ORCID Profile
0009-0005-6883-0558
Current Organisation
Children's Medical Research Institute
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Publisher: Public Library of Science (PLoS)
Date: 22-05-2009
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.PBIOMOLBIO.2019.04.002
Abstract: DNA replication plays a central role in genome health. Deleterious alteration of replication dynamics, or "replication stress", is a key driver of genome instability and oncogenesis. The replication stress response is regulated by the ATR kinase, which functions to mitigate replication abnormalities through coordinated efforts that arrest the cell cycle and repair damaged replication forks. mTOR kinase regulates signaling networks that control cell growth and metabolism in response to environmental cues and cell stress. In this review, we discuss interconnectivity between the ATR and mTOR pathways, and provide putative mechanisms for mTOR engagement in DNA replication and the replication stress response. Finally, we describe how connectivity between mTOR and replication stress may be exploited for cancer therapy.
Publisher: Springer Science and Business Media LLC
Date: 30-11-2020
DOI: 10.1038/S41556-020-00605-6
Abstract: Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.
Publisher: Springer Science and Business Media LLC
Date: 06-10-2012
DOI: 10.1007/S00412-012-0385-6
Abstract: In most eukaryotes, telomeres are composed of tandem arrays of species-specific DNA repeats ending with a G-rich 3' overhang. In budding yeast, Cdc13 binds this overhang and recruits Ten1-Stn1 and the telomerase protein Est1 to protect (cap) and elongate the telomeres, respectively. To dissect and study the various pathways employed to cap and maintain the telomere end, we engineered telomerase to incorporate Tetrahymena telomeric repeats (G₄T₂) onto the telomeres of the budding yeast Kluyveromyces lactis. These heterologous repeats caused telomere-telomere fusions, cell cycle arrest at G2/M, and severely reduced viability--the hallmarks of telomere uncapping. Fusing Cdc13 or Est1 to universal minicircle sequence binding protein (UMSBP), a small protein that binds the single-stranded G₄T₂ repeats, rescued the cell viability and restored telomere capping, but not telomerase-mediated telomere maintenance. Surprisingly, Cdc13-UMSBP-mediated telomere capping was dependent on the homologous recombination factor Rad52, while Est1-UMSBP was not. Thus, our results distinguish between two, redundant, telomere capping pathways.
Publisher: Proceedings of the National Academy of Sciences
Date: 19-08-2013
Abstract: Telomeres protect the ends of eukaryotic chromosomes. Telomeres shorten with age and serve as a biological clock that limits cell proliferation. Excessive telomere shortening accelerates aging, but telomere elongation may facilitate cancer. We found inherited mutations in the regulator of telomere elongation helicase 1 (RTEL1), which cause Hoyeraal–Hreidarsson syndrome, a fatal disease characterized by accelerated telomere shortening, immunodeficiency, and developmental defects. Introducing a normal RTEL1 gene into affected cells prevented telomere shortening and extended their lifespan in culture. The telomere defects, genomic instability, and growth arrest observed in RTEL1-deficient cells help in our understanding the central roles of telomeres in aging and cancer.
Publisher: Cold Spring Harbor Laboratory
Date: 24-06-2022
DOI: 10.1101/2022.06.24.497492
Abstract: Faithful DNA replication requires the orderly firing of replication origins across the genome. At present, we lack details around how origins are selected for activation and the subsequent impact of this on replication dynamics. Here, we have investigated how chromatin organisation contributes to replication initiation and dynamics by intersecting ChIP-seq, Hi-C, Repli-seq, and OK-seq data from primary and tumour cells lines. We found replication initiation is significantly enriched at TAD boundaries, co-localizing with CTCF and cohesin in early and mid S-phase. Strong replication fork directionality (RFD) from initiation zones in TAD boundaries could occur in a bi- or uni-directional manner, which highly correlated with replication timing. While TAD boundaries were largely invariant, a minority of initiation zones were shared across cell lines, indicative of cell type specific regulation. These data are consistent with chromatin structure organizing replication initiation and dynamics, ensuring orderly completion of replication from TAD boundaries into TAD internal regions.
Publisher: S. Karger AG
Date: 2005
DOI: 10.1159/000086427
Abstract: Peripheral acetylcholine levels notably control the synthesis in macrophages of pro-inflammatory cytokines however, it remains unclear whether this peripheral regulatory pathway affects central nervous system neurons. To explore the interrelationship between neuronal cholinergic homeostasis and peripheral inflammatory responses in primates, we used spinal cord sections from cynomolgus monkeys after 7 days oral or intravenous treatment with Monarsen oligonucleotide. Monarsen is an antisense oligonucleotide 3′-protected by 2′-oxymethylation, which was proved to induce selective destruction of the stress-induced acetylcholinesterase splice variant AChE-R mRNA. Handling stress predictably suppressed neuronal choline acetyl transferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in all treated monkeys. In Monarsen-treated animals, we further observed suppression of stress-induced increases in plasma AChE activities. Corresponding decreases in AChE-R mRNA were seen in spinal cord neurons, associated with parallel decline patterns in the mRNA encoding for the splice factor SC35 (the levels of which co-increase with those of AChE-R) as well as in the neuronal pro-inflammatory interleukins IL-1β and IL-6. The antisense effects showed direct dose dependence and were inversely associated with neuronal cell size. These findings suggest a causal association between neuronal cholinergic allostasis and inflammatory reactions in primates and support the peripheral use of RNA-targeted intervention with AChE-R accumulation for the management of both stress and inflammatory responses.
