Biology subjects

Masahara-Negishi, Y.

Publications and source records attributed to Masahara-Negishi, Y..

3 recordsLinked to original sources

bioRxiv2025

Reconstitution of phospho-regulated mitotic chromatid assembly and disassembly

Exactly how cell cycle regulators control a sequential series of mitotic events is not fully understood. Here we report reconstitution assays that recapitulate the assembly and disassembly of mitotic chromatids in vitro using a minimal set of recombinant proteins, including condensin I. By incorporating cyclin B-Cdk1 and PP2A-B55, this system enables us to dissect the phospho-regulation of condensin I in these processes. We provide evidence that the terminal intrinsically disordered regions (tIDRs) of the non-SMC subunits suppress condensin I activity, and that this self-suppression is relieved by Cdk1 phosphorylation. Importantly, full activation of condensin I requires the phosphorylation of a conserved residue located in the central region of the kleisin subunit CAP-H. Conversely, PP2A-B55 induces dissociation of condensin I from reconstituted chromatids, leading to their disassembly. Complementary analyses using Xenopus egg extracts reveal that the tIDRs and the kleisin central region are phosphorylated and dephosphorylated with distinct kinetics during mitotic entry and exit. Together, these findings define an intricate regulatory network that coordinates chromatid assembly and disassembly with mitotic progression.

molecular biology↗

bioRxiv2025

Functional interplay between condensin I and topoisomerase IIα in single-molecule DNA compaction

Condensin I and topoisomerase II (topo II) are chromosomal ATPases essential for mitotic chromosome assembly. Mechanistically how the two ATPases cooperate to assemble mitotic chromosomes remains unknown. Here we use total internal reflection fluorescence microscopy to analyze the interplay between condensin I and topo II at single-molecule resolution. As observed in previous studies, condensin I alone predominantly forms DNA loops in an ATP-dependent manner. However, when topo II is included in the reaction, condensin I forms stable compact structures (termed "lumps") in a manner dependent on the C-terminal domain of topo II. Each of the stable lumps contains a single condensin I complex and a single topo II dimer. Remarkably, we find that topo II, when catalytically active, renders the lumps resistant to protease treatment. Several lines of evidence show that the protease-resistant lumps contain knotted DNA. A mutant condensin I complex defective in ATP hydrolysis, together with topo II, forms smaller lumps in which the probability of DNA knotting is greatly reduced. Our results demonstrate how topo II-mediated strand passage is coupled with condensin I-mediated loop extrusion to generate a compact DNA structure. Together with recent studies, we discuss the functional implications of these observations in mitotic chromosome assembly and stabilization.

molecular biology↗

bioRxiv2023

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

cell biology↗