Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase.

During meiosis, recombination between homologous chromosomes (homologs) generates crossovers that promote proper segregation at the first meiotic division. Recombination is initiated by Spo11-catalyzed DNA double strand breaks (DSBs). 5' end resection of the DSBs creates 3' single strand tails that two recombinases, Rad51 and Dmc1, bind to form presynaptic filaments that search for homology, mediate strand invasion and generate displacement loops (D-loops).

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination in Saccharomyces cerevisiae.

The DNA double-strand breaks that initiate meiotic recombination are formed in the context of the meiotic chromosome axis, which in Saccharomyces cerevisiae contains a meiosis-specific cohesin isoform and the meiosis-specific proteins Hop1 and Red1. Hop1 and Red1 are important for double-strand break formation; double-strand break levels are reduced in their absence and their levels, which vary along the lengths of chromosomes, are positively correlated with double-strand break levels. How axis protein levels influence double-strand break formation and recombination remains unclear.

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination.

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus.

C-Terminal HA Tags Compromise Function and Exacerbate Phenotypes of Bloom's Helicase Homolog Sgs1 SUMOylation-Associated Mutants.

The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA).

Unresolved Recombination Intermediates Cause a -Dependent Cell Cycle Arrest in .

In , the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are crossover precursors. studies have suggested that this may be due to dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation. To ask whether dissolution occurs , we conditionally depleted Sgs1 and/or Rmi1 during return to growth (RTG), a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle.

S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates.

We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate.

Noncanonical Contributions of MutLγ to VDE-Initiated Crossovers During Meiosis.

In , the meiosis-specific axis proteins Hop1 and Red1 are present nonuniformly across the genome. In a previous study, the meiosis-specific VMA1-derived endonuclease (VDE) was used to examine Spo11-independent recombination in a recombination reporter inserted in a Hop1/Red1-enriched region () and in a Hop1/Red1-poor region (). VDE-initiated crossovers at 4 were mostly dependent on Mlh3, a component of the MutLγ meiotic recombination intermediate resolvase, while VDE-initiated crossovers at were mostly Mlh3-independent.

Methods for Controlled Protein Depletion to Study Protein Function during Meiosis.

Proteins with potential roles in meiotic recombination often have essential or important functions during the mitotic cell cycle. In addition, proteins may have different functions at different times during meiosis. In such cases, it can be challenging to precisely determine protein function during meiosis using null or hypomorphic mutants.

Proteasomes on the chromosome.

Targeted proteolysis plays an important role in the execution and regulation of many cellular events. Two recent papers in Science identify novel roles for proteasome-mediated proteolysis in homologous chromosome pairing, recombination, and segregation during meiosis.

Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis.

The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined.

Differential regulation of the anti-crossover and replication fork regression activities of Mph1 by Mte1.

We identified Mte1 (Mph1-associated telomere maintenance protein 1) as a multifunctional regulator of Saccharomyces cerevisiae Mph1, a member of the FANCM family of DNA motor proteins important for DNA replication fork repair and crossover suppression during homologous recombination. We show that Mte1 interacts with Mph1 and DNA species that resemble a DNA replication fork and the D loop formed during recombination. Biochemically, Mte1 stimulates Mph1-mediated DNA replication fork regression and branch migration in a model substrate.

Generation of an inducible system to express polo-like kinase, Cdc5 as TAP fusion protein during meiosis in Saccharomyces cerevisiae.

Tandem affinity purification (TAP) is a highly efficient method for isolation of protein complexes from endogenous biological macromolecules. TAP system consists of dual affinity tags that facilitates the sequential purification of the desired proteins expressed at their low levels in vivo. Polo-like kinases (PLK) are serine/threonine protein kinases that are the crucial regulators of cell cycle.

Top3-Rmi1 DNA single-strand decatenase is integral to the formation and resolution of meiotic recombination intermediates.

The topoisomerase III (Top3)-Rmi1 heterodimer, which catalyzes DNA single-strand passage, forms a conserved complex with the Bloom's helicase (BLM, Sgs1 in budding yeast). This complex has been proposed to regulate recombination by disassembling double Holliday junctions in a process called dissolution. Top3-Rmi1 has been suggested to act at the end of this process, resolving hemicatenanes produced by earlier BLM/Sgs1 activity.

Tetrad, random spore, and molecular analysis of meiotic segregation and recombination.

The power of Saccharomyces cerevisiae as an experimental organism derives from its genetic tractability. Mutant variants can be isolated or constructed and phenotypically characterized with relative ease. In addition, the ability to recover and characterize all four products of meiosis, as haploid spores in a tetrad ascus, greatly facilitates determining the allelic composition of variants, measuring linkage relationships between alleles, and constructing new allele combinations for the analysis of genetic interactions.

A timeless but timely connection between replication and recombination.

Initiation of meiotic recombination by DNA double-strand break formation is temporally coordinated with replication. Murakami and Keeney show that this coordination requires recruitment of the Dbf4-dependent kinase to the replication fork by the conserved TIM-TIPIN complex. The same mechanism may regulate other important replication-associated processes.

BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism.

The BLM helicase has been shown to maintain genome stability by preventing accumulation of aberrant recombination intermediates. We show here that the Saccharomyces cerevisiae BLM ortholog, Sgs1, plays an integral role in normal meiotic recombination, beyond its documented activity limiting aberrant recombination intermediates. In wild-type meiosis, temporally and mechanistically distinct pathways produce crossover and noncrossover recombinants.

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.

Meiotic recombination intermediates are resolved with minimal crossover formation during return-to-growth, an analogue of the mitotic cell cycle.

Accurate segregation of homologous chromosomes of different parental origin (homologs) during the first division of meiosis (meiosis I) requires inter-homolog crossovers (COs). These are produced at the end of meiosis I prophase, when recombination intermediates that contain Holliday junctions (joint molecules, JMs) are resolved, predominantly as COs. JM resolution during the mitotic cell cycle is less well understood, mainly due to low levels of inter-homolog JMs.

Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs.

Stabilization and electrophoretic analysis of meiotic recombination intermediates in Saccharomyces cerevisiae.

Joint Molecule (JM) recombination intermediates result from DNA strand-exchange between homologous chromosomes. Physical monitoring of JM formation in budding yeast has provided a wealth of information about the timing and mechanism of meiotic recombination. These assays are especially informative when applied to the analysis of mutants for which genetic analysis of recombination is impossible, i.

Genome-wide mapping of meiotic DNA double-strand breaks in Saccharomyces cerevisiae.

DNA double-strand breaks (DSBs) initiate meiotic recombination in eukaryotes. We describe two strategies that use microarrays to determine the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) approach that targets the Spo11 protein, which remains covalently attached to DSB ends in certain mutant backgrounds.

Ipl1/Aurora B kinase coordinates synaptonemal complex disassembly with cell cycle progression and crossover formation in budding yeast meiosis.

Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. We identified Ipl1/Aurora B kinase as the main regulator of SC disassembly.

Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis.

In budding yeast, exit from the pachytene stage of meiosis requires the mid-meiosis transcription factor Ndt80, which promotes expression of approximately 200 genes. Ndt80 is required for meiotic function of polo-like kinase (PLK, Cdc5) and cyclin-dependent kinase (CDK), two cell cycle kinases previously implicated in pachytene exit. We show that ongoing CDK activity is dispensable for two events that accompany exit from pachytene: crossover formation and synaptonemal complex breakdown.