RESUMEN
Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Recombinación HomólogaRESUMEN
Efficient DNA repair and limitation of genome rearrangements rely on crosstalk between different DNA double-strand break (DSB) repair pathways, and their synchronization with the cell cycle. The selection, timing and efficacy of DSB repair pathways are influenced by post-translational modifications of histones and DNA damage repair (DDR) proteins, such as phosphorylation. While the importance of kinases and serine/threonine phosphatases in DDR have been extensively studied, the role of tyrosine phosphatases in DNA repair remains poorly understood. In this study, we have identified EYA4 as the protein phosphatase that dephosphorylates RAD51 on residue Tyr315. Through its Tyr phosphatase activity, EYA4 regulates RAD51 localization, presynaptic filament formation, foci formation, and activity. Thus, it is essential for homologous recombination (HR) at DSBs. DNA binding stimulates EYA4 phosphatase activity. Depletion of EYA4 decreases single-stranded DNA accumulation following DNA damage and impairs HR, while overexpression of EYA4 in cells promotes dephosphorylation and stabilization of RAD51, and thereby nucleoprotein filament formation. Our data have implications for a pathological version of RAD51 in EYA4-overexpressing cancers.
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Recombinasa Rad51 , Transactivadores , ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/genética , Fosfoproteínas Fosfatasas/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Tirosina/genética , Humanos , Transactivadores/metabolismoRESUMEN
Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.
Asunto(s)
Proteína BRCA1/metabolismo , Neoplasias Óseas/metabolismo , Cromatina/metabolismo , Reparación del ADN , Proteínas Nucleares/metabolismo , Osteosarcoma/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/genética , Neoplasias Óseas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cromatina/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Inestabilidad Genómica , Células HEK293 , Chaperonas de Histonas , Histonas/genética , Histonas/metabolismo , Recombinación Homóloga , Humanos , Proteínas Nucleares/genética , Osteosarcoma/genética , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The ubiquitin signaling pathway is crucial for the DNA damage response pathway. More specifically, RNF168 is integral in regulating DNA repair proteins at damaged chromatin. However, the detailed mechanism by which RNF168 is regulated in cells is not fully understood. Here, we identify the ubiquitin-ribosomal fusion proteins UBA80 (also known as RPS27A) and UBA52 (also known as RPL40) as interacting proteins for H2A/H2AX histones and RNF168. Both UBA80 and UBA52 are recruited to laser-induced micro-irradiation DNA damage sites and are required for DNA repair. Ectopic expression of UBA80 and UBA52 inhibits RNF168-mediated H2A/H2AX ubiquitination at K13/15 and impairs 53BP1 recruitment to DNA lesions. Mechanistically, the C-terminal ribosomal fragments of UBA80 and UBA52, S27A and L40, respectively, limit RNF168-nucleosome engagement by masking the regulatory acidic residues at E143/E144 and the nucleosome acidic patch. Together, our results reveal that UBA80 and UBA52 antagonize the ubiquitination signaling pathway and fine-tune the spatiotemporal regulation of DNA repair proteins at DNA damage sites.
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Reparación del ADN , Histonas , Nucleosomas , Proteínas Ribosómicas , Ubiquitina-Proteína Ligasas , Daño del ADN , Histonas/metabolismo , Nucleosomas/genética , Proteínas Ribosómicas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , HumanosRESUMEN
An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.
Asunto(s)
Proteína BRCA1 , Reparación del ADN , Recombinación Homóloga , Factores de Transcripción , Proteína 1 de Unión al Supresor Tumoral P53 , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Mamíferos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.
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Replicación del ADN , ADN/química , ADN/metabolismo , G-Cuádruplex , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Línea Celular , ADN Polimerasa Dirigida por ADN/metabolismo , Genes myc , Humanos , Modelos Moleculares , Mutación , Motivos de Nucleótidos , Nucleotidiltransferasas/genética , Unión ProteicaRESUMEN
DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B-EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.
Asunto(s)
Cromatina , Reparación del ADN/genética , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Transcripción Genética/genética , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Silenciador del Gen , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary "readers" of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.
