Biology subjects

Weir, J. R.

Publications and source records attributed to Weir, J. R..

9 recordsLinked to original sources

bioRxiv2026

Assembly principles of a SYCP2-HORMAD1-HORMAD2 mammalian meiotic axis complex

During meiotic prophase I, the chromosome axis orchestrates programmed DNA double-strand break formation, repair and synapsis between homologous chromosomes. In mammals, the axis is assembled from the coiled-coil elements SYCP2 and SYCP3 that come together with the HORMA-domain proteins HORMAD1 and HORMAD2, but how these components associate into a coherent structural unit remains incompletely understood. Combining recombinant reconstitution, mass photometry, SEC-MALS, AlphaFold modelling and crosslinking mass spectrometry, we show that the HORMA domains of HORMAD1 and HORMAD2 form a selective pseudosymmetric heterodimer independently of either proteins own closure motif, with interface determinants conserved across vertebrates. We identify a previously unrecognised second closure motif (CM2) in SYCP2 that preferentially binds HORMAD1, distinct from the previously described HORMAD2-binding closure motif (CM1). Together, these results revise the current model of mammalian axis assembly and define a tripartite SYCP2-HORMAD1-HORMAD2 module as a fundamental structural unit of the mammalian meiotic chromosome axis.

cell biology↗

bioRxiv2026

G-quadruplexes regulate chromatin accessibility and gene expression in Bloom Syndrome

Bloom Syndrome (BS) is a recessive genetic disorder characterized by hyper-recombination and genome instability. It is caused by mutations in BLM, which encodes a conserved RecQ helicase that unwinds various aberrant DNA structures. One such structure is DNA G-quadruplexes (G4s), which have versatile regulatory potential in chromatin organization and gene expression. However, whether G4 profiles are altered in BS and how G4s contribute to disease-associated molecular changes remain unclear. Here, we profiled chromatin accessibility and gene expression using ATAC-seq and RNA-seq and mapped endogenous G4 by ChIP-seq in wild type (WT) and BS cell lines. We observed that in BS cell lines, differential G4 formation positively correlated with both differential chromatin accessibility and gene expression. To test the direct involvement of G4s in the molecular phenotypes in BS, we applied pyridostatin, a G4-stabilizing molecule, in WT cells and showed that G4 stabilization partially recapitulated BS-associated molecular phenotypes. Additionally, we found that regions with increased chromatin accessibility in BS individuals in a family were also enriched for G4-forming sequences. Together, our data substantiate a regulatory role for G4s in Bloom syndrome and support a molecular model in which unresolved G4s in BLM- /- cells enhance chromatin accessibility, thereby promoting gene expression. These findings reveal an expanded regulatory function of BLM mediated through G4 structures and previously underappreciated role of G4s in the molecular etiology of BS.

genomics↗

bioRxiv2025

RPA directly stimulates Mer3/HFM1 helicase processivity to ensure normal crossover formation in meiosis

Meiotic crossover formation is critical for generating viable gametes and enhancing genetic diversity. The helicase Mer3 (HFM1 in humans) is a highly conserved factor essential for promoting crossovers and ensuring their proper distribution. Here, we identify replication protein A (RPA) as a direct interactor of budding yeast Mer3. We demonstrate that this interaction is conserved between human HFM1 and RPA. Cross-linking mass spectrometry and structural modelling with AlphaFold2 reveal a conserved and specific Mer3-RPA interface. Single-molecule magnetic tweezers assays demonstrate that direct RPA interaction is required for Mer3 helicase processivity under conditions of low DNA tension. Consistently, a mer3 mutant deficient in RPA binding exhibit reduced crossover frequencies and accumulate unresolved recombination intermediates during budding yeast meiosis. Via genome-wide localisation experiments, we link this effect to a weakened recruitment to double-strand break sites of the mer3 mutant. Our findings provide mechanistic insights into coordination of meiotic recombination by the Mer3 helicase through interactions with the canonical DNA repair machinery, highlighting a conserved mechanism underlying crossover control during sexual reproduction.

biochemistry↗

bioRxiv2024

Exploring Meiosis in Brown Algae: Meiotic Axis Proteins in the model brown alga Ectocarpus

Most extant eukaryotic systems share core meiosis-specific genes, suggesting meiosis evolved only once in the last eukaryotic common ancestor (LECA). These genes have been characterized as master regulators of meiotic recombination, ensuring genetically diverse lineages. However, our understanding is limited as eukaryotic organisms beyond the animal, plant, and yeast lineages remain poorly understood. Recently, core meiotic genes have been identified in the genome of the model brown alga Ectocarpus, but currently lack proper characterization. Here, we combine bioinformatic, structural, and biochemical approaches to characterise the axial element orthologs, meiotic Ectocarpus HORMA domain protein (ecHOP1) and its interactor, reductional division protein 1 (ecRED1), in order to elucidate the molecular mechanisms of synaptonemal complex (SC) and double strand break (DSB) formation in brown algae. We highlight novel domain architecture within ecHOP1 and ecRED1 that support HORMA domain conformational switches and quantify the thermodynamic parameters of these interactions. Together, our data suggests that brown algae may employ alternative HORMA domain regulation mechanisms compared with animal, plant and yeast systems, and provide clues for future studies on the evolutionary constraints and adaptation of meiosis across the tree of life.

