Functional analysis of pathological mutations in DNA topoisomerase 3A.

DNA topoisomerase IIIα (TOP3A) is a highly conserved type IA topoisomerase critical for genome maintenance. Its deletion causes embryonic lethality in many organisms, which has hampered attempts to understand its physiological role. Recently, human subjects with TOP3A mutations were identified who display a Bloom's syndrome (BS)-like phenotype and mitochondrial dysfunction, consistent with TOP3A's roles in the nucleus, alongside the Bloom's helicase, and in mitochondria.

Mechanistic insight into anaphase bridge signaling to the abscission checkpoint.

During cytokinesis in human cells, a failure to resolve persistent DNA bridges that span the cell-division plane maintains the Aurora B-dependent abscission checkpoint in an active state. However, the molecular mechanism by which unresolved sister-chromatid bridging signals to this checkpoint is poorly defined. Here, we define an essential role for the Bloom's syndrome helicase, BLM, in signaling to the abscission-checkpoint machinery in response to replication stress through the conversion of dsDNA bridges into RPA-coated ssDNA.

Replication stress promotes cellular transformation in Drosophila epithelium.

The accurate control of DNA replication is crucial for the maintenance of genomic stability and cell viability. In this study, we explore the consequences of depleting the replicative DNA Polymerase α (POLA) in the wing disc of Drosophila melanogaster. Our findings reveal that reduced POLA activity induces DNA replication stress and activates the replication checkpoint in vivo.

Mouse ZGRF1 helicase facilitates DNA repair and maintains efficient fertility.

The recently characterised human ZGRF1 helicase promotes genomic stability by facilitating DNA interstrand crosslink repair. In its absence, human cells exhibit greater sensitivity towards anti-cancer drugs such as mitomycin C and camptothecin. Moreover, the downregulation of expression is associated with increased survival in cancer patients.

Quantitative Chromatin Protein Dynamics During Replication Origin Firing in Human Cells.

Accurate genome duplication requires a tightly regulated DNA replication program that relies on the fine regulation of origin firing. While the molecular steps involved in origin firing have been determined predominantly in budding yeast, the complexity of this process in human cells has yet to be fully elucidated. Here, we describe a straightforward proteomics approach to systematically analyze protein recruitment to the chromatin during induced origin firing in human cells.

RECQL4 affects MHC class II-mediated signalling and favours an immune-evasive signature that limits response to immune checkpoint inhibitor therapy in patients with malignant melanoma.

Background: Cancer immunotherapy has transformed metastatic cancer treatment, yet challenges persist regarding therapeutic efficacy. RECQL4, a RecQ-like helicase, plays a central role in DNA replication and repair as part of the DNA damage response, a pathway implicated in enhancing efficacy of immune checkpoint inhibitor (ICI) therapies. However, its role in patient response to ICI remains unclear.

Ion-mediated condensation controls the mechanics of mitotic chromosomes.

During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. How do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? Here we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone depletion and theory to show that chromosomal elasticity originates from the chromatin fibre behaving as a flexible polymer, whereas energy dissipation can be explained by modelling chromatin loops as an entangled polymer solution.

Replication fork barriers to study site-specific DNA replication perturbation.

DNA replication ensures the complete and accurate duplication of the genome. The traditional approach to analysing perturbation of DNA replication is to use chemical inhibitors, such as hydroxyurea or aphidicolin, that slow or stall replication fork progression throughout the genome. An alternative approach is to perturb replication at a single site in the genome that permits a more forensic investigation of the cellular response to the stalling or disruption of a replication fork.

PICH deficiency limits the progression of MYC-induced B-cell lymphoma.

Plk1-interacting checkpoint helicase (PICH) is a DNA translocase involved in resolving ultrafine anaphase DNA bridges and, therefore, is important to safeguard chromosome segregation and stability. PICH is overexpressed in various human cancers, particularly in lymphomas such as Burkitt lymphoma, which is caused by MYC translocations. To investigate the relevance of PICH in cancer development and progression, we have combined novel PICH-deficient mouse models with the Eμ-Myc transgenic mouse model, which recapitulates B-cell lymphoma development.

Completing genome replication outside of S phase.

Mitotic DNA synthesis (MiDAS) is an unusual form of DNA replication that occurs during mitosis. Initially, MiDAS was characterized as a process associated with intrinsically unstable loci known as common fragile sites that occurs after cells experience DNA replication stress (RS). However, it is now believed to be a more widespread "salvage" mechanism that is called upon to complete the duplication of any under-replicated genomic region.

BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures.

Bloom's syndrome (BLM) protein is a known nuclear helicase that is able to unwind DNA secondary structures such as G-quadruplexes (G4s). However, its role in the regulation of cytoplasmic processes that involve RNA G-quadruplexes (rG4s) has not been previously studied. Here, we demonstrate that BLM is recruited to stress granules (SGs), which are cytoplasmic biomolecular condensates composed of RNAs and RNA-binding proteins.

The SUMO-NIP45 pathway processes toxic DNA catenanes to prevent mitotic failure.

SUMOylation regulates numerous cellular processes, but what represents the essential functions of this protein modification remains unclear. To address this, we performed genome-scale CRISPR-Cas9-based screens, revealing that the BLM-TOP3A-RMI1-RMI2 (BTRR)-PICH pathway, which resolves ultrafine anaphase DNA bridges (UFBs) arising from catenated DNA structures, and the poorly characterized protein NIP45/NFATC2IP become indispensable for cell proliferation when SUMOylation is inhibited. We demonstrate that NIP45 and SUMOylation orchestrate an interphase pathway for converting DNA catenanes into double-strand breaks (DSBs) that activate the G2 DNA-damage checkpoint, thereby preventing cytokinesis failure and binucleation when BTRR-PICH-dependent UFB resolution is defective.

Cyclin E overexpression in the accessory gland induces tissue dysplasia.

The regulation of the cell division cycle is governed by a complex network of factors that together ensure that growing or proliferating cells maintain a stable genome. Defects in this system can lead to genomic instability that can affect tissue homeostasis and thus compromise human health. Variations in ploidy and cell heterogeneity are observed frequently in human cancers.

PICH acts as a force-dependent nucleosome remodeler.

In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes.

RAD51 protects human cells from transcription-replication conflicts.

Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS.

MicroRNA-449a Inhibits Triple Negative Breast Cancer by Disturbing DNA Repair and Chromatid Separation.

Chromosomal instability (CIN) can be a driver of tumorigenesis but is also a promising therapeutic target for cancer associated with poor prognosis such as triple negative breast cancer (TNBC). The treatment of TNBC cells with defects in DNA repair genes with poly(ADP-ribose) polymerase inhibitor (PARPi) massively increases CIN, resulting in apoptosis. Here, we identified a previously unknown role of microRNA-449a in CIN.

Nonlinear mechanics of human mitotic chromosomes.

In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A), and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome.

Duplex DNA and BLM regulate gate opening by the human TopoIIIα-RMI1-RMI2 complex.

Topoisomerase IIIα is a type 1A topoisomerase that forms a complex with RMI1 and RMI2 called TRR in human cells. TRR plays an essential role in resolving DNA replication and recombination intermediates, often alongside the helicase BLM. While the TRR catalytic cycle is known to involve a protein-mediated single-stranded (ss)DNA gate, the detailed mechanism is not fully understood.

PICH Supports Embryonic Hematopoiesis by Suppressing a cGAS-STING-Mediated Interferon Response.

The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase DNA bridges in mitosis along with a complex of DNA repair proteins. Previous studies show PICH deficiency-induced embryonic lethality in mice. However, the function of PICH that is required to suppress embryonic lethality in PICH-deficient mammals remains to be determined.

Simvastatin improves mitochondrial respiration in peripheral blood cells.

Statins are prescribed to treat hypercholesterolemia and to reduce the risk of cardiovascular disease. However, statin users frequently report myalgia, which can discourage physical activity or cause patients to discontinue statin use, negating the potential benefit of the treatment. Although a proposed mechanism responsible for Statin-Associated Myopathy (SAM) suggests a correlation with impairment of mitochondrial function, the relationship is still poorly understood.

The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells.

Replication-blocking DNA lesions are particularly toxic to proliferating cells because they can lead to chromosome mis-segregation if not repaired prior to mitosis. In this study, we report that ZGRF1 null cells accumulate chromosome aberrations following replication perturbation and show sensitivity to two potent replication-blocking anticancer drugs: mitomycin C and camptothecin. Moreover, ZGRF1 null cells are defective in catalyzing DNA damage-induced sister chromatid exchange despite accumulating excessive FANCD2, RAD51, and γ-H2AX foci upon induction of interstrand DNA crosslinks.

Folate stress induces SLX1- and RAD51-dependent mitotic DNA synthesis at the fragile X locus in human cells.

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity.

High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing.

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin.