terminal residues.     

Multiple mechanisms of meiotic recombination

In this issue, reveal that different meiotic recombination mechanisms predominate in fission yeast and budding yeast. Budding yeast usually form crossover recombinants through double Holliday junctions, whereas fission yeast unexpectedly appear to form crossover recombinants through single junctions.     

Targeted stimulation of meiotic recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein. DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure. We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites. At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control.     

A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination

Genome stability relies on faithful DNA repair both in mitosis and in meiosis. Here, we report on a Caenorhabditis elegans protein that we found to be homologous to the mammalian repair-related protein CtIP and to the budding yeast Com1/Sae2 recombination protein. A com-1 mutant displays normal meiotic chromosome pairing but forms irregular chromatin aggregates instead of diakinesis bivalents. While meiotic DNA double-strand breaks (DSBs) are formed, they appear to persist or undergo improper repair. Despite the presence of DSBs, the recombination protein RAD-51, which is known to associate with single-stranded DNA (ssDNA) flanking DSBs, does not localize to meiotic chromosomes in the com-1 mutant. Exposure of the mutant to gamma-radiation, however, induces RAD-51 foci, which suggests that the failure of RAD-51 to load is specific to meiotic (SPO-11-generated) DSBs. These results suggest that C. elegans COM-1 plays a role in the generation of ssDNA tails that can load RAD-51, invade homologous DNA tracts and thereby initiate recombination. Extrapolating from the worm homolog, we expect similar phenotypes for mutations in the mammalian tumor suppressor CtIP.     

Double duty for Exo1 during meiotic recombination

Comment on: Zakharyevich K, et al. Mol Cell 2010; 40:1001-15.     

The impressionistic landscape of meiotic recombination

Two high-resolution maps of meiotic recombination initiation sites across the genomes of budding yeast and mice illuminate broad similarities in the control of meiotic recombination in these diverse species but also highlight key differences. These studies offer new insights into the relationships between recombination, chromosome structure, and genome evolution.     

Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination

Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation.     

DNA sequence-mediated, evolutionarily rapid redistribution of meiotic recombination hotspots

Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor.     

Maximal power tests for detecting defects in meiotic recombination

We have determined the marker separations (genetic distances) that maximize the probability, or power, of detecting meiotic recombination deficiency when only a limited number of meiotic progeny can be assayed. We find that the optimal marker separation is as large as 30-100 cM in many cases. Provided the appropriate marker separation is used, small reductions in recombination potential (as little as 50%) can be detected by assaying a single interval in as few as 100 progeny. If recombination is uniformly altered across the genomic region of interest, the same sensitivity can be obtained by assaying multiple independent intervals in correspondingly fewer progeny. A reduction or abolition of crossover interference, with or without a reduction of recombination proficiency, can be detected with similar sensitivity. We present a set of graphs that display the optimal marker separation and the number of meiotic progeny that must be assayed to detect a given recombination deficiency in the presence of various levels of crossover interference. These results will aid the optimal design of experiments to detect meiotic recombination deficiency in any organism.     

A physical assay for meiotic recombination in Coprinus cinereus

We have used the polymerase chain reaction (PCR) method to monitor meiotic recombination in the basidiomycete Coprinus cinereus. We used DNA-mediated transformation to recover strains with modifications of the trp1 locus. The modifications were designed to introduce unique PCR priming sites separated by a homologous 2.4?kb region in which crossing over could occur. We showed that exchange occurred in this region at the frequency expected for a typical region of this genome (2.4?kb should correspond to a genetic length of 0.08?cM). We also detected products resulting from crossing over in DNAs extracted from cells in meiotic prophase. The assay should be useful for monitoring exchange in mutants that cannot complete meiosis.     

