Andreas Hochwagen

A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation

The nonrandom distribution of meiotic recombination influences patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in the accumulation of Spo11 protein covalently bound to small DNA fragments. By sequencing these fragments, we uncover a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore how DSB distribution is influenced by large-scale chromosome structures, chromatin, transcription factors, and local sequence composition. Our analysis offers mechanistic insight into DSB formation and early processing steps, supporting the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We also discuss implications for evolutionary dynamics of recombination hot spots.

Pds5 cooperates with cohesin in maintaining sister chromatid cohesion

BACKGROUND Sister chromatid cohesion depends on a complex called cohesin, which contains at least four subunits: Smc1, Smc3, Scc1 and Scc3. Cohesion is established during DNA replication, is partially dismantled in many, but not all, organisms during prophase, and is finally destroyed at the metaphase-to-anaphase transition. A quite separate protein called Spo76 is required for sister chromatid cohesion during meiosis in the ascomycete Sordaria. Spo76-like proteins are highly conserved amongst eukaryotes and a homologue in Aspergillus nidulans, called BimD, is required for the completion of mitosis. The isolation of the cohesin subunit Smc3 as a suppressor of BimD mutations suggests that Spo76/BimD might function in the same process as cohesin. RESULTS We show here that the yeast homologue of Spo76, called Pds5, is essential for establishing sister chromatid cohesion and maintaining it during metaphase. We also show that Pds5 co-localizes with cohesin on chromosomes, that the chromosomal association of Pds5 and cohesin is interdependent, that Scc1 recruits Pds5 to chromosomes in G1 and that its cleavage causes dissociation of Pds5 from chromosomes at the metaphase-to-anaphase transition. CONCLUSIONS Our data show that Pds5 functions as part of the same process as cohesin. Sequence similarities and secondary structure predictions indicate that Pds5 consists of tandemly repeated HEAT repeats, and might therefore function as a protein-protein interaction scaffold, possibly in the cohesin-DNA complex assembly.

Global Analysis of the Meiotic Crossover Landscape

Tight control of the number and distribution of crossovers is of great importance for meiosis. Crossovers establish chiasmata, which are physical connections between homologous chromosomes that provide the tension necessary to align chromosomes on the meiotic spindle. Understanding the mechanisms underlying crossover control has been hampered by the difficulty in determining crossover distributions. Here, we present a microarray-based method to analyze multiple aspects of crossover control simultaneously and rapidly, at high resolution, genome-wide, and on a cell-by-cell basis. Using this approach, we show that loss of interference in zip2 and zip4/spo22 mutants is accompanied by a reduction in crossover homeostasis, thus connecting these two levels of crossover control. We also provide evidence to suggest that repression of crossing over at telomeres and centromeres arises from different mechanisms. Lastly, we uncover a surprising role for the synaptonemal complex component Zip1 in repressing crossing over at the centromere.

Checking your breaks: Surveillance mechanisms of meiotic recombination

Numerous DNA double-strand breaks (DSBs) are introduced into the genome in the course of meiotic recombination. This poses a significant hazard to the genomic integrity of the cell. Studies in a number of organisms have unveiled the existence of surveillance mechanisms or checkpoints that couple the formation and repair of DSBs to cell cycle progression. Through these mechanisms, aberrant meiocytes are delayed in their meiotic progression, thereby facilitating repair of meiotic DSBs, or are culled through programmed cell death, thereby protecting the germline from aneuploidies that could lead to spontaneous abortions, birth defects and cancer predisposition in the offspring. Here we summarize recent progress in our understanding of these checkpoints. This review focuses on the surveillance mechanisms of the budding yeast S. cerevisiae, where the molecular details are best understood, but will frequently compare and contrast these mechanisms with observations in other organisms.

