RESUMO
Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular , Centríolos , Animais , Humanos , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo , Proteínas de Ligação ao Cálcio/metabolismoRESUMO
Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.
Assuntos
Degeneração Retiniana , Retinose Pigmentar , Animais , Cílios , Proteínas do Olho , Camundongos , MicrotúbulosRESUMO
Mutations in genes that encode centrosomal/ciliary proteins cause severe cognitive deficits, while common single-nucleotide polymorphisms in these genes are associated with schizophrenia (SZ) and cognition in genome-wide association studies. The role of these genes in neuropsychiatric disorders is unknown. The ciliopathy gene SDCCAG8 is associated with SZ and educational attainment (EA). Genome editing of SDCCAG8 caused defects in primary ciliogenesis and cilium-dependent cell signalling. Transcriptomic analysis of SDCCAG8-deficient cells identified differentially expressed genes that are enriched in neurodevelopmental processes such as generation of neurons and synapse organization. These processes are enriched for genes associated with SZ, human intelligence (IQ) and EA. Phenotypic analysis of SDCCAG8-deficent neuronal cells revealed impaired migration and neuronal differentiation. These data implicate ciliary signalling in the aetiology of SZ and cognitive dysfunction. We found that centrosomal/ciliary genes are enriched for association with IQ, suggesting altered gene regulation as a general model for neurodevelopmental impacts of centrosomal/ciliary genes.
Assuntos
Autoantígenos/genética , Transtornos Cognitivos/patologia , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Mutação , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Esquizofrenia/patologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/genética , Humanos , Esquizofrenia/etiologia , Esquizofrenia/genéticaRESUMO
The International University of Andalucía (UNIA) Current Trends in Biomedicine Workshop on Molecular Causes of Primary Microcephaly and Related Diseases took place in Baeza, Spain, November 18-20, 2019. This meeting brought together scientists from Europe, the USA and China to discuss recent advances in our molecular and genetic understanding of a group of rare neurodevelopmental diseases characterised by primary microcephaly, a condition in which head circumference is smaller than normal at birth. Microcephaly can be caused by inherited mutations that affect key cellular processes, or environmental exposure to radiation or other toxins. It can also result from viral infection, as exemplified by the recent Zika virus outbreak in South America. Here we summarise a number of the scientific advances presented and topics discussed at the meeting.
Assuntos
Suscetibilidade a Doenças , Microcefalia/etiologia , Transtornos do Neurodesenvolvimento/etiologia , Centrossomo , Cílios , Congressos como Assunto , Dano ao DNA , Replicação do DNA , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Humanos , Microcefalia/diagnóstico , Transtornos do Neurodesenvolvimento/diagnóstico , Neurogênese/genética , Fenótipo , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Centríolos/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , DNA Complementar/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The maintenance of genome stability involves integrated biochemical activities that detect DNA damage or incomplete replication, delay the cell cycle, and direct DNA repair activities on the affected chromatin. These processes, collectively termed the DNA damage response (DDR), are crucial for cell survival and to avoid disease, particularly cancer. Recent work has highlighted links between the DDR and the primary cilium, an antenna-like, microtubule-based signalling structure that extends from a centriole docked at the cell surface. Ciliary dysfunction gives rise to a range of complex human developmental disorders termed the ciliopathies. Mutations in ciliopathy genes have been shown to impact on several functions that relate to centrosome integrity, DNA damage signalling, responses to problems in DNA replication and the control of gene expression. This review covers recent findings that link cilia and the DDR and explores the various roles played by key genes in these two contexts. It outlines how proteins encoded by ciliary genes impact checkpoint signalling, DNA replication and repair, gene expression and chromatin remodelling. It discusses how these diverse activities may integrate nuclear responses with those that affect a structure of the cell periphery. Additional directions for exploration of the interplay between these pathways are highlighted, with a focus on new ciliary gene candidates that alter genome stability.