Publisher: Cold Spring Harbor Laboratory
Date: 24-09-2018
DOI: 10.1101/425264
Abstract: Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death promotes genome instability in replication stressed cells through erse mechanisms depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and genome instability.
Publisher: Cold Spring Harbor Laboratory
Date: 29-10-2020
Abstract: Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.
Publisher: Oxford University Press (OUP)
Date: 15-06-2020
DOI: 10.1093/NAR/GKAA503
Abstract: Telomeres cap the ends of eukaryotic chromosomes and distinguish them from broken DNA ends to suppress DNA damage response, cell cycle arrest and genomic instability. Telomeres are elongated by telomerase to compensate for incomplete replication and nuclease degradation and to extend the proliferation potential of germ and stem cells and most cancers. However, telomeres in somatic cells gradually shorten with age, ultimately leading to cellular senescence. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and erse symptoms including bone marrow failure, immunodeficiency, and neurodevelopmental defects. HHS is caused by germline mutations in telomerase subunits, factors essential for its biogenesis and recruitment to telomeres, and in the helicase RTEL1. While erse phenotypes were associated with RTEL1 deficiency, the telomeric role of RTEL1 affected in HHS is yet unknown. Inducible ectopic expression of wild-type RTEL1 in patient fibroblasts rescued the cells, enabled telomerase-dependent telomere elongation and suppressed the abnormal cellular phenotypes, while silencing its expression resulted in gradual telomere shortening. Our observations reveal an essential role of the RTEL1 C-terminus in facilitating telomerase action at the telomeric 3′ overhang. Thus, the common etiology for HHS is the compromised telomerase action, resulting in telomere shortening and reduced lifespan of telomerase positive cells.
Publisher: Elsevier BV
Date: 10-2021
DOI: 10.1016/J.TCB.2021.06.002
Abstract: The nucleus is a dynamic environment containing chromatin, membraneless organelles, and specialized molecular structures at the nuclear membrane. Within the spectrum of DNA repair activities are observations of increased mobility of damaged chromatin and the displacement of DNA lesions to specific nuclear environments. Here, we focus on the role that nuclear-specific filamentous actin plays in mobilizing damaged chromatin in response to DNA double-strand breaks and replication stress. We also examine nuclear pore complexes and promyelocytic leukemia-nuclear bodies as specialized platforms for homology-directed repair. The literature suggests an emerging model where specific types of DNA lesions are subjected to nuclear-derived forces that mobilize damaged chromatin and promote interaction with repair hubs to facilitate specialized repair reactions.
Publisher: Cold Spring Harbor Laboratory
Date: 24-10-2018
DOI: 10.1101/451708
Abstract: “Replication stress” describes phenomena that alter DNA replication rates 1–3 . Multiple architectural challenges within the confined nuclear volume must be navigated during replication to prevent or repair replication stress. Cellular mechanisms potentiating changes in nuclear architecture that facilitate DNA replication remain unclear. Here we show that the ATR, IPMK and mTOR kinases regulate actin polymerisation in human cells to alter nuclear architecture and promote replication fork repair. We demonstrate that replication stress activates mTOR, in an ATR and IPMK-dependent manner, to induce polymerisation of nuclear filamentous actin (F-actin). mTOR and ATR then counteract replication stress-induced nuclear envelope deformation and increase nuclear volume through their regulation of actin dynamics. Additionally, we reveal that FANCD2 labelled replication forks colocalise with actin filaments in late S-phase. mTOR and ATR then regulate the mobility, speed and directionality of stalled replication foci within the three-dimensional nuclear architecture. Importantly, we find nuclear F-actin also acts as a substrate for the directed migration of stalled replication foci to the nuclear periphery. Suppressing mTOR and ATR-dependent actin forces prevents replication fork restart and promotes chromosome segregation errors in primary and cancer cell lines. Together, these data reveal that ATR and mTOR regulate actin dynamics in the replication stress response to alter nuclear architecture and maintain genome stability.
Publisher: Elsevier BV
Date: 02-2016
DOI: 10.1016/J.STEM.2015.11.003
Abstract: Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations such as aneuploidy in culture. These aberrations progressively increase over time and may compromise the properties and clinical utility of the cells. The underlying mechanisms that drive initial genomic instability and its continued progression are largely unknown. Here, we show that aneuploid hPSCs undergo DNA replication stress, resulting in defective chromosome condensation and segregation. Aneuploid hPSCs show altered levels of actin cytoskeletal genes controlled by the transcription factor SRF, and overexpression of SRF rescues impaired chromosome condensation and segregation defects in aneuploid hPSCs. Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate during initiation of instability in diploid cells.
Publisher: EMBO
Date: 21-07-2015
Publisher: Informa UK Limited
Date: 17-06-2016
Publisher: Cold Spring Harbor Laboratory
Date: 31-01-2020
DOI: 10.1101/2020.01.31.929059
Abstract: Mutations in the telomere binding protein, POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we investigated the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR-interference and biotin-based proximity labelling, respectively. These screens revealed that replication stress is a major vulnerability in cells expressing mutant POT1 and manifest in increased mitotic DNA synthesis (MiDAS) at telomeres. Our study also unveiled a role for the nuclear pore complex (NPC) in resolving replication defects at telomeres. Depletion of NPC subunits in the context of POT1 dysfunction increased DNA damage signaling and telomere fragility. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.
No related grants have been discovered for Noa Lamm-Shalem.