APE1 condensation in nucleoli of non-cancer cells depends on rRNA transcription and forming G-quadruplex RNA structures

Nucleic Acids Research, Год журнала: 2025, Номер 53(5)

Опубликована: Фев. 27, 2025

Abstract APE1 [apurinic/apyrimidinic (AP) endodeoxyribonuclease 1] is the main endonuclease of base excision repair pathway acting on abasic sites in DNA. an abundant nuclear protein, and improper expression or localization this factor could lead to accumulation toxic DNA intermediates. Altered subcellular distribution are associated with cancer development, suggesting importance a fine-tuning mechanism for activities. Recent works highlighted presence within nucleoli cells ability form biomolecular condensate. However, whether secondary structures ribosomal RNA (rRNA) influence nucleolar remains poorly understood. Since protein overexpression can result artificial accumulation, it imperative have appropriate cellular models study trafficking under physiological conditions. To address issue, we generated murine embryonic stem cell line expressing endogenous fluorescent-tagged APE1. Live-cell imaging demonstrates that requires active rRNA transcription modulated by different genotoxicants. In vitro experiments showed condensate formation depends RNA-forming G-quadruplex relies critical lysine residues. This sheds light mechanisms underlying nucleolus RNA-dependent condensates.

Язык: Английский

---

Spatial organization and functions of Chk1 activation by TopBP1 biomolecular condensates

Cell Reports, Год журнала: 2024, Номер 43(4), С. 114064 - 114064

Опубликована: Апрель 1, 2024

Assembly of TopBP1 biomolecular condensates triggers activation the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics reverse genetics investigate role sequence-specific motifs in formation functions condensates. We propose that BACH1/FANCJ is involved partitioning BRCA1 within compartments. show Chk1 activated at interface provide evidence these structures arise sites damage primary human fibroblasts. phosphorylation depends on integrity a conserved arginine motif TopBP1's ATR domain (AAD). Its mutation uncouples from condensation, revealing optogenetically induced cycle checkpoints slows down replication forks absence damage. Together with previous work, data suggest intrinsically disordered AAD encodes distinct molecular steps ATR/Chk1 pathway.

Язык: Английский

---

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 Using Proximity Labeling and Affinity Purification

Journal of Proteome Research, Год журнала: 2023, Номер 22(6), С. 1660 - 1681

Опубликована: Апрель 18, 2023

The DNA repair scaffold SLX4 has pivotal roles in cellular processes that maintain genome stability, most notably homologous recombination. Germline mutations are associated with Fanconi anemia, a disease characterized by chromosome instability and cancer susceptibility. role of mammalian recombination depends critically on binding activating structure-selective endonucleases, namely SLX1, MUS81-EME1, XPF-ERCC1. Increasing evidence indicates cells rely distinct SLX4-dependent complexes to remove lesions specific regions the genome. Despite our understanding as for proteins, detailed repertoire interactors never been reported. Here, we provide comprehensive map human interactome using proximity-dependent biotin identification (BioID) affinity purification coupled mass spectrometry (AP-MS). We identified 221 unique high-confidence interactors, which vast majority represent novel SLX4-binding proteins. Network analysis these hits revealed pathways known involvement SLX4, such repair, several emerging interest, including RNA metabolism chromatin remodeling. In summary, report here provides deeper how functions while revealing new may involve SLX4.

Язык: Английский

---

Technologies for studying phase-separated biomolecular condensates

Advanced Biotechnology, Год журнала: 2024, Номер 2(1)

Опубликована: Март 7, 2024

Biomolecular condensates, also referred to as membrane-less organelles, function fundamental organizational units within cells. These structures primarily form through liquid-liquid phase separation, a process in which proteins and nucleic acids segregate from the surrounding milieu assemble into micron-scale structures. By concentrating functionally related acids, these biomolecular condensates regulate myriad of essential cellular processes. To study significant intricate range technologies have been either adapted or developed. In this review, we provide an overview most utilized rapidly evolving field. include methods used identify new explore their components, investigate properties spatiotemporal regulation, understand principles governing condensates. We discuss potential challenges review current advancements applying development technologies, such those synthetic biology.

Язык: Английский

---

In silico analysis of several frequent SLX4 mutations appearing in human cancers.