Assuntos
Núcleo Celular/genética , Cílios/genética , Ciliopatias/genética , Dano ao DNA , Reparo do DNA/genética , Transdução de Sinais/genética , Animais , Ciclo Celular/genética , Núcleo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Ciliopatias/metabolismo , Regulação da Expressão Gênica , Humanos , Microtúbulos/metabolismo , MutaçãoRESUMO
Melanin granules cluster within supra-nuclear caps in basal keratinocytes (KCs) of the human epidermis, where they protect KC genomic DNA against ultraviolet radiation (UVR) damage. While much is known about melanogenesis in melanocytes (MCs) and a moderate amount about melanin transfer from MC to KC, we know little about the fate of melanin once inside KCs. We recently reported that melanin fate in progenitor KCs is regulated by rare asymmetric organelle movement during mitosis. Here, we explore the role of actin, microtubules, and centrosome-associated machinery in distributing melanin within KCs. Short-term cultures of human skin explants were treated with cytochalasin-B and nocodazole to target actin filaments and microtubules, respectively. Treatment effects on melanin distribution were assessed by the Warthin-Starry stain, on centrosome-associated proteins by immunofluorescence microscopy, and on co-localisation with melanin granules by brightfield microscopy. Cytochalasin-B treatment disassembled supra-nuclear melanin caps, while nocodazole treatment moved melanin from the apical to basal KC domain. Centrosome and centriolar satellite-associated proteins showed a high degree of co-localisation with melanin. Thus, once melanin granules are transferred to KCs, their preferred apical distribution appears to be facilitated by coordinated movement of centrosomes and centriolar satellites. This mechanism may control melanin's strategic position within UVR-exposed KCs.
Assuntos
Melaninas/metabolismo , Pele/metabolismo , Actinas/metabolismo , Biomarcadores , Polaridade Celular , Células Cultivadas , Centrossomo/metabolismo , Grânulos Citoplasmáticos/metabolismo , Citoesqueleto/metabolismo , Imunofluorescência , Humanos , Hibridização In Situ , Queratinócitos/metabolismo , Melanócitos/metabolismo , FenótipoRESUMO
Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.
Assuntos
Senescência Celular/fisiologia , Replicação do DNA/fisiologia , Células-Tronco Hematopoéticas/patologia , Estresse Fisiológico , Animais , Proliferação de Células , Senescência Celular/genética , Dano ao DNA/genética , DNA Ribossômico/genética , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Manutenção de Minicromossomo/genéticaRESUMO
Primary cilia are microtubule structures that extend from the distal end of the mature, mother centriole. CEP164 is a component of the distal appendages carried by the mother centriole that is required for primary cilium formation. Recent data have implicated CEP164 as a ciliopathy gene and suggest that CEP164 plays some roles in the DNA damage response (DDR). We used reverse genetics to test the role of CEP164 in the DDR. We found that conditional depletion of CEP164 in chicken DT40 cells using an auxin-inducible degron led to no increase in sensitivity to DNA damage induced by ionising or ultraviolet irradiation. Disruption of CEP164 in human retinal pigmented epithelial cells blocked primary cilium formation but did not affect cellular proliferation or cellular responses to ionising or ultraviolet irradiation. Furthermore, we observed no localisation of CEP164 to the nucleus using immunofluorescence microscopy and analysis of multiple tagged forms of CEP164. Our data suggest that CEP164 is not required in the DDR.
Assuntos
Núcleo Celular/metabolismo , Reparo do DNA , Proteínas dos Microtúbulos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Núcleo Celular/patologia , Galinhas , Cílios/genética , Cílios/metabolismo , Dano ao DNA , Edição de Genes , Células HeLa , Humanos , Células Jurkat , Proteínas dos Microtúbulos/genética , Epitélio Pigmentado da Retina/patologia , Raios Ultravioleta/efeitos adversosRESUMO
Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression.
Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos/genética , Mitose , Proteômica/métodos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Marcação por Isótopo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , CoesinasRESUMO
Here, we review how DNA damage affects the centrosome and how centrosomes communicate with the DNA damage response (DDR) apparatus. We discuss how several proteins of the DDR are found at centrosomes, including the ATM, ATR, CHK1 and CHK2 kinases, the BRCA1 ubiquitin ligase complex and several members of the poly(ADP-ribose) polymerase family. Stereotypical centrosome organisation, in which two centriole barrels are orthogonally arranged in a roughly toroidal pericentriolar material (PCM), is strongly affected by exposure to DNA-damaging agents. We describe the genetic dependencies and mechanisms for how the centrioles lose their close association, and the PCM both expands and distorts after DNA damage. Another consequence of genotoxic stress is that centrosomes undergo duplication outside the normal cell cycle stage, meaning that centrosome amplification is commonly seen after DNA damage. We discuss several potential mechanisms for how centrosome numbers become dysregulated after DNA damage and explore the links between the DDR and the PLK1- and separase-dependent mechanisms that drive centriole separation and reduplication. We also describe how centrosome components, such as centrin2, are directly involved in responding to DNA damage. This review outlines current questions on the involvement of centrosomes in the DDR.