Mapping of Meiotic Single-Stranded DNA Reveals Double-Strand-Break Hotspots near Centromeres and Telomeres

BACKGROUND Every chromosome requires at least one crossover to be faithfully segregated during meiosis. At least two levels of regulation govern crossover distribution: where the initiating DNA double-strand breaks (DSBs) occur and whether those DSBs are repaired as crossovers. RESULTS We mapped meiotic DSBs in budding yeast by identifying sites of DSB-associated single-stranded DNA (ssDNA) accumulation. These analyses revealed substantial DSB activity in pericentrometric regions, in which crossover formation is largely absent. Our data suggest that centromeric suppression of recombination occurs at the level of break repair rather than DSB formation. Additionally, we found an enrichment of DSBs within a approximately 100 kb region near the ends of all chromosomes. Introduction of new telomeres was sufficient for inducing large ectopic regions of increased DSB formation, thereby revealing a remarkable long-range effect of telomeres on DSB formation. The concentration of DSBs close to chromosome ends increases the relative DSB density on small chromosomes, providing an interference-independent mechanism that ensures that all chromosomes receive at least one crossover per homolog pair. CONCLUSIONS Together, our results indicate that selective DSB repair accounts for crossover suppression near centromeres and suggest a simple telomere-guided mechanism that ensures sufficient DSB activity on all chromosomes.

The FK506 Binding Protein Fpr3 Counteracts Protein Phosphatase 1 to Maintain Meiotic Recombination Checkpoint Activity

The meiotic recombination checkpoint delays gamete precursors in G2 until DNA breaks created during recombination are repaired and chromosome structure has been restored. Here, we show that the FK506 binding protein Fpr3 prevents premature adaptation to damage and thus serves to maintain recombination checkpoint activity. Impaired checkpoint function is observed both in cells lacking FPR3 and in cells treated with rapamycin, a small molecule inhibitor that binds to the proline isomerase (PPIase) domain of Fpr3. FPR3 functions in the checkpoint through controlling protein phosphatase 1 (PP1). Fpr3 interacts with PP1 through its PPIase domain, regulates PP1 localization, and counteracts the activity of PP1 in vivo. Our findings define a branch of the recombination checkpoint involved in the adaptation to persistent chromosomal damage and a critical function for FK506 binding proteins during meiosis.

RNA Methylation by the MIS Complex Regulates a Cell Fate Decision in Yeast

For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions.

A Mec1- and PP4-Dependent Checkpoint Couples Centromere Pairing to Meiotic Recombination

The faithful alignment of homologous chromosomes during meiotic prophase requires the coordination of DNA double-strand break (DSB) repair with large-scale chromosome reorganization. Here we identify the phosphatase PP4 (Pph3/Psy2) as a mediator of this process in Saccharomyces cerevisiae. In pp4 mutants, early stages of crossover repair and homology-independent pairing of centromeres are coordinately blocked. We traced the loss of centromere pairing to the persistent phosphorylation of the chromosomal protein Zip1 on serine 75. Zip1-S75 is a consensus site for the ATR-like checkpoint kinase Mec1, and centromere pairing is restored in mec1 mutants. Importantly, Zip1-S75 phosphorylation does not alter chromosome synapsis or DSB repair, indicating that Mec1 separates centromere pairing from the other functions of Zip1. The centromeric localization and persistent activity of PP4 during meiotic prophase suggest a model whereby Zip1-S75 phosphorylation dynamically destabilizes homology-independent centromere pairing in response to recombination initiation, thereby coupling meiotic chromosome dynamics to DSB repair.

Checkpoint mechanisms: the puppet masters of meiotic prophase

The coordinated execution of cell cycle processes during meiosis is essential for the production of viable gametes and fertility. Coordination is particularly important during meiotic prophase, when nuclei undergo a dramatic reorganization that requires the precise choreography of chromosome movements, pairing interactions and DNA double-strand break (DSB) repair. Analysis of the underlying regulatory mechanisms has revealed crucial and widespread roles for DNA-damage checkpoint proteins, not only in cell cycle surveillance, but also in controlling many processes uniquely characteristic of meiosis. The resulting regulatory network uses checkpoint machinery to provide an integral coordinating mechanism during every meiotic division and enables cells to safely maintain an error-prone event such as DSB formation as an essential part of the meiotic program.

The Multiple Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing

Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. Previous studies indicated that, in addition to their cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation, and assembly of the synaptonemal complex (SC). Using mutants in the cohesin subunit Rec8, in which phosphorylated residues were mutated to alanines, we show that cohesin phosphorylation is not only important for cohesin removal, but that cohesins meiotic prophase functions are distinct from each other. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutations in REC8 that differentially affect Rec8s cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a key determinant of meiotic chromosome morphogenesis and a central player in multiple meiotic events.