RESUMEN
Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur during transcription and DNA replication. Coordination of these processes across different time and length scales involves dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers, mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates may cooperate to coordinate genome maintenance in space and time.
Asunto(s)
Replicación del ADN , Homeostasis del Telómero , Humanos , Reparación del ADN , ADN/metabolismo , Daño del ADN , Telómero/metabolismoRESUMEN
The Nucleosome Remodeling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. Whether these different complexes serve distinct functional activities during differentiation is not fully explored. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD2 and MBD3 variants for their potential to rescue the differentiation block observed for mouse embryonic stem cells (ESCs) lacking MBD3. While MBD3 is indeed crucial for ESC differentiation to neuronal cells, it functions independently of its MBD domain. We further identify that MBD2 isoforms can replace MBD3 during lineage commitment, however with different potential. Full-length MBD2a only partially rescues the differentiation block, while MBD2b, an isoform lacking an N-terminal GR-rich repeat, fully rescues the Mbd3 KO phenotype. In case of MBD2a, we further show that removing the methylated DNA binding capacity or the GR-rich repeat enables full redundancy to MBD3, highlighting the synergistic requirements for these domains in diversifying NuRD complex function.
Asunto(s)
Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Nucleosomas , Animales , Ratones , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Isoformas de Proteínas/genética , Diferenciación Celular , Células Madre Embrionarias de Ratones , MamíferosRESUMEN
RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability.
Asunto(s)
Proteína de Replicación A , Telómero , Proteína de Replicación A/química , Telómero/metabolismo , ARN/metabolismo , ADN de Cadena Simple , Unión Proteica , Replicación del ADNRESUMEN
PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently pose limitations to the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression to be generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Recurrencia Local de Neoplasia , ADP-Ribosilación , Mutación , Proteínas Portadoras/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Endodesoxirribonucleasas , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Genes Supresores de Tumor , Recombinación Homóloga , Humanos , Interferencia de ARN , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.
Asunto(s)
Glutamato-Cisteína Ligasa , Factor 2 Relacionado con NF-E2 , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antioxidantes/farmacología , Bortezomib/farmacología , Glutamato-Cisteína Ligasa/metabolismo , Glutamato-Cisteína Ligasa/farmacología , Glutatión/metabolismo , Hepatocitos/metabolismo , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Nitrofurantoína/metabolismo , Nitrofurantoína/farmacología , Estrés Oxidativo , Paraquat/metabolismo , Paraquat/farmacología , Inhibidores de Proteasoma/farmacología , ARN Interferente Pequeño/metabolismo , Rotenona/metabolismo , Rotenona/farmacologíaRESUMEN
Protein recruitment to DNA break sites is an integral part of the DNA damage response (DDR). Elucidation of the hierarchy and temporal order with which DNA damage sensors as well as repair and signaling factors assemble around chromosome breaks has painted a complex picture of tightly regulated macromolecular interactions that build specialized compartments to facilitate repair and maintenance of genome integrity. While many of the underlying interactions, e.g. between repair factors and damage-induced histone marks, can be explained by lock-and-key or induced fit binding models assuming fixed stoichiometries, structurally less well defined interactions, such as the highly dynamic multivalent interactions implicated in phase separation, also participate in the formation of multi-protein assemblies in response to genotoxic stress. Although much remains to be learned about these types of cooperative and highly dynamic interactions and their functional roles, the rapidly growing interest in material properties of biomolecular condensates and in concepts from polymer chemistry and soft matter physics to understand biological processes at different scales holds great promises. Here, we discuss nuclear condensates in the context of genome integrity maintenance, highlighting the cooperative potential between clustered stoichiometric binding and phase separation. Rather than viewing them as opposing scenarios, their combined effects can balance structural specificity with favorable physicochemical properties relevant for the regulation and function of multilayered nuclear condensates.
Asunto(s)
Núcleo Celular/metabolismo , Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Animales , Ensamble y Desensamble de Cromatina , ADN/metabolismo , Eucariontes/genética , Eucariontes/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.
Asunto(s)
Endodesoxirribonucleasas , Exodesoxirribonucleasas , ADN , Daño del ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Enzimas Multifuncionales/genéticaRESUMEN
DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1's binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.
Asunto(s)
Proteína BRCA1/genética , Reparación del ADN/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Cromatina/genética , ADN/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Genes BRCA1 , Células HeLa , Histonas/metabolismo , Recombinación Homóloga/genética , Humanos , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.