Asunto(s)
Cromatina/metabolismo , Daño del ADN , Recombinación Homóloga/genética , Receptores de Superficie Celular/metabolismo , Autoantígenos/metabolismo , Línea Celular Tumoral , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Unión Proteica , Transporte de Proteínas/genética , Receptores de Cinasa C Activada , Receptores de Superficie Celular/genética , Proteínas Supresoras de TumorRESUMEN
Precise determination of boundaries in co-culture systems is difficult to achieve with scanning electrochemical microscopy alone. Thus, biological scanning electrochemical microscope platforms generally consist of a scanning electrochemical microscope positioner mounted on the stage of an inverted microscope for correlated electrochemical and optical imaging. Use of a fluorescence microscope allows for site-specific fluorescence labeling to obtain more clearly resolved spatial and electrochemical data. Here, we construct a unique hyperspectral assisted-biological scanning electrochemical microscope platform to widen the scope of biological imaging. Specifically, we incorporate a variable fluorescence bandpass source into a biological scanning electrochemical microscope platform for simultaneous optical, spectral, and electrochemical imaging. Not only does this platform serve as a cost-effective alternative to white light laser imaging, but additionally it provides multi-functional analysis of biological samples. Here, we demonstrate the efficacy of our platform to discern the electrochemical contribution of site-specific cells by optically and spectroscopically resolving boundaries as well as cell types within a complex biological system.
Asunto(s)
Rayos Láser , Imagen Óptica , Técnicas de Cocultivo , Microscopía Electroquímica de Rastreo , Microscopía Fluorescente/métodosRESUMEN
Correction for 'Enhancing scanning electrochemical microscopy's potential to probe dynamic co-culture systems via hyperspectral assisted-imaging' by Sondrica Goines et al., Analyst, 2022, 147, 2396-2404, https://doi.org/10.1039/D2AN00319H.
RESUMEN
Antibiotic-induced microbial imbalance, or dysbiosis, has systemic and long-lasting effects on the host and response to cancer therapies. However, the effects on tumor endothelial cells are largely unknown. Therefore, the goal of the current study was to generate matched B16-F10 melanoma associated endothelial cell lines isolated from mice with and without antibiotic-induced dysbiosis. After validating endothelial cell markers on a genomic and proteomic level, functional angiogenesis assays (i.e., migration and tube formation) also confirmed their vasculature origin. Subsequently, we found that tumor endothelial cells derived from dysbiotic mice (TEC-Dys) were more sensitive to ionizing radiotherapy in the range of clinically-relevant hypofractionated doses, as compared to tumor endothelial cells derived from orthobiotic mice (TEC-Ortho). In order to identify tumor vasculature-associated drug targets during dysbiosis, we used tandem mass tag mass spectroscopy and focused on the statistically significant cellular membrane proteins overexpressed in TEC-Dys. By these criteria c-Met was the most differentially expressed protein, which was validated histologically by comparing tumors with or without dysbiosis. Moreover, in vitro, c-Met inhibitors Foretinib, Crizotinib and Cabozantinib were significantly more effective against TEC-Dys than TEC-Ortho. In vivo, Foretinib inhibited tumor growth to a greater extent during dysbiosis as compared to orthobiotic conditions. Thus, we surmise that tumor response in dysbiotic patients may be greatly improved by targeting dysbiosis-induced pathways, such as c-Met, distinct from the many targets suppressed due to dysbiosis.
Asunto(s)
Disbiosis , Células Endoteliales/enzimología , Melanoma Experimental , Neovascularización Patológica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met , Animales , Disbiosis/enzimología , Disbiosis/microbiología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/enzimología , Melanoma Experimental/microbiología , Melanoma Experimental/terapia , Ratones , Neovascularización Patológica/enzimología , Neovascularización Patológica/microbiología , Neovascularización Patológica/terapia , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , RadioterapiaRESUMEN
The DNA remodeling enzyme FANCM and its DNA-binding partner, FAAP24, constitute a complex involved in the activation of Fanconi anemia (FA) DNA damage response mechanism, but neither gene has distinct patient mutants. In this study, we created isogenic models for both FANCM and FAAP24 and investigated their integrated functions in DNA damage response. We found that FANCM and FAAP24 coordinately facilitate FA pathway activation and suppress sister chromatid exchange. Importantly, we show that FANCM and FAAP24 possess nonoverlapping functions such that FAAP24 promotes ATR-mediated checkpoint activation particularly in response to DNA crosslinking agents, whereas FANCM participates in recombination-independent interstrand crosslink repair by facilitating recruitment of lesion incision activities, which requires its translocase activity. Our data suggest that FANCM and FAAP24 play multiple, while not fully epistatic, roles in maintaining genomic integrity.
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ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Genoma Humano , Inestabilidad Genómica , ADN/metabolismo , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Células HCT116 , Células HEK293 , HumanosRESUMEN
The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.
Asunto(s)
Dineínas Citoplasmáticas/fisiología , Roturas del ADN de Doble Cadena , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína BRCA1/genética , Línea Celular , Cromatina/metabolismo , Dineínas Citoplasmáticas/química , Dineínas Citoplasmáticas/metabolismo , Reparación del ADN , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Radiación IonizanteRESUMEN
Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Secuencia de Aminoácidos , Animales , Supervivencia Celular , Células Cultivadas , Cromatina/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Datos de Secuencia Molecular , ARN Interferente Pequeño/genética , Elementos de Respuesta , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.