biochemistry↗

bioRxiv2024

Kinetic and structure-based comparisons of silent and stimulatory flagellin interactions with TLR5

The bacterial protein flagellin is the sole ligand of the innate immune receptor Toll- like receptor 5 (TLR5). Flagellins with strong agonism bind TLR5 at their D1 and D0 domains, while poor agonist "silent" flagellins bind with D1 only. However, D0-TLR5 interactions insufficiently explain the silent phenotype. Here, we characterize the D1 domain binding kinetics of the silent flagellin RhFlaB compared to the strong canonical agonist StFliC. Using Surface Plasmon Resonance, we show that the RhFlaB-D1 binds more strongly than StFliC-D1, but forms shorter-lived complexes. Cryo-EM analysis of RhFlaB showed its D1 primary interface (PI) contains two distinct hydrophobic pockets, which should facilitate rapid association, while its D1s secondary interface (SI) is dominated by negatively charged residues, which likely impede residue-residue interactions. Comparisons with an existing StFliC-D1 structure suggest that strong agonism requires strong binding at both the PI and SI of the D1, and silent flagellins evade detection partly through weaker SI interactions. These findings offer a foundation for designing targeted interventions that can modulate TLR5 activity to influence immunity, inflammation, or even gut microbiome composition.

biochemistry↗

bioRxiv2022

Mer3 helicase protects early crossover intermediates from STR complex disassembly during meiosis

During meiosis I it is necessary that homologous chromosomes are linked to one another so that they can be faithfully separated. S. cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of class I crossovers during meiosis. Here we describe the structural organisation of Mer3 and, using AlphaFold modelling and XL-MS, we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the recombination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase in a phospho-dependent manner. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1/Top3/Rmi1 (STR) complex. Thus we provide a mechanism whereby Mer3 downregulates the anti-crossover activity of the STR complex, hence promoting the formation of crossovers during meiosis I.

biochemistry↗

bioRxiv2022

Studying meiosis in mitosis: Activating the meiosis-specific Red1-Hop1-Mek1 complex in mitotic budding yeast cells

In mitosis, sister chromatids are preferred repair templates for homologous recombination, whereas in meiosis interhomolog-based repair is promoted. How this switch, which is a defining event in sexual reproduction, is accomplished remains poorly understood. In budding yeast, a meiosis-specific complex consisting of Red1, Hop1 and Mek1 (RHMc) enforces meiotic interhomolog bias, potentially through inhibition of intersister-based repair. The current data points to a linear assembly governing RHMc formation: the HORMA protein Hop1 associates with Red1 via a closure-motif-HORMA domain interaction, and Mek1 kinase is recruited through phospho-mediated interactions with Hop1. Here, via expression in mitotic cells we autonomously establish the RHM complex. In vivo analysis complemented with in vitro biochemical reconstitution shows that Mek1 associates with Red1, in a manner that might resemble binding of other kinases with scaffolding activators. The NH2-terminus of Red1 contributes to Hop1 binding, suggesting cooperative binding between Red1 and the HORMA domain of Hop1, beyond closure motif-based interactions. Meiotic activation of Mek1 kinase is dictated by complex formation and upstream DNA break-dependent signaling. We find Mek1 can be activated under DNA damaging conditions in mitotically dividing cells, where activation depends on upstream Mec1 kinase function and RHMc integrity. We perform a structure-function analysis of RHMc formation and Mek1 activation. Finally, we show that activation of Mek1 in mitosis leads to rad51{Delta}-like DNA break sensitivity, providing evidence for the model that RHMc instates meiotic interhomolog-based repair by inhibiting mitotic homologous recombination. Our analysis enables querying downstream effects of RHMc action on DNA repair. Because aberrant re-expression of homologs of Red1 and Hop1 leads to DNA repair defects in human cancer, our system can be used to study roles of these genes during tumorigenesis.

genetics↗

bioRxiv2020

Mer2 binds directly to both nucleosomes and axial proteins as the keystone of meiotic recombination.

In meiosis, DNA double strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific ?axis-tethered loop? chromosome organization. Through in vitro reconstitution and budding yeast genetics we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates to nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 establishes an interaction with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.

biochemistry↗

bioRxiv2019

A meiosis-specific AAA+ assembly reveals repurposing of ORC during budding yeast gametogenesis

ORC (Orc1-6) is an AAA+ complex that loads the AAA+ MCM helicase to replication origins. Orc1, a subunit of ORC, functionally interacts with budding yeast Pch2, a meiosis-specific AAA+ protein. Pch2 regulates several chromosomal events of gametogenesis, but mechanisms that dictate Pch2 function remain poorly understood. We demonstrate that ORC directly interacts with an AAA+ Pch2 hexamer. The ORC-Pch2 assembly is established without Cdc6, a factor crucial for ORC-MCM binding. Biochemical analysis suggests that Pch2 utilizes ORCs Cdc6-binding interface and employs its non-enzymatic NH2-terminal domain and AAA+ core to engage ORC. In contrast to phenotypes observed upon Orc1 impairment, nuclear depletion of other subunits of ORC does not lead to Pch2-like phenotypes, indicating that ORC integrity per se is not required to support Pch2 function. We thus reveal functional interplay between Pch2 and ORC, and uncover the repurposing of ORC to establish a non-canonical and meiosis-specific AAA+ assembly.

biochemistry↗