Biology subjects

MacQueen, A. J.

Publications and source records attributed to MacQueen, A. J..

7 recordsLinked to original sources

bioRxiv2026

Two distinct modes of meiotic chromosome synapsis

The pairwise alignment of homologous chromosomes within the synaptonemal complex (SC) is important for meiotic crossover recombination and fertility. However, chromosomes do not need sequence homology to synapse, with meiotic recombination defects often leading to synapsis of non-homologous chromosome segments. Here we show that such heterologous synapsis reflects a distinctly regulated mode of meiotic chromosome synapsis that also happens during the early stages of wild-type yeast meiosis and occurs in parallel to the well-known synapsis initiation at crossover-designated sites. Heterologous synapsis initiates along chromosome arms after double-strand break resection and is accompanied by canonical markers of crossover repair, but does not need recombinase-dependent strand invasion. Instead, it requires the DNA-damage sensor kinase ATR/Mec1, which phosphorylates of a specific amino acid in Zip1, the major transverse filament protein of the SC. Phospho-mimetic mutants in ZIP1 rescue the synapsis defect of mec1 mutants and also partially restore gamete viability, indicating that this particular MEC1 function is important for the faithful completion of meiosis. Importantly, crossover repair quickly rectifies heterologous synapsis, allowing successful completion of meiosis even when most of the genome initially synapses independently of homology. Our data identify meiotic chromosome synapsis as a dynamic and reversible process that becomes optimized as result of recombination-dependent chromosome pairing.

genetics↗

bioRxiv2025

Crossover formation and coordinated assembly of synaptonemal complex relies on a direct interaction between Zip1 and Zip3

Several proteins collaborate to promote the crossover recombination events critical for accurate chromosome segregation during meiosis. How these "ZMM" factors (Zip2, Zip3, Zip4, Spo16, Mer3 and MutS{gamma}) collaboratively function remains incompletely understood. We previously reported that Zip3s abundance and activity rely on the synaptonemal complex (SC) component Zip1, and specifically on Zip1s N-terminal residues associated with crossovers and coupling SC assembly to the crossover pathway. Here, we demonstrate that Zip3 co-immunoprecipitates Zip1 from meiotic cells independent of recombination initiation and other ZMMs, and that Zip3s interaction with Zip1 relies on Zip1s N terminal residues. Co-expression and pull-down experiments in bacterial cells demonstrate that Zip1 and Zip3 interact directly. Experiments to identify Zip3 regions required for the Zip1 interaction unexpectedly revealed an incorrectly annotated translational start; we also determined that Zip3s N-terminal structured region is necessary and sufficient for the interaction, and a predicted coil downstream of Zip3s RING domain is essential for specific activities attributed to Zip1s N-terminal tip such as proximity labeling of Zip3 by Zip2 and the coupling of crossover recombination to SC assembly. Finally, we discovered that interaction with Zip1 protects Zip3 not only from proteasome-mediated degradation but also from post-translational modification when another ZMM is absent. We propose that direct interaction with Zip1s N terminus orients Zip3 within a nascent ZMM ensemble in a manner that facilitates crossover formation and the coupling of crossover intermediates to SC assembly, and furthermore ensures Zip3 remains both abundant and unmodified until all requisite ZMMs have joined the group.