Assuntos
Centrossomo/metabolismo , Dano ao DNA , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mutations in breast cancer susceptibility gene BRCA1 (breast cancer early-onset 1) are associated with increased risk of developing breast and ovarian cancers. BRCA1 is a large protein of 1863 residues with two small structured domains at its termini: a RING domain at the N-terminus and a BRCT (BRCA1 C-terminus domain) repeat domain at the C-terminus. Previously, we quantified the effects of missense mutations on the thermodynamic stability of the BRCT domains, and we showed that many are so destabilizing that the folded functional state is drastically depopulated at physiological temperature. In the present study, we ask whether and how reduced thermodynamic stability of the isolated BRCT mutants translates into loss of function of the full-length protein in the cell. We assessed the effects of missense mutants on different stages of BRCA1-mediated DNA repair by homologous recombination using chicken lymphoblastoid DT40 cells as a model system. We found that all of the mutations, regardless of how profound their destabilizing effects, retained some DNA repair activity and thereby partially rescued the chicken BRCA1 knockout. By contrast, the mutation R1699L, which disrupts the binding of phosphorylated proteins (but which is not destabilizing), was completely inactive. It is likely that both protein context (location of the BRCT domains at the C-terminus of the large BRCA1 protein) and cellular environment (binding partners, molecular chaperones) buffer these destabilizing effects such that at least some mutant protein is able to adopt the folded functional state.
Assuntos
Reparo do DNA/fisiologia , Mutação de Sentido Incorreto/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Galinhas , Feminino , Humanos , Estabilidade Proteica , Estrutura Secundária de Proteína , Ubiquitina-Proteína Ligases/químicaRESUMO
Centrosome duplication is licensed by the disengagement, or 'uncoupling', of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the anaphase-promoting complex (APC/C), leading to securin degradation and release of active separase. Although APC/C activation during G2 arrest is dependent on polo-like kinase 1 (Plk1)-mediated degradation of the APC/C inhibitor, early mitotic inhibitor 1 (Emi1), Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulates centriole disengagement in response to hydroxyurea or DNA damage-induced cell-cycle arrest and this leads to centrosome amplification. However, the reduplication of disengaged centrioles is dependent on cyclin-dependent kinase 2 (Cdk2) activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Although release from these arrests leads to mitotic entry, the presence of disengaged and/or amplified centrosomes results in the formation of abnormal mitotic spindles that lead to chromosome mis-segregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability.
Assuntos
Pontos de Checagem do Ciclo Celular , Centrossomo/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Centríolos/efeitos dos fármacos , Centríolos/metabolismo , Centríolos/efeitos da radiação , Centrossomo/efeitos dos fármacos , Centrossomo/efeitos da radiação , Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Células HeLa , Humanos , Hidroxiureia/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Radiação Ionizante , Separase , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/efeitos da radiação , Quinase 1 Polo-LikeRESUMO
Centrins are small, highly conserved members of the EF-hand superfamily of calcium-binding proteins that are found throughout eukaryotes. They play a major role in ensuring the duplication and appropriate functioning of the ciliary basal bodies in ciliated cells. They have also been localised to the centrosome, which is the major microtubule organising centre in animal somatic cells. We describe the identification, cloning and characterisation of centrins in multiple eukaryotic species. Although centrins have been implicated in centriole biogenesis, recent results have indicated that centrosome duplication can, in fact, occur in the absence of centrins. We discuss these data and the non-centrosomal functions that are emerging for the centrins. In particular, we discuss the involvement of centrins in nucleotide excision repair, a process that repairs the DNA lesions that are induced primarily by ultraviolet irradiation. We discuss how centrin may be involved in these diverse processes and contribute to nuclear and cytoplasmic events.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Ciclo Celular/fisiologia , Centríolos/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Reparo do DNA , Genoma , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular/química , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Motivos EF Hand , Evolução Molecular , Humanos , Conformação Proteica , Fuso Acromático/genética , Vertebrados/metabolismoRESUMO
CEP164 recruits TTBK2 to the distal end of centrioles to allow primary cilium formation. In this issue of Structure, Rosa e Silva et al. (2022) present co-crystallized structures that show the molecular basis of this recruitment and define how ciliopathy mutations in CEP164 disrupt the CEP164-TTBK2 complex.
Assuntos
Cílios , Ciliopatias , Centríolos/genética , Ciliopatias/genética , Humanos , MutaçãoRESUMO
Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates