Biology subjects

Terakawa, T.

Publications and source records attributed to Terakawa, T..

11 recordsLinked to original sources

bioRxiv2026

YY1-concentration-dependent formation of mechanically distinct DNA condensates through different interaction mechanisms

The transcription factor Yin Yang 1 (YY1) plays roles in chromatin organization, combining sequence-specific DNA recognition via zinc finger domains with multivalent interactions mediated by intrinsically disordered regions. While YY1 has been implicated in phase separation and enhancer-promoter communication, how its structured and intrinsically disordered regions cooperate to shape DNA-protein assemblies remains unclear. Here, we used single-molecule DNA curtain fluorescence imaging to dissect the molecular basis of YY1-DNA assembly. We found that YY1 induces higher-order assembly in a concentration-dependent manner. At moderately high concentrations, YY1 formed weakly-linked DNA condensates in which dynamic, liquid-like YY1 molecules are scaffolded by relatively immobile, solid-like DNA. At high concentrations, zinc finger-mediated bridging dominates, producing strongly-linked condensates. Distinct domain-deletion mutants selectively impaired either weakly-(soft) or strongly-linked (hard) DNA condensate formation, indicating that these architectures are generated by separate domain-dependent mechanisms. Our findings establish a domain-level framework for DNA condensate formation and highlight how transcription factors can integrate specific and non-specific DNA interactions to control the material state of chromatin and would influence genome regulation.

biophysics↗

bioRxiv2026

A DNA deliverer-receiver mechanism for DNA recruitment in phase-separated transcriptional condensates

DNA transcription is a complex process involving numerous components that can assemble into phase-separated transcriptional condensates. However, whether condensates formed by multiple transcription factors behave through simple additive effects or instead exhibit non-additive, emergent properties remains unclear. Here, we use large-scale molecular dynamics simulations to investigate how three core transcription factors regulating pluripotency and early embryonic development--Nanog, Oct4, and Sox2--organize biomolecular condensates in the absence and presence of DNA. We find that condensate formation is primarily driven by intrinsically-disordered-region-mediated interactions of Nanog and Sox2, each of which individually promotes Oct4 phase separation; by contrast, when Nanog and Sox2 coexist in the absence of DNA, Oct4 is less efficiently incorporated into condensates. In the presence of DNA, condensates display a distinct spatial organization: Nanog and Sox2 form dense, well-mixed clusters, whereas Oct4 remains more dispersed in interstitial regions where DNA preferentially localizes, resulting in [~]20% higher DNA content in Oct4-containing condensates. Notably, phase separation reshapes DNA-protein interaction landscapes, altering the intramolecular regions that engage DNA. Together, these results support a synergistic DNA deliverer-receiver mechanism and suggest that non-additive, multi-component condensate organization constitutes an additional layer of gene expression regulation beyond canonical transcription factor-DNA binding.

biophysics↗

bioRxiv2025

Mechanistic models of asymmetric hand-over-hand translocation and nucleosome navigation by CMG helicase

Faithful replication of eukaryotic chromatin requires the CMG helicase to translocate directionally along single-stranded DNA (ssDNA) while unwinding double-stranded DNA (dsDNA) and navigating nucleosomes. However, the mechanism by which CMG achieves processive translocation and deals with nucleosomal barriers remains incompletely understood. Here, using coarse-grained molecular dynamics simulations with ATP-driven conformational switching, we showed that asymmetric rotational transitions among four distinct ssDNA-binding states enable CMG to achieve directional translocation and DNA unwinding. We further demonstrated that the fork protection complex (Csm3/Tof1) and RPA enhance processivity through distinct mechanisms: Csm3/Tof1 grips the parental duplex to suppress backtracking, while RPA alleviates lagging-strand clogging. Upon nucleosome encounter, Csm3/Tof1 promoted partial unwrapping of the entry DNA, but further progression was energetically restricted near the nucleosomal dyad. The histone chaperone FACT lowered this barrier and simultaneously prevented inappropriate histone transfer to the lagging strand. Our results provide mechanistic insights into how the eukaryotic replisome coordinates helicase activity, nucleosome navigation, histone chaperon function, and histone recycling during eukaryotic DNA replication.

biophysics↗

bioRxiv2025

Solution AFM imaging and coarse-grained molecular modeling of yeast condensin structural variation coupled to the ATP hydrolysis cycle

Condensin is a protein complex that regulates chromatin structural changes during mitosis. It varies the molecular conformation through the ATP hydrolysis cycle and extrudes DNA loops into its ring-like structure as a molecular motor. Condensin contains Smc2 and Smc4, in which a coiled-coil arm tethers the hinge and head domains and dimerizes at the hinge. ATPs bind between the heads, induce their engagement, and are hydrolyzed to promote their disengagement. In the previous study, we performed solution atomic force microscopy (AFM) imaging of yeast condensin holo-complex in the presence of ATP and conducted flexible molecular fitting to the AFM image, obtaining the hinge structure with open conformation. However, it has yet to be clarified how the opening/closing of the hinge is coupled to the ATP hydrolysis cycle. In this study, we performed solution AFM imaging in the presence and absence of varying nucleotides, including AMP-PNP, ATP{gamma}S, and ADP. Furthermore, we conducted coarse-grained molecular dynamics simulations of a Smc2/4 heterodimer and selected the molecular structure that best represented each AFM image. Our results suggested that head engagement upon ATP binding is coupled to hinge opening. Also, the results indicated that the N-terminal region of Brn1, one of the accessory subunits, re-associates to the Smc2 head after ADP release. This study deepens our understanding of the conformational variation of yeast condensin driven by the ATP hydrolysis cycle.

biophysics↗

bioRxiv2024

Minimal protein list for chromatin replication-coupled symmetric histone recycling revealed through in vitro reconstitution

Upon eukaryotic DNA replication, symmetric histone recycling from parental to daughter strands is vital for transmitting epigenetic information to the next generations. Recent genome-wide sequencing studies have identified several protein regions contributing to symmetric histone recycling in a cell. However, a minimal list of proteins for symmetric histone recycling remains unknown. Here, we successfully reconstituted histone recycling with [~]30 purified proteins and analyzed the products digested by Micrococcal nuclease with the newly developed pipeline called a Repli-pore-seq in which nanopore sequencing and deep-learning-based classification were combined. As a result, we confirmed that the histones were recycled symmetrically to the lagging and leading strands. The recycled histones form tetrasomes or hexasomes and are deposited on the DNA sequences energetically favorable for forming nucleosomes. We also observed the discordance of the recycled position between lagging and leading strands on the GC-rich DNA sequences. Among proteins dispensable for chromatin replication, the lagging-strand maturation factors, Fen1/Cdc9, were also dispensable for symmetric recycling. The removal of Pol {delta} disrupted the recycling symmetry, and that of Ctf4 or Csm3/Tof1 altered the recycling position. These findings provide critical insights into the molecular players and mechanisms underlying symmetric histone recycling.

biophysics↗

bioRxiv2024

Molecular dynamics simulations of human cohesin subunits identify DNA binding sites and their potential roles in DNA loop extrusion

The SMC complex cohesin mediates interphase chromatin structural formation in eukaryotic cells through DNA loop extrusion. Here, we sought to investigate its mechanism using molecular dynamics simulations. To achieve this, we first constructed the amino-acid-residue-resolution structural models of the cohesin subunits, SMC1, SMC3, STAG1, and NIPBL. By simulating these subunits with double-stranded DNA molecules, we predicted DNA binding patches on each subunit and quantified the affinities of these patches to DNA using their dissociation rate constants as a proxy. Then, we constructed the structural model of the whole cohesin complex and mapped the predicted high-affinity DNA binding patches on the structure. From the spatial relations of the predicted patches, we identified that multiple patches on the SMC1, SMC3, STAG1, and NIPBL subunits form a DNA clamping patch group. The simulations of the whole complex with double-stranded DNA molecules suggest that this patch group facilitates DNA bending and helps capture a DNA segment in the cohesin ring formed by the SMC1 and SMC3 subunits. In previous studies, these have been identified as critical steps in DNA loop extrusion. Therefore, this study provides experimentally testable predictions of DNA binding sites implicated in previously proposed DNA loop extrusion mechanisms and highlights the essential roles of the accessory subunits STAG1 and NIPBL in the mechanism.

molecular biology↗

bioRxiv2024

SMC complex unidirectionally translocates DNA by coupling segment capture with an asymmetric kleisin path

SMC (structural maintenance of chromosomes) protein complexes are ring-shaped molecular motors essential for genome folding. Despite recent progress, the detailed molecular mechanism of DNA translocation in concert with the ATP-driven conformational changes of the complex remains to be clarified. In this study, we elucidated the mechanisms of SMC action on DNA using all-atom and coarse-grained molecular dynamics simulations. We first created a near-atomic full-length model of a prokaryotic SMC-kleisin complex based on experimental structures and implemented ATP-dependent conformational changes using a structure-based coarse-grained model. We further incorporated key protein-DNA hydrogen bond interactions derived from fully atomistic simulations. Extensive simulations of the SMC complex with 800 base pairs of duplex DNA over the ATP cycle observed unidirectional DNA translocation by the SMC complex. The process exhibited a step size of [~]200 base pairs, wherein the SMC complex captured a DNA segment of about the same size within the SMC ring in the engaged state, followed by its pumping into the kleisin ring as ATP was hydrolyzed. Analysis of trajectories identified the asymmetric path of the kleisin as a critical factor for the observed unidirectionality. SIGNIFICANCE STATEMENTRing-shaped SMC (structural maintenance of chromosomes) protein complexes, which are highly conserved across all three domains of life, play an essential role in chromosome organization through a process called DNA loop extrusion. However, the molecular mechanism underlying the ATP-dependent motor activity of SMC complexes remains unclear. Using all-atom and residue-resolution coarse-grained molecular dynamics simulations, we revealed that prokaryotic SMC complexes translocate unidirectionally along DNA via a segment capture mechanism. We found that the unidirectionality arises from the kleisin subunit breaking the symmetry of the ring-shaped SMC complex structure. Our findings provide insights into the molecular motor mechanisms shared by SMC complexes.