PubMed, Год журнала: 2024, Номер 2024

Опубликована: Янв. 1, 2024

SLX4 is an interactor and activator of structure-specific exonuclease that helps resolve tangled recombination intermediates arising at stalled replication forks. It one the many factors assist with homologous recombination, major mechanism for restarting replication. mutations have been reported in cancers but a pan cancer map all has not undertaken. Here, using data from Catalogue Somatic Mutations Cancers (COSMIC), we show occur almost every them truncate protein which should severely alter function enzyme. We identified frequent R1779W point mutation occurs domain required heterodimerization its partner, SLX1. In silico structure analysis this shows it significantly alters likely to destabilize interaction Although brief communication limited only

Язык: Английский

---

Preserving genome integrity: The vital role of SUMO-targeted ubiquitin ligases

Cell Insight, Год журнала: 2023, Номер 2(6), С. 100128 - 100128

Опубликована: Окт. 23, 2023

Various post-translational modifications (PTMs) collaboratively fine-tune protein activities. SUMO-targeted ubiquitin E3 ligases (STUbLs) emerge as specialized enzymes that recognize SUMO-modified substrates through SUMO-interaction motifs and subsequently ubiquitinate them via the RING domain, thereby bridging SUMO signaling pathways. STUbLs participate in a wide array of molecular processes, including cell cycle regulation, DNA repair, replication, mitosis, operating under both normal conditions response to challenges such genotoxic stress. Their ability catalyze various types chains results diverse proteolytic non-proteolytic outcomes for target substrates. Importantly, are strategically positioned close proximity proteases deubiquitinases (DUBs), ensuring precise dynamic control over their proteins. In this review, we provide insights into unique properties indispensable roles STUbLs, with particular emphasis on significance preserving genome integrity humans.

Язык: Английский

---

Nucleolar accumulation of APE1 through condensates is mediated by rRNA forming G-quadruplex structures

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 5, 2024

Abstract APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is the main endonuclease of base excision repair (BER) pathway acting on abasic (AP)-sites in damaged DNA. an abundant nuclear protein with a higher concentration than other BER enzymes, and therefore, improper expression localization this factor could lead to accumulation toxic DNA intermediates. Altered sub-cellular localization, levels, or hyper-acetylation are associated cancer development suggesting importance fine-tuning mechanism for nuclear-associated processes. Recent work highlighted multi-functional roles APE1, including rRNA quality control. However, how influences activity remains poorly understood, but previously underappreciated APE1-RNA interactions may influence ability form biomolecular condensates tune partitioning into nucleoli. Since nucleolar ectopic proteins be result overexpression strategies, it imperative have cellular models study trafficking under physiological conditions. Here we created first cell line express fluorescently tagged at its endogenous locus, enabling live-cell imaging. Live-cell imaging demonstrates that requires active transcription. When modeled vitro, condensate formation depends RNA G-quadruplex (rG4) structures modulated by critical lysine residues APE1. This sheds light mechanisms underlying nucleolus RNA-dependent modulate switch between processing damage repair. Significance Statement We characterized endogenous, subcellular stress Using line, show enrichment occurs conditions and, performing vitro droplet assays, associate transcription G-quadruplexes, abundantly present healthy deepens our understanding APE1’s role cells absence provide novel responds stress. Our results suggest phase separation important part their normal functions correct lesions.

Язык: Английский

---

Lighting ATR/Chk1 by mesoscale TopBP1 condensates

Trends in Cell Biology, Год журнала: 2024, Номер 34(6), С. 440 - 441

Опубликована: Май 6, 2024

Язык: Английский

---

The Role of SUMOylation in Biomolecular Condensate Dynamics and Protein Localization

Cell Insight, Год журнала: 2024, Номер 3(6), С. 100199 - 100199

Опубликована: Сен. 10, 2024

Язык: Английский

---

Multifaceted roles of SUMO in DNA metabolism

Nucleus, Год журнала: 2024, Номер 15(1)

Опубликована: Сен. 17, 2024

Sumoylation, a process in which SUMO (small ubiquitin like modifier) is conjugated to target proteins, emerges as post-translational modification that mediates protein-protein interactions, protein complex assembly, and localization of proteins. The coordinated actions ligases, proteases, SUMO-targeted ligases determine the net result sumoylation. It well established sumoylation can somewhat promiscuously proteins groups selectively individual Through changing dynamics, orchestrates multi-step processes chromatin biology. Sumoylation influences various steps mitosis, DNA replication, damage repair, pathways protecting chromosome integrity. This review highlights examples SUMO-regulated nuclear provide mechanistic views metabolism.

Язык: Английский

---