Biology subjects

Minamino, M.

Publications and source records attributed to Minamino, M..

4 recordsLinked to original sources

bioRxiv2025

Biochemical reconstitution of sister chromatid cohesion establishment during DNA replication

Concomitant with DNA replication, the ring-shaped cohesin complex encircles both newly synthesized sister chromatids, enabling their faithful segregation during cell divisions. Our molecular understanding of how cohesin co-entraps both replication products remains incomplete. Here, we reconstitute sister chromatid cohesion establishment using purified budding yeast proteins. Cohesin rings, initially loaded onto template DNA, remain DNA-bound during DNA synthesis and encircle both replication products. Additionally, DNA replication instigates new cohesin recruitment, as a second pathway that generates sister chromatid cohesion. In both scenarios, cohesin often embraces only one of the two replication products, suggestive of a two-step sister chromatid capture mechanism. Sister chromatid co-entrapment occurs independently of replication fork-associated cohesion establishment factors, suggesting a role for the latter during chromatin replication or in facilitating the subsequent cohesin acetylation reaction. Our results make sister chromatid cohesion establishment amenable to direct experimental exploration.

molecular biology↗

bioRxiv2024

DNA replication fork stabilization and restart mechanisms revealed by biochemical reconstitution

Understanding how DNA replication forks stall and restart and how the DNA damage checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading strand DNA synthesis quickly stops but CMG helicase continues to unwind and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC and DNA polymerases {delta} and {varepsilon} which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA damage checkpoint limits this sequestration by restraining fork progression, which protects stalled forks from collapse and ensures restart.

biochemistry↗

bioRxiv2022

The Response of the Eukaryotic Replisome to G-quadruplex- and i-motif-forming Sequences

Sequences that can form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play a range of physiological roles. However, they can also pose a challenge to the replication machinery and in turn threaten genome stability. Multiple lines of evidence suggest G4s interfere with replication, but the underlying mechanism remains unclear. Moreover, there is a lack of evidence of how iMs affect the replisome. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM is sufficient to arrest DNA replication. Direct single molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. A combination of genetic and biophysical characterisation establishes that structure forming capacity is a key determinant of replisome arrest. Mechanistically, replication fork arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Sgs1 or Chl1. Altogether, this study provides a potential mechanism for quadruplex structure formation and resolution during replication and highlights G4s and iMs as endogenous sources of replication stress, which may explain their genomic instability and mutation frequencies in cancer.

molecular biology↗

bioRxiv2020

Phase separation induced by cohesin SMC protein complexes

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization. One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

biophysics↗