genetics↗

bioRxiv2025

The conserved SEN1 DNA/RNA helicase has multiple functions during yeast meiosis

Diploid Saccharomyces cerevisiae cells undergo meiosis when they are starved of nitrogen in the presence of a non-fermentable carbon source. Nutrient starvation triggers expression of Ime1, a master regulatory protein required to activate transcription of meiotic "early genes" that mediate premeiotic S phase and prophase I processes, including recombination and chromosome synapsis. During prophase I, the highly conserved, toposomerase-like protein, Spo11, creates double strand breaks that are used to identify homologous chromosomes and generate crossovers between them. DNA:RNA hybrids are formed when an RNA molecule anneals to a complementary strand of DNA and are present at the ends of double strand breaks during prophase I of meiosis in a variety of organisms. DNA:RNA hybrids can be removed by degradation of the RNA by RNase H or by unwinding of the RNA by an essential, multi-functional DNA:RNA helicase called Sen1. Sen1 is orthologous to the mammalian Senataxin (SETX) helicase. Phenotypic characterization of mouse mutants lacking either Senataxin or RNase H activity exhibit male infertility and defects in double strand break repair. SETX is also required for meiotic sex chromosome inactivation, making it unclear whether SETXs role in meiotic recombination is direct or an indirect consequence due to defects in SETX functions that affect transcription. Using a variety of orthogonal approaches, this work demonstrates that SEN1 has multiple, temporally distinct roles that promote yeast meiosis. First, it enables the timely expression of IME1-regulated early genes. Second, it helps prevent and/or remove DNA:RNA hybrids that form during premeiotic S phase. Third, it facilitates both repair of Spo11 generated double strand breaks generated during prophase I and chromosome synapsis. AUTHOR SUMMARYDNA:RNA hybrids are unusual structures found throughout the genomes of many species, including yeast and mammals. While DNA:RNA hybrids may promote various cellular functions, persistent hybrids lead to double strand breaks, resulting in genomic instability. DNA:RNA hybrid formation and removal are therefore highly regulated by enzymes that either degrade or unwind RNA from the hybrid. Meiosis is the specialized cell division that creates haploid gametes for sexual reproduction. Previous work in yeast and mammals showed that elimination of DNA:RNA hybrids by RNase H facilitates meiotic recombination. This work demonstrates that the conserved Sen1 DNA:RNA helicase regulates the presence of DNA:RNA hybrids in three temporally distinct processes during yeast meiosis. First, SEN1 allows for meiosis-specific genes to be expressed at the proper time to allow entry into meiosis. Second, SEN1 prevents the accumulation of hybrids during premeiotic DNA replication. Third, SEN1 promotes the repair of programmed meiotic double strand breaks that are necessary to form crossovers between homologous chromosomes to allow their proper segregation at the first meiotic division. Given the evolutionary conservation of Sen1 with its mammalian counterpart, Senataxin, studies of Sen1 function in yeast are likely to be informative about the regulation of DNA:RNA hybrids during humans as well.

genetics↗

bioRxiv2023

Proximity labeling reveals new insights into the relationships between meiotic recombination proteins in S. cerevisiae

Several protein ensembles facilitate MutS{gamma} crossover recombination and the associated process of synaptonemal complex (SC) assembly during meiosis, but the physical and functional relationships between the components involved remain obscure. We have employed proximity labeling as a phenotypic tool to discern functional relationships between meiotic recombination and SC proteins in S. cerevisiae, and to gain deeper insight into molecular deficits of crossover-defective mutants. We find that recombination initiation (Spo11) and the Mer3 helicase are dispensable for proximity labeling of the Zip3 E3 ligase by components of the ZZS ensemble (Zip2, Zip4 and Spo16) but are required for proximity labeling of Zip3 by Msh4, consistent with the possibility that MutS{gamma} joins Zip3 only after a specific recombination intermediate has been generated. Proximity labeling analysis of crossover-defective zip1 mutants suggests a key shared defect is a failure to assemble an early recombination ensemble where ZZS can properly engage Zip3. We furthermore discovered that Zip3s abundance within the meiotic cell is uniquely dependent on the presence of Zip1, and that the post-translational modification of Zip3 is promoted by most MutS{gamma} pathway proteins but countered by Zip1. Based on this and additional data, we propose a model whereby Zip1 stabilizes a functional, unmodified form of Zip3 until intermediate steps in recombination are complete. We also find that SC structural protein Ecm11 is proximity labeled by ZZS complex proteins in a Zip4-dependent manner, but by Zip3 and Msh4, at least in part, via a distinct pathway. Finally, streptavidin pulldowns followed by mass spectrometry on eleven different proximity labeling strains uncovers shared proximity targets of MutS{gamma}-associated proteins, some with known meiotic functions and others not yet implicated in a meiotic activity, highlighting the potential power of proximity labeling as a discovery tool.

