Processing and Visualization of Protec-Seq Data for the Generation of Calibrated, Genome-Wide Double Double-Strand Break (dDSB) Maps.

This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.

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. To address this question, we developed a novel approach that uses a bacterial ParB-parS partition system to recruit axis proteins at high levels to inserts at recombination coldspots where Hop1 and Red1 levels are normally low. Recruiting Hop1 markedly increased double-strand breaks and homologous recombination at target loci, to levels equivalent to those observed at endogenous recombination hotspots. This local increase in double-strand breaks did not require Red1 or the meiosis-specific cohesin component Rec8, indicating that, of the axis proteins, Hop1 is sufficient to promote double-strand break formation. However, while most crossovers at endogenous recombination hotspots are formed by the meiosis-specific MutLγ resolvase, crossovers that formed at an insert locus were only modestly reduced in the absence of MutLγ, regardless of whether or not Hop1 was recruited to that locus. Thus, while local Hop1 levels determine local double-strand break levels, the recombination pathways that repair these breaks can be determined by other factors, raising the intriguing possibility that different recombination pathways operate in different parts of the genome.

Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure.

Rec8 is a prominent component of the meiotic prophase chromosome axis that mediates sister chromatid cohesion, homologous recombination and chromosome synapsis. Here, we explore the prophase roles of Rec8. (i) During the meiotic divisions, Rec8 phosphorylation mediates its separase-mediated cleavage. We show here that such cleavage plays no detectable role for chromosomal events of prophase. (ii) We have analyzed in detail three rec8 phospho-mutants, with 6, 24 or 29 alanine substitutions. A distinct 'separation of function' phenotype is revealed. In the mutants, axis formation and recombination initiation are normal, as is non-crossover recombination; in contrast, crossover (CO)-related events are defective. Moreover, the severities of these defects increase coordinately with the number of substitution mutations, consistent with the possibility that global phosphorylation of Rec8 is important for these effects. (iii) We have analyzed the roles of three kinases that phosphorylate Rec8 during prophase. Timed inhibition of Dbf4-dependent Cdc7 kinase confers defects concordant with rec8 phospho-mutant phenotypes. Inhibition of Hrr25 or Cdc5/polo-like kinase does not. Our results suggest that Rec8's prophase function, independently of cohesin cleavage, contributes to CO-specific events in conjunction with the maintenance of homolog bias at the leptotene/zygotene transition of meiotic prophase.

Ubc9 sumoylation controls SUMO chain formation and meiotic synapsis in Saccharomyces cerevisiae.

Posttranslational modification with the small ubiquitin-related modifier SUMO depends on the sequential activities of E1, E2, and E3 enzymes. While regulation by E3 ligases and SUMO proteases is well understood, current knowledge of E2 regulation is very limited. Here, we describe modification of the budding yeast E2 enzyme Ubc9 by sumoylation (Ubc9(*)SUMO). Although less than 1% of Ubc9 is sumoylated at Lys153 at steady state, a sumoylation-deficient mutant showed significantly reduced meiotic SUMO conjugates and abrogates synaptonemal complex formation. Biochemical analysis revealed that Ubc9(*)SUMO is severely impaired in its classical activity but promoted SUMO chain assembly in the presence of Ubc9. Ubc9(*)SUMO cooperates with charged Ubc9 (Ubc9~SUMO) by noncovalent backside SUMO binding and by positioning the donor SUMO for optimal transfer. Thus, sumoylation of Ubc9 converts an active enzyme into a cofactor and reveals a mechanism for E2 regulation that orchestrates catalytic (Ubc9~SUMO) and noncatalytic (Ubc9(*)SUMO) functions of Ubc9.

Smc5/6-Mms21 prevents and eliminates inappropriate recombination intermediates in meiosis.

Repairing broken chromosomes via joint molecule (JM) intermediates is hazardous and therefore strictly controlled in most organisms. Also in budding yeast meiosis, where production of enough crossovers via JMs is imperative, only a subset of DNA breaks are repaired via JMs, closely regulated by the ZMM pathway. The other breaks are repaired to non-crossovers, avoiding JM formation, through pathways that require the BLM/Sgs1 helicase. "Rogue" JMs that escape the ZMM pathway and BLM/Sgs1 are eliminated before metaphase by resolvases like Mus81-Mms4 to prevent chromosome nondisjunction. Here, we report the requirement of Smc5/6-Mms21 for antagonizing rogue JMs via two mechanisms; destabilizing early intermediates and resolving JMs. Elimination of the Mms21 SUMO E3-ligase domain leads to transient JM accumulation, depending on Mus81-Mms4 for resolution. Absence of Smc6 leads to persistent rogue JMs accumulation, preventing chromatin separation. We propose that the Smc5/6-Mms21 complex antagonizes toxic JMs by coordinating helicases and resolvases at D-Loops and HJs, respectively.

Separation of roles of Zip1 in meiosis revealed in heterozygous mutants of Saccharomyces cerevisiae.

Synapsis of homologs during meiotic prophase I is associated with a protein complex built along the bivalents--the synaptonemal complex (SC). Mutations in the SC-component gene ZIP1 diminish SC formation, leading to reduced recombination levels and low spore viability. Here we show that in SK1 strains heterozygous for a deletion of ZIP1 in certain regions meiotic interference are impaired with no decrease in recombination levels. The extent of synapsis is over all reduced and NDJ levels of a large endogenous chromosome and of artificial chromosomes (YACs) rise to twice the level of wild type strains. A substantial proportion of mis-segregating YACs had undergone crossing over. This demonstrates that different functions of Zip1 display differential sensitivities to changes in expression levels.