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. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

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). D-loop processing then forms crossover and non-crossover recombinants. Meiotic recombination occurs in two temporally distinct phases. During Phase 1, Rad51 is inhibited and Dmc1 mediates the interhomolog recombination that promotes homolog synapsis. In Phase 2, Rad51 becomes active and functions with Rad54 to repair residual DSBs, making increasing use of sister chromatids. The transition from Phase 1 to Phase 2 is controlled by the meiotic recombination checkpoint through the meiosis-specific effector kinase Mek1. This work shows that constitutive activation of Rad51 in Phase 1 results in a subset of DSBs being repaired by a Rad51-mediated interhomolog recombination pathway that is distinct from that of Dmc1. Strand invasion intermediates generated by Rad51 require more time to be processed into recombinants, resulting in a meiotic recombination checkpoint delay in prophase I. Without the checkpoint, Rad51-generated intermediates are more likely to involve a sister chromatid, thereby increasing Meiosis I chromosome nondisjunction. This Rad51 interhomolog recombination pathway is specifically promoted by the conserved 5'-3' helicase PIF1 and its paralog, RRM3 and requires Pif1 helicase activity and its interaction with PCNA. This work demonstrates that (1) inhibition of Rad51 during Phase 1 is important to prevent competition with Dmc1 for DSB repair, (2) Rad51-mediated meiotic recombination intermediates are initially processed differently than those made by Dmc1, and (3) the meiotic recombination checkpoint provides time during prophase 1 for processing of Rad51-generated recombination intermediates.

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. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

Temporal and Functional Relationship between Synaptonemal Complex Morphogenesis and Recombination during Meiosis.

During prophase of meiosis I, programmed double strand breaks (DSBs) are processed into crossovers, a critical requirement for segregation of homologous chromosomes (homologs) and genome haploidization in sexually reproducing organisms. Crossovers form via homologous recombination in close temporospatial association with morphogenesis of the synaptonemal complex (SC), a proteinaceous structure that connects paired homologs along their length during the pachytene stage. Synapsis and recombination are a paradigm for the interplay between higher order chromosome structure and DNA metabolism, yet their temporal and functional relationship remains poorly understood. Probing linkage between these processes in budding yeast, we show that SC assembly is associated with a distinct threshold number of unstable D-loops. The transition from bona fide paranemic D-loops to plectonemic DSB single end invasions (SEIs) is completed during midpachynema, when the SC is fully assembled. Double Holliday junctions (dHJs) form at the time of desynapsis and are resolved into crossovers during diplonema. The SC central element component Zip1 shepherds recombination through three transitions, including DSB first end strand exchange and second end capture, as well as dHJ resolution. Zip1 mediates SEI formation independent of its polymerization whereas precocious Zip1 assembly interferes with double Holliday junction resolution. Together, our findings indicate that the synaptonemal complex controls recombination while assembled but also beyond its disassembly, possibly by establishing spatial constraints at recombination sites.

Genome sequencing of Pakistani families with male infertility identifies deleterious genotypes in SPAG6, CCDC9, TKTL1, TUBA3C, and M1AP.

BACKGROUND: There are likely to be hundreds of monogenic forms of human male infertility. Whole genome sequencing (WGS) is the most efficient way to make progress in mapping the causative genetic variants, and ultimately improve clinical management of the disease in each patient. Recruitment of consanguineous families is an effective approach to ascertain the genetic forms of many diseases. OBJECTIVES: To apply WGS to large consanguineous families with likely hereditary male infertility and identify potential genetic cases. MATERIALS AND METHODS: We recruited seven large families with clinically diagnosed male infertility from rural Pakistan, including five with a history of consanguinity. We generated WGS data on 26 individuals (3-5 per family) and analyzed the resulting data with a computational pipeline to identify potentially causal single nucleotide variants, indels, and copy number variants. RESULTS: We identified plausible genetic causes in five of the seven families, including a homozygous 10 kb deletion of exon 2 in a well-established male infertility gene (M1AP), and biallelic missense substitutions (SPAG6, CCDC9, TUBA3C) and an in-frame hemizygous deletion (TKTL1) in genes with emerging relevance. DISCUSSION AND CONCLUSION: The rate of genetic findings using the current approach (71%) was much higher than what we recently achieved using whole-exome sequencing (WES) of unrelated singleton cases (20%). Furthermore, we identified a pathogenic single-exon deletion in M1AP that would be undetectable by WES. Screening more families with WGS, especially in underrepresented populations, will further reveal the types of variants underlying male infertility and accelerate the use of genetics in the patient management.

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. One example is the Sgs1-Top3-Rmi1 helicase-decatenase complex, which is required for normal vegetative growth and genome stability. In such cases, conditional loss-of-function mutants can be useful. In this chapter, we describe the construction of two types of conditional mutants, meiotic depletion alleles and auxin-induced degradation alleles, that allow protein depletion specifically during budding yeast meiosis, and illustrate their use with Sgs1. We also describe a modified method for the isolation of meiotic recombination intermediates that combines previous psoralen cross-linking and cetyltrimethylammonium bromide isolation methods.

Control of meiotic pairing and recombination by chromosomally tethered 26S proteasome.

During meiosis, paired homologous chromosomes (homologs) become linked via the synaptonemal complex (SC) and crossovers. Crossovers mediate homolog segregation and arise from self-inflicted double-strand breaks (DSBs). Here, we identified a role for the proteasome, the multisubunit protease that degrades proteins in the nucleus and cytoplasm, in homolog juxtaposition and crossing over. Without proteasome function, homologs failed to pair and instead remained associated with nonhomologous chromosomes. Although dispensable for noncrossover formation, a functional proteasome was required for a coordinated transition that entails SC assembly between longitudinally organized chromosome axes and stable strand exchange of crossover-designated DSBs. Notably, proteolytic core and regulatory proteasome particles were recruited to chromosomes by Zip3, the ortholog of mammalian E3 ligase RNF212, and SC protein Zip1 . We conclude that proteasome functions along meiotic chromosomes are evolutionarily conserved.

Analysis of meiotic recombination intermediates by two-dimensional gel electrophoresis.

During meiosis, programmed double strand breaks give rise to crossover and non-crossover recombination products. Meiotic recombination products are formed via several branched intermediates, including single end invasions and double Holliday junctions. Two-dimensional gel electrophoresis provides a sensitive and specific approach for detecting branched recombination intermediates, determining their genetic requirements, and enriching intermediates for further analysis. Here, we describe analysis of branched recombination intermediates in the yeast Saccharomyces cerevisiae by two-dimensional gel electrophoresis. We also provide an introduction to meiotic time-course procedures, stabilization of branched DNA molecules by interstrand crosslinking, extraction of genomic DNA from meiotic cultures, and quantitative analysis of two-dimensional gel blots.