Epigenetic control of meiotic recombination in plants

Meiotic recombination is a deeply conserved process within eukaryotes that has a profound effect on patterns of natural genetic variation. During meiosis homologous chromosomes pair and undergo DNA double strand breaks generated by the Spo11 endonuclease. These breaks can be repaired as crossovers that result in reciprocal exchange between chromosomes. The frequency of recombination along chromosomes is highly variable, for example, crossovers are rarely observed in heterochromatin and the centromeric regions. Recent work in plants has shown that crossover hotspots occur in gene promoters and are associated with specific chromatin modifications, including H2 A.Z. Meiotic chromosomes are also organized in loop-base arrays connected to an underlying chromosome axis, which likely interacts with chromatin to organize patterns of recombination.Therefore, epigenetic information exerts a major influence on patterns of meiotic recombination along chromosomes, genetic variation within populations and evolution of plant genomes.     

Persistence and loss of meiotic recombination hotspots

The contradiction between the long-term persistence of the chromosomal hotspots that initiate meiotic recombination and the self-destructive mechanism by which they act strongly suggests that our understanding of recombination is incomplete. This "hotspot paradox" has been reinforced by the finding that biased gene conversion also removes active hotspots from human sperm. To investigate the requirements for hotspot persistence, we developed a detailed computer simulation model of their activity and its evolutionary consequences. With this model, unopposed hotspot activity could drive strong hotspots from 50% representation to extinction within 70 generations. Although the crossing over that hotspots cause can increase population fitness, this benefit was always too small to slow the loss of hotspots. Hotspots could not be maintained by plausible rates of de novo mutation, nor by crossover interference, which alters the frequency and/or spacing of crossovers. Competition among hotspots for activity-limiting factors also did not prevent their extinction, although the rate of hotspot loss was slowed. Key factors were the probability that the initiating hotspot allele is destroyed and the nonmeiotic contributions hotspots make to fitness. Experimental investigation of these deserves high priority, because until the paradox is resolved all components of the mechanism are open to doubt.     

Molecular features of meiotic recombination hot spots

Meiotic recombination occurs preferentially at certain regions called hot spots and is important for generating genetic diversity and proper segregation of chromosomes during meiosis. Hot spots have been characterized most extensively in yeast, mice and humans. The development of methods based on sperm typing and population genetics has facilitated rapid and high-resolution mapping of hot spots in mice and humans in recent years. With increasing information becoming available on meiotic recombination in different species, it is now possible to compare several molecular features associated with hot-spot loci. Further, there have been advances in our knowledge of the factors influencing hot-spot activity and the role that they play in structuring the genome into haplotype blocks. We review the molecular features associated with hot spots in terms of their properties and mechanisms underlying their function and distribution. A large number of these features seem to be shared among hot spots from different species suggesting common mechanisms for their formation and function.     

Role of ubiquitination in meiotic recombination repair

Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associa...     

SCF ensures meiotic chromosome segregation through a resolution of meiotic recombination intermediates

The SCF (Skp1-Cul1-F-box) complex contributes to a variety of cellular events including meiotic cell cycle control, but its function during meiosis is not understood well. Here we describe a novel function of SCF/Skp1 in meiotic recombination and subsequent chromosome segregation. The skp1 temperature-sensitive mutant exhibited abnormal distribution of spindle microtubules in meiosis II, which turned out to originate from abnormal bending of the spindle in meiosis I. Bent spindles were reported in mitosis of this mutant, but it remained unknown how SCF could affect spindle morphology. We found that the meiotic bent spindle in skp1 cells was due to a hypertension generated by chromosome entanglement. The spindle bending was suppressed by inhibiting double strand break (DSB) formation, indicating that the entanglement was generated by the meiotic recombination machinery. Consistently, Rhp51/Rad51-Rad22/Rad52 foci persisted until meiosis I in skp1 cells, proving accumulation of recombination intermediates. Intriguingly bent spindles were also observed in the mutant of Fbh1, an F-box protein containing the DNA helicase domain, which is involved in meiotic recombination. Genetic evidence suggested its cooperation with SCF/Skp1. Thus, SCF/Skp1 together with Fbh1 is likely to function in the resolution of meiotic recombination intermediates, thereby ensuring proper chromosome segregation.     

Gene conversion, recombination nodules, and the initiation of meiotic synapsis

The nature of the relationship between the two types of meiotic recombination outcomes, exchange (crossing-over) and simple gene conversion, has been debated for years. I here propose that these two types of events are not necessarily causally related and hypothesize that the primary role of events detected as simple gene conversion is in the recognition of homology during synapsis.