Biology subjects

Chistol, G.

Publications and source records attributed to Chistol, G..

6 recordsLinked to original sources

bioRxiv2026

TOPBP1-Dependent DNA Damage Processing Promotes Centromere Loss in an Inviable Xenopus Hybrid

Hybrid incompatibility is associated with chromosome instability, DNA damage, and embryonic lethality, yet how cells respond to genome instability in hybrids is unclear. In inviable hybrids generated by fertilizing Xenopus tropicalis eggs with Xenopus laevis sperm, CENP-A is lost from paternal chromosomes, leading to elimination of two chromosome arms. Using hybrid Xenopus egg extract reactions, we show that CENP-A is removed from X. laevis chromosomes exposed to X. tropicalis cytoplasm through an RNA polymerase II-dependent process associated with DNA damage. Topoisomerase II Binding Protein 1 (TOPBP1) localized to damaged acentric chromosomes during metaphase, and its depletion impaired chromosome alignment, increased DNA damage, and blocked CENP-A removal. Inhibition of DNA polymerase theta increased DNA damage while promoting CENP-A retention, indicating that DNA damage processing rather than damage itself promotes CENP-A loss. Together, our findings reveal separable roles for TOPBP1 in chromosome organization and centromere destabilization, showing how a protective genome surveillance pathway can instead drive chromosome instability in an inviable hybrid.

cell biology↗

bioRxiv2025

Mcm10 and RecQL4 Synergize to Activate the Eukaryotic Replicative DNA Helicase

Chromosomes are copied from thousands of origins. At each origin, two replicative DNA helicases are first assembled, then activated to begin unwinding DNA. Several replication proteins are subsequently recruited to the active helicase, forming a replisome. The helicase must undergo dramatic conformational changes during its activation and this process is poorly understood, especially in metazoa. How the metazoan replicative helicase is activated, and which proteins promote this essential process are long-standing questions. Using a combination of single-molecule imaging and ensemble biochemistry, we show that Mcm10 and RecQL4 act in a concerted manner to activate replicative helicases. Mcm10 first binds to inactive helicases, then recruits RecQL4, which synergizes with Mcm10 to promote helicase activation. Mcm10 is not incorporated into replisomes and dissociates from origins during replication initiation. In the absence of Mcm10, RecQL4 is recruited to origins via an interaction with the Mcm7 subunit of the helicase. Our data reveal that Mcm10 and RecQL4 play partially redundant roles during helicase activation, help resolve long-standing controversies about the roles of Mcm10 and RecQL4 in DNA replication, and reveal replication initiation defects caused by pathologic RecQL4 mutations.

molecular biology↗

bioRxiv2024

Single-Molecule Imaging Reveals the Mechanism of Bidirectional Replication Initiation in Metazoa

Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG (Cdc45*Mcm2-7*GINS) helicases at each origin. This requires several firing factors (including TopBP1, RecQL4, DONSON) whose exact roles remain unclear. How two helicases are correctly assembled and activated at every single origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered a surprisingly dynamic picture of initiation. Firing factors transiently bind origins but do not travel with replisomes. Two Cdc45 simultaneously arrive at each origin and two GINS are recruited together, even though neither protein can dimerize. The synchronized delivery of two GINS is mediated by DONSON, which acts as a dimerization scaffold. We show that RecQL4 promotes DONSON dissociation and facilitates helicase activation. The high fidelity of bidirectional origin firing can be explained by a Hopfield-style kinetic proofreading mechanism.

molecular biology↗

bioRxiv2022

Visualizing the Dynamics of DNA Replication and Repair at the Single-Molecule Molecule Level

During cell division, the genome of each eukaryotic cell is copied by thousands of replisomes - large protein complexes consisting of several dozen proteins. Recent studies suggest that the eukaryotic replisome is much more dynamic than previously thought. To directly visualize replisome dynamics in a physiological context, we recently developed a single-molecule approach for imaging replication proteins in Xenopus egg extracts. These extracts contain all the soluble nuclear proteins and faithfully recapitulate DNA replication and repair in vitro, serving as a powerful platform for studying the mechanisms of genome maintenance. Here we present detailed protocols for conducting single-molecule experiments in nuclear egg extracts and preparing key reagents. This workflow can be easily adapted to visualize the dynamics and function of other proteins implicated in DNA replication and repair.

biophysics↗

bioRxiv2020

Single-Strand DNA Breaks Cause Replisome Disassembly

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet CMG eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks", which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates beyond the nick on double-stranded DNA and is then actively removed from chromatin by the p97 ATPase. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they argue that CMG cannot be deposited on dsDNA while cells resolve replication stress. HighlightsO_LIThe structures of leading and lagging strand collapsed forks are different C_LIO_LICMG passively "runs off" the broken DNA end during leading strand fork collapse C_LIO_LICMG is unloaded from duplex DNA after lag collapse in a p97-dependent manner C_LIO_LINicks are uniquely toxic lesions that cause fork collapse and replisome disassembly C_LI

molecular biology↗

bioRxiv2017

Mechanochemical Coupling and Bi-Phasic Force-Velocity Dependence in the Ultra-Fast Ring ATPase SpoIIIE

Multi-subunit ring-shaped ATPases are molecular motors that harness chemical free energy to perform vital mechanical tasks such as polypeptide translocation, DNA unwinding, and chromosome segregation. Previously we reported the intersubunit coordination and stepping behavior of the hexameric ring-shaped ATPase SpoIIIE (Liu et al., 2015). Here we use optical tweezers to characterize the motors mechanochemistry. Analysis of the motor response to external force at various nucleotide concentrations identifies phosphate release as the likely force-generating step. Analysis of SpoIIIE pausing indicates that pauses are off-pathway events. Characterization of SpoIIIE slipping behavior reveals that individual motor subunits engage DNA upon ATP binding. Furthermore, we find that SpolIIEs velocity exhibits an intriguing bi-phasic dependence on force. We hypothesize that this behavior is an adaptation of ultra-fast motors tasked with translocating DNA from which they must also remove DNA-bound protein roadblocks. Based on these results, we formulate a comprehensive mechanochemical model for SpoIIIE.

biochemistry↗