Biology subjects

Muir, K. W.

Publications and source records attributed to Muir, K. W..

5 recordsLinked to original sources

bioRxiv2025

Models for the architecture of the human inner kinetochore CCAN complex on centromeric α-satellite CENP-A nucleosome arrays

Human kinetochores assemble onto centromeric DNA defined by tandem copies of thousands of the 171 bp -satellite repeat sequence. The centromere-specific CENP-A nucleosome (CENP-ANuc) recruits the inner kinetochore constitutive centromere associated network (CCAN) complex. A previous cryo-EM structure of CCAN bound to a CENP-ANuc reconstituted with a 171 bp -satellite repeat sequence showed how extranucleosomal linker DNA threads through an internal tunnel in the CCAN complex. To understand the higher-order architecture of the inner kinetochore assembled onto -satellite repeat arrays, we have determined cryo-EM structures of CCAN with longer DNA sequences. These include free DNA and single and dimeric -satellite repeats with CENP-A nucleosomes. We show from the structures of CCAN bound to both free DNA and monomeric CENP-ANuc that CCAN engages 65-70 bp of DNA comprising 30-35 bp of an upstream -satellite repeat. This upstream DNA interacts with the histone-fold domain subunits of the CENP-TWSX module in a manner resembling how DNA is wrapped in nucleosomes. A complex of CCAN assembled onto a dimeric -satellite repeat with two CENP-A nucleosomes showed that CCAN can only be accommodated on the linker DNA by unwrapping DNA from the CENP-TWSX module together with 25 bp of DNA from the upstream nucleosome. We discuss the implications of these results for models of CCAN assembly on arrays of -satellite chromatin containing CENP-ANuc.

molecular biology↗

bioRxiv2025

Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex

During mitosis and meiosis kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation in all eukaryotes, however recent structural, functional, and evolutionary studies have revealed divergent mechanisms of kinetochore assembly. In this study, we use cryo-EM to understand the structural mechanisms by which the budding yeast microtubule-binding outer kinetochore KMN complex assembles, and how its interactions with the centromere-binding inner kinetochore are regulated. The ten-subunit KMN complex comprises three subcomplexes: Knl1c, Mis12cMtw1c and Ndc80c. We show that -helical motifs in the C-termini of the Mis12cMtw1c subunits Dsn1, Mis12Mtw1 and Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12cMtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1AI) folds into two -helices that engage the Mis12cMtw1c head 1 domain, thereby occluding binding sites for the inner kinetochore subunits CENP-CMif2 and CENP-UAme1, reducing their affinity for Mis12cMtw1. Our structure reveals how Aurora BIpl1 phosphorylation of Dsn1AI would release this auto-inhibition to substantially strengthen pre-existing connections between the inner and outer kinetochore. SummaryKinetochore-localised KMN complexes mediate chromosome segregation by attaching chromosomes to spindle microtubules. Cryo-EM, biochemical and genetic experiments reveal conserved underlying mechanisms of complex assembly, auto-inhibition and phospho-regulation that ensure precise control of KMN assembly onto centromeres to enable genome division.

biochemistry↗

bioRxiv2023

Mechanism of outer kinetochore assembly on microtubules and its regulation by mitotic error correction

Kinetochores couple chromosomes to the mitotic spindle and transduce the energy of microtubule depolymerization to segregate the genome during cell division. Kinetochore - microtubule attachments are often initially erroneous and subject to a mitotic error correction (EC) mechanism that drives their turnover until biorientation is achieved. How this is accomplished and regulated, and how kinetochore-mediated chromosome segregation occurs at a molecular level remain major outstanding questions. Here we describe the cryo-electron microscopy (cryo-EM) structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. We observe coordinated interactions of the outer kinetochore complexes through multiple interfaces, in addition to a short staple within the Dam1 subunit that facilitates Dam1c ring assembly. Perturbation of these interfaces results in loss of yeast viability. Force-rupture assays indicated this is a consequence of substantial reductions in kinetochore - microtubule binding strength. EC-mediated phosphorylation of Ndc80c-Dam1c interfaces would drive complex disassembly, whereas Dam1 staple phosphorylation would promote Dam1c ring disassembly, explaining how kinetochore - microtubule attachments are destabilized and reset by the EC mechanism. One-Sentence SummaryPhosphorylation disrupts the outer kinetochore to regulate kinetochore-microtubule attachments in mitotic error correction.

molecular biology↗

bioRxiv2022

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome

Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an [~]171 bp -satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto -satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the -satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the -satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces. One-Sentence SummaryThe human inner kinetochore CCAN complex tightly grips the linker DNA of the -satellite CENP-A nucleosome.

biochemistry↗

bioRxiv2020

Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP

The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 [A]. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

molecular biology↗