Asunto(s)
Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Reparación del ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Histonas/genética , Humanos , Mutación , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Unión Proteica/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Histone ubiquitinations are critical for the activation of the DNA damage response (DDR). In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub). The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.
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Histonas/genética , Complejo Represivo Polycomb 1/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Daño del ADN/genética , Reparación del ADN , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Nucleosomas/genética , Unión Proteica/genética , Transducción de Señal/genética , UbiquitinaRESUMEN
Tousled-like kinases 1 and 2 (TLK1 and 2) are cell cycle-regulated serine/threonine kinases that are involved in multiple biological processes. Mutation of TLK1 and 2 confer neurodegenerative diseases. Recent studies demonstrate that TLK1 and 2 are involved in DNA repair. However, there is no direct evidence that TLK1 and 2 function at DNA damage sites. Here, we show that both TLK1 and TLK2 are hyper-autophosphorylated at their N-termini, at least in part, mediated by their homo- or hetero-dimerization. We found that TLK1 and 2 hyper-autophosphorylation suppresses their recruitment to damaged chromatin. Furthermore, both TLK1 and 2 associate with PCNA specifically through their evolutionarily conserved non-canonical PCNA-interacting protein (PIP) box at the N-terminus, and mutation of the PIP-box abolishes their recruitment to DNA damage sites. Mechanistically, the TLK1 and 2 hyper-autophosphorylation masks the PIP-box and negatively regulates their recruitment to the DNA damage site. Overall, our study dissects the detailed genetic regulation of TLK1 and 2 at damaged chromatin, which provides important insights into their emerging roles in DNA repair.
RESUMEN
HELB is a human helicase involved in initiation of DNA replication, the replication stress response, and regulation of double-strand DNA break repair. rs75770066 is a rare SNP in the HELB gene that affects age at natural menopause. rs75770066 results in a D506G substitution in an acidic patch within the 1A domain of the helicase that is known to interact with RPA. We found that this amino acid change dramatically impairs the cellular function of HELB. D506G-HELB exhibits impaired interaction with RPA, which likely results in the effects of rs75770066 as this reduces recruitment of HELB to sites of DNA damage. Reduced recruitment of D506G-HELB to double-strand DNA breaks and the concomitant increase in homologous recombination likely alters the levels of meiotic recombination, which affects the viability of gametes. Because menopause occurs when oocyte levels drop below a minimum threshold, altered repair of meiotic double-stranded DNA breaks has the potential to directly affect the age at natural menopause.
RESUMEN
The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.
Asunto(s)
Cromatina , Daño del ADN , Histonas , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitinación , Humanos , Proteínas Adaptadoras Transductoras de Señales , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Camptotecina/farmacología , Proteínas de Ciclo Celular , Cromatina/metabolismo , Reparación del ADN , Células HEK293 , Histonas/metabolismo , Histonas/genética , Fosforilación , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Primary microcephaly is an autosomal recessive disorder characterized by marked reduction in human brain size. Microcephalin (MCPH1), one of the genes mutated in primary microcephaly, plays an important role in DNA damage checkpoint control and mitotic entry. Additionally, MCPH1 ensures the proper temporal activation of chromosome condensation during mitosis, by acting as a negative regulator of the condensin II complex. We previously found that deletion of the of the MCPH1 N terminus leads to the premature chromosome condensation (PCC) phenotype. In the present study, we unexpectedly observed that a truncated form of MCPH1 appears to be expressed in MCPH1(S25X/S25X) patient cells. This likely results from utilization of an alternative translational start codon, which would produce a mutant MCPH1 protein with a small deletion of its N-terminal BRCT domain. Furthermore, missense mutations in the MCPH1 cluster at its N terminus, suggesting that intact function of this BRCT protein-interaction domain is required both for coordinating chromosome condensation and human brain development. Subsequently, we identified the SET nuclear oncogene as a direct binding partner of the MCPH1 N-terminal BRCT domain. Cells with SET knockdown exhibited abnormal condensed chromosomes similar to those observed in MCPH1-deficient mouse embryonic fibroblasts. Condensin II knockdown rescued the abnormal chromosome condensation phenotype in SET-depleted cells. In addition, MCPH1 V50G/I51V missense mutations, impair binding to SET and fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1(-/-) mouse embryonic fibroblasts. Collectively, our findings suggest that SET is an important regulator of chromosome condensation/decondensation and that disruption of the MCPH1-SET interaction might be important for the pathogenesis of primary microcephaly.