genetics↗

bioRxiv2021

A Role for Synaptonemal Complex in Meiotic Mismatch Repair

During meiosis a large subset of interhomolog recombination repair intermediates form within the physical context of the synaptonemal complex (SC), a protein-rich structure assembled at the interface of aligned homologous chromosomes. However, the functional relationship between SC structure and homologous recombination remains poorly defined. In prior work we determined that tripartite SC is dispensable for recombination in S. cerevisiae; SC central element proteins Ecm11 and Gmc2 instead limit the number of recombination events. Here we report that while dispensable for recombination per se, SC central element proteins influence the processing of interhomolog recombination intermediates in a manner that minimizes errors in mismatch correction. Failure to correct mis-paired bases within heteroduplex at meiotic recombination sites leads to genotypically sectored colonies (post meiotic segregation events) arising from mitotic proliferation of mismatch-containing spores. We discovered an increase in post-meiotic segregation at the THR1 locus in cells lacking Ecm11 or Gmc2, or in the SC-deficient but crossover recombination-proficient zip1[{Delta}21-163] mutant. High-throughput sequencing of octad meiotic products revealed a genome-wide increase in recombination events with uncorrected mismatches in ecm11 mutants relative to wild type. Meiotic cells missing Ecm11 also display longer gene conversion tracts, but tract length alone does not account for the higher frequency of uncorrected mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 mutants when analyzing all gene conversion tracts, but is similar between wild type and ecm11 if one considers only those events with uncorrected mismatches. Our data suggest that a subset of recombination events is similarly susceptible to mismatch repair errors in both wild type and ecm11 strains, but in ecm11 mutants many more recombination events fall into this inefficient repair category. Finally, we observe elevated post-meiotic segregation at THR1 in mutants with a dual deficiency in MutS{gamma}-mediated crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutS{gamma} crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates resolved via both MutS{gamma}-associated and MutS{gamma}-independent pathways, and is the molecular basis for elevated post-meiotic segregation in both MutS{gamma} crossover-proficient (ecm11, gmc2) and MutS{gamma} crossover-deficient (msh4, zip3) strains.

genetics↗

bioRxiv2019

SYCP2 translocation-mediated dysregulation and frameshift variants cause human male infertility

Infertility is one of the most common disorders for men of reproductive age. To identify novel genetic etiologies, we studied a male with severe oligozoospermia and 46, XY,t(20;22)(q13.3;q11.2). We identified exclusive overexpression of SYCP2 from the der(20) allele that is hypothesized to result from enhancer adoption. Modeling the dysregulation in budding yeast resulted in disruption of the synaptonemal complex, a common cause of defective spermatogenesis in mammals. Exome sequencing of infertile males revealed three novel heterozygous SYCP2 frameshift variants in additional subjects with cryptozoospermia and azoospermia. This study provides the first evidence of SYCP2-mediated male infertility in humans.

genetics↗

bioRxiv2018

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes, which is often associated with the meiosis-specific MutS{gamma} complex. The recombination intermediates that give rise to MutS{gamma} interhomolog crossovers are embedded within a hallmark meiotic prophase structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known central region building blocks of the budding yeast SC, the Zip1 protein is unique for its SC-independent role in promoting MutS{gamma} crossovers. Here we report that adjacent regions within Zip1s unstructured N terminus encompass its crossover and SC assembly functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents the robust SUMOylation of the SC central element component, Ecm11, but allows excess MutS{gamma} crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutS{gamma} crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutS{gamma} crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3s localization at Zip1 polycomplex and supports some MutS{gamma} crossing over but prevents normal SC assembly and robust Ecm11 SUMOylation. These results highlight distinct N terminal regions that are differentially critical for Zip1s roles in crossover recombination and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors in close proximity to one another.

genetics↗