Biology subjects

King, G. A.

Publications and source records attributed to King, G. A..

6 recordsLinked to original sources

bioRxiv2026

Negative supercoiling facilitates human TOP3α-mediated decatenation of DNA braids via catenation junction melting

DNA catenation between sister chromatids arises during replication and must be efficiently resolved to ensure faithful chromosome segregation. The removal of DNA catenation is generally attributed to the type II topoisomerase TOP2, which catalyses double-stranded DNA passage reactions and can therefore directly remove inter-DNA linkages. By contrast, the type IA topoisomerase TOP3 catalyses strand passage through transient breaks in single-stranded DNA. Here, using single-molecule optical tweezers and fluorescence microscopy, we show that negative supercoiling promotes local melting of duplex DNA at DNA-DNA crossings within braided molecules, generating single-stranded DNA that can be engaged by TOP3. These supercoiled substrates can be efficiently resolved by TOP3-RMI1-RMI2 (TRR), whereas relaxed DNA is resistant to TRR decatenation. Moreover, we find that RMI2 and the ssDNA binding Replication Protein A (RPA) stimulates TOP3 decatenation and demonstrate that decatenation remains viable up to 30pN of tension on the substrate. These findings reveal a mechanism by which torsional stress converts an otherwise inaccessible duplex DNA crossing into a substrate for a single-strand-specific topoisomerase. We propose that negative supercoiling can therefore direct type IA topoisomerases to topological DNA linkages by promoting local duplex melting, providing a physical mechanism that couples DNA supercoiling to the resolution of DNA entanglements.

molecular biology↗

bioRxiv2025

Tom70-mediated mitochondria-nuclear envelope contacts regulate nuclear pore complex inheritance during gametogenesis

Gametogenesis rejuvenates the cellular lineage and excludes senescence-associated factors from gametes. In Saccharomyces cerevisiae, this involves sequestration of nuclear constituents into the Gametogenesis-Uninherited Nuclear Compartment (GUNC), which is excluded from gametes. Here we identify the conserved mitochondrial import receptor Tom70 as a key regulator of GUNC-mediated exclusion. Loss of TOM70 disrupts the sequestration of nuclear pore complexes, but not senescence-associated aggregates and nucleolar components, into the GUNC. Tom70s role appears independent of its canonical function in mitochondrial import and instead reflects a meiosis-specific requirement for mitochondria-nuclear envelope tethering. During meiosis II, Tom70 concentrates around the GUNC, where it recruits the nuclear envelope tethering protein Cnm1. Loss of CNM1 partially phenocopies tom70{Delta}, consistent with parallel tethering interactions. These findings uncover a previously unrecognized organelle contact-dependent pathway that remodels the nuclear envelope to support selective nuclear inheritance. More broadly, they highlight how organelle contacts integrate with nuclear quality control to safeguard gamete integrity.

cell biology↗

bioRxiv2025

A Conserved Disruption of the Nuclear Permeability Barrier in Meiosis is Controlled by a Kinase-Phosphatase Pair in Saccharomyces cerevisiae

In eukaryotic organisms, the nucleus is remodeled to accommodate the space required for chromosome segregation. Remodeling strategies range from closed division, where the nuclear envelope remains intact, to open divisions, where the nuclear envelope is temporarily disassembled. While the budding yeast Saccharomyces cerevisiae undergoes closed mitosis, its meiotic nuclear division strategy is less understood. Here we investigate nuclear permeability during meiosis in budding yeast and discover that meiosis II represents a semi-closed division marked by bidirectional mixing between the nucleus and cytoplasm. This includes nuclear entry of the Ran GTPase activating protein (RanGAP), typically cytoplasmic, although RanGAP relocalization appears to be a consequence, rather than a cause of permeability changes. This intercompartmental mixing occurs without nuclear envelope breakdown or dispersal of nucleoporins and is independent of known nuclear pore complex remodeling events. This phenomenon, termed virtual nuclear envelope breakdown (vNEBD), represents a unique mechanism distinct from other semi-closed divisions. We demonstrate that vNEBD is integrated into the meiotic program and regulated by the conserved meiotic kinase Ime2 and the meiosis-specific protein phosphatase 1 regulatory subunit, Gip1. Remarkably, the vNEBD event is conserved between S. cerevisiae and the distantly related Schizosaccharomyces pombe, indicating a conserved, critical role in meiosis.

cell biology↗

bioRxiv2022

Meiotic Nuclear Pore Complex Remodeling Provides Key Insights into Nuclear Basket Organization

Nuclear pore complexes (NPCs) are large proteinaceous assemblies that mediate nuclear compartmentalization. NPCs undergo largescale structural rearrangements during mitosis in metazoans and some fungi. However, our understanding of NPC remodeling beyond mitosis remains limited. Using time-lapse fluorescence microscopy, we discovered that NPCs undergo two mechanistically-separable remodeling events during budding yeast meiosis whereby parts or all of the nuclear basket transiently dissociate from the NPC core during meiosis I and II, respectively. Meiosis I detachment, observed for Nup60 and Nup2, is driven by Polo kinase-mediated phosphorylation of Nup60 at its interface with the Y-complex. Subsequent reattachment of Nup60-Nup2 to the NPC core is mediated by a lipid-binding amphipathic helix in Nup60. Preventing Nup60-Nup2 reattachment causes misorganization of the entire nuclear basket in gametes. Strikingly, meiotic nuclear basket remodeling also occurs in the distantly related fission yeast, Schizosaccharomyces pombe. Our study reveals a conserved and developmentally programmed aspect of NPC plasticity, providing key mechanistic insights into nuclear basket organization. SUMMARYKing and Wettstein et al. reveal that nuclear pore complexes undergo two distinct remodeling events during budding yeast meiosis: partial and full nuclear basket detachment. By dissecting the regulation of these events, the study provides mechanistic insights into NPC organization.

cell biology↗

bioRxiv2019

Supercoiling DNA optically

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures.1,2,3 For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic,4,5,6 angular-optical,7,8,9 micro-pipette,10 and magneto-optical tweezers.11 However, in order to address many open questions, there is a growing need for new techniques that can combine rapid torque control with both spatial manipulation and fluorescence microscopy. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. Supercoiled DNA formed in this way is amenable to fast buffer exchange, and can be interrogated with both force spectroscopy and fluorescence imaging of the whole DNA. We establish this method as a powerful platform to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

biophysics↗

bioRxiv2019

Meiotic cellular rejuvenation is coupled to nuclear remodeling in budding yeast

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and old cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation.

cell biology↗