biophysics↗

bioRxiv2024

Molecular mechanism of parental H3/H4 recycling at a replication fork

In eukaryotic chromatin replication, faithful recycling of histones from parental DNA to replicated leading and lagging strands is essential for maintaining epigenetic information across generations. A recent experimental study has revealed that disrupting interactions between the N-terminal disordered tail of Mcm2, a subunit in DNA replication machinery, and a histone H3/H4 tetramer, a carrier of epigenetic modifications, perturbs their faithful recycling. However, the molecular pathways via which the H3/H4 tetramer attached to Mcm2 is recycled to the replicated strands have yet to be deciphered. Furthermore, the factors that regulate the ratio recycled to each strand and the destination location still need to be discovered. The elucidation requires visualization of a structural trajectory from H3/H4 bound to Mcm2 until recycled to the replicated strands. In this study, we performed molecular dynamics simulations of yeast DNA replication machinery (Mcm2-7, Cdc45, GINS, Pol {varepsilon}, and RPA), an H3/H4 tetramer, and replicated DNA strands. The simulations demonstrated that histones are recycled via Cdc45-mediated and unmediated pathways even without histone chaperones, as our in vitro biochemical assays supported. Also, RPA binding to the ssDNA portion of the lagging strand regulated the ratio recycled to each strand, whereas DNA bending by Pol {varepsilon} modulated the destination location. Together, the simulations in this study provided testable hypotheses, which are vital for clarifying intracellular histone recycling controlled by the cooperation of many histone chaperones.

biophysics↗

bioRxiv2021

Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the so-called hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown, potentially due to its conformational plasticity. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. The conformational change of the hinge domain might be essential for the dsDNA binding regulation and play important roles in condensin-mediated DNA-loop extrusion.

biophysics↗

bioRxiv2021

DNA translocase repositions a nucleosome by the lane-switch mechanism

Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone core complexes and forms nucleosomes, translocases inevitably encounter nucleosomes. Previous studies have shown that a histone core complex repositions upstream (downstream) when SP6RNA or T7 RNA polymerase (bacterial exonuclease, RecBCD) partially unwraps nucleosomal DNA. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identify the lane-shift mechanism for downstream nucleosome repositioning via coarse-grained molecular dynamics simulations, which we validated by restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding region (lane) to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have crucial implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling. SIGNIFICANCEEukaryotic chromosomes are composed of repeating subunits termed nucleosomes. Thus, proteins that translocate along the chromosome, DNA translocases, inevitably collide with nucleosomes. Previous studies revealed that a translocase repositions a nucleosome upstream or downstream upon their collision. Though the molecular mechanisms of the upstream repositioning have been extensively studied, that of downstream repositioning remains elusive. In this study, we performed coarse-grained molecular dynamics simulations, proposed the lane-shift mechanism for downstream repositioning, and validated this mechanism by restriction enzyme digestion assays and deep sequencing assays. This mechanism has broad implications for how translocases deal with nucleosomes for their functions.

biophysics↗

bioRxiv2017

Sequential Eviction Of Crowded Nucleoprotein Complexes By The RecBCD Molecular Motor

In physiological settings, all nucleic acids motor proteins must travel along substrates that are crowded with other proteins. However, the physical basis for how motor proteins behave in these highly crowded environments remains unknown. Here we use real-time single molecule imaging, kinetic Monte Carlo simulations, and Molecular dynamics simulations to determine how the ATP-dependent translocase RecBCD travels along DNA occupied by tandem arrays of high affinity DNA-binding proteins. We demonstrate that RecBCD forces each protein into its nearest adjacent neighbor, causing rapid disruption of the underlying protein-nucleic acid interface. This mechanism is not simply the same way that RecBCD disrupts isolated nucleoprotein complexes on otherwise naked DNA. Instead, molecular crowding itself completely alters the mechanism by which RecBCD removes tightly bound protein obstacles from DNA.\n\nSignificance statementChromosomes are crowded places, and any nucleic acid motor proteins that act upon DNA must function within these crowded environments. How crowded environments affect motor protein behaviors remains largely unexplored. Here, we use single molecule fluorescence microscopy visualize the ATP-dependent motor protein RecBCD as it travels along crowded DNA molecules bearing long tandem arrays of DNA-binding proteins. Our findings show that RecBCD can push through highly crowded protein arrays while evicting the proteins from DNA. Molecular dynamics simulations suggest that RecBCD forces the proteins into once another, causing rapid disruption of the protein-DNA interface. These findings may provide insights into how other types of motor proteins travel along crowded nucleic acids.

biophysics↗