Biology subjects

Nagae, F.

Publications and source records attributed to Nagae, F..

5 recordsLinked to original sources

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↗

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

Submolecular-scale Hairpin DNA Folding Dynamics Studied by High-Speed AFM with Optical Tweezers

Optical tweezers have contributed to elucidate the folding mechanisms associated with biomolecules. By combining single-molecule or super-resolution techniques, imaging can also be performed while measuring or inducing force coupling with biochemical reactions; however, they cannot capture structural information beyond the fluorophore spatial resolution. To overcome this problem, here, we developed a technique that combines optical tweezers with high-speed atomic force microscopy (AFM). To solve the problem of incompatible instrumental configurations, we developed a unique optical tweezers measurement system that is specialized for high-speed AFM. Upon applying an external force to a synthesized DNA secondary structure, we successfully visualized the dissociation of the duplex structure. Furthermore, we succeeded in observing spontaneous reannealing of the duplex structure upon releasing the force, which demonstrates that the folding reaction can be reversibly controlled. We also reveal that along with duplex unfolding, a metastable secondary structure is generated and its topology changes transiently over time. The results indicate that this technique provides structural insights that cannot be obtained by conventional fluorescence techniques.

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

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↗