Biology subjects

Vader, G.

Publications and source records attributed to Vader, G..

6 recordsLinked to original sources

bioRxiv2026

The SKA complex is a mitotic vulnerability of chromosomally unstable cancers

Aneuploidy and chromosomal instability are common characteristics of cancer but remain underexploited therapeutically. Because chromosomally unstable cells exhibit altered mitotic control, they are selectively vulnerable to inhibition of the kinesin KIF18A. Here, we identify the spindle and kinetochore-associated (SKA) complex as a mitotic regulator that establishes a similar dependency. SKA loss is tolerated in cells with a defective mitotic checkpoint, but when mitosis is prolonged, it leads to mitotic errors and loss of viability. Genetic analysis reveals that SKA and KIF18A are epistatic, although they function through mechanistically distinct mitotic pathways. SKA depletion selectively impacts chromosomally unstable cancer cells and mirrors the effects of KIF18A inhibition. We thus identify a cellular vulnerability associated with prolonged mitosis in chromosomally unstable cancers.

cell biology↗

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

Homeostatic control of meiotic G2/prophase checkpoint function by Pch2 and Hop1

Checkpoints cascades coordinate cell cycle progression with essential chromosomal processes. During meiotic G2/prophase, recombination and chromosome synapsis are monitored by what are considered distinct checkpoints [1-3]. In budding yeast, the AAA+ ATPase Pch2 is thought to specifically promote cell cycle delay in response to synapsis defects [4-6]. However, unperturbed pch2{Delta} cells are delayed in meiotic G2/prophase [6], suggesting paradoxical roles for Pch2 in cell cycle progression. Here, we provide insight into the checkpoint roles of Pch2 and its connection to Hop1, a HORMA domain-containing client protein. Contrary to current understanding, we find that the Pch2-Hop1 module is crucial for checkpoint function in response to both recombination and synapsis defects, thus revealing a shared meiotic checkpoint cascade. Meiotic checkpoint responses are transduced by DNA break-dependent phosphorylation of Hop1 [7, 8]. Based on our data and on the effect of Pch2 on HORMA topology [9-11], we propose that Pch2 promotes checkpoint proficiency by catalyzing the availability of signaling-competent Hop1. Conversely, we demonstrate that Pch2 can act as a checkpoint silencer, also in the face of persistent DNA repair defects. We establish a framework in which Pch2 and Hop1 form a homeostatic module that governs general meiotic checkpoint function. We show that this module can - depending on the cellular context - fuel or extinguish meiotic checkpoint function, which explains the contradictory roles of Pch2 in cell cycle control. Within the meiotic checkpoint, the Pch2-Hop1 module thus operates analogous to the Pch2/TRIP13-Mad2 module in the spindle assembly checkpoint that monitors chromosome segregation [12-16].

genetics↗

bioRxiv2020

A dCas9/CRISPR-based targeting system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination

In meiosis, crossover formation between homologous chromosomes is essential for faithful segregation. However, improperly controlled or placed meiotic recombination can have catastrophic consequences on genome stability. Specifically, within centromeres and surrounding regions (i.e. pericentromeres), crossovers are associated with chromosome missegregation and developmental aneuploidy. In organisms ranging from yeast to humans, crossovers are repressed within (peri)centromeric regions. We previously identified a key role for the multi-subunit, kinetochore-associated Ctf19 complex (Ctf19c; the budding yeast equivalent of the human CCAN) in regulating pericentromeric crossover formation. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits to a non-centromeric locus during meiosis. Using this approach, we query sufficiency in meiotic crossover suppression, and identify Ctf19 (the budding yeast homologue of vertebrate CENP-P) as a central mediator of kinetochore-associated crossover control. We show that the effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues known to be important for the recruitment of the Scc2-Scc4 cohesin regulator to kinetochores. We thus reveal a crucial determinant that links kinetochores to meiotic recombinational control. This work provides insight into localized control of meiotic recombination. Furthermore, our approach establishes a dCas9/CRISPR-based experimental platform that can be utilized to investigate and locally manipulate meiotic crossover control. This platform can easily be adapted in order to investigate other aspects of localized chromosome biology.

genetics↗

bioRxiv2019

Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase

Pch2 is an AAA+ protein that controls DNA break formation, recombination and checkpoint signaling during meiotic G2/prophase. Chromosomal association of Pch2 is linked to these processes, and several factors influence the association of Pch2 to euchromatin and the specialized chromatin of the ribosomal (r)DNA array of budding yeast. Here, we describe a comprehensive mapping of Pch2 localization across the budding yeast genome during meiotic G2/prophase. Within non-rDNA chromatin, Pch2 associates with a subset of actively RNA Polymerase II (RNAPII)-dependent transcribed genes. Chromatin immunoprecipitation (ChIP)- and microscopy-based analysis reveals that active transcription is required for chromosomal recruitment of Pch2. Similar to what was previously established for association of Pch2 with rDNA chromatin, we find that Orc1, a component of the Origin Recognition Complex (ORC), is required for the association of Pch2 to these euchromatic, transcribed regions, revealing a broad connection between chromosomal association of Pch2 and Orc1/ORC function. Ectopic mitotic expression is insufficient to drive recruitment of Pch2, despite the presence of active transcription and Orc1/ORC in mitotic cells. This suggests meiosis-specific licensing of Pch2 recruitment to sites of transcription, and accordingly, we find that the synaptonemal complex (SC) component Zip1 is required for the recruitment of Pch2 to transcription-associated binding regions. Interestingly, Pch2 binding patterns are distinct from meiotic axis enrichment sites (as defined by Red1, Hop1 and Rec8). This suggests that although Pch2 is linked to axis/SC-directed recruitment and function, the chromosomal population of Pch2 described here is not directly associated with chromosomal axis sites. In line with this observation, interfering with the pool of Pch2 that associates with active RNAPII transcription does not lead to effects on the chromosomal abundance of Hop1, a known axial client of Pch2. We thus report characteristics and dependencies for Pch2 recruitment to meiotic chromosomes, and reveal an unexpected link between Pch2, SC formation, chromatin and active transcription.

genetics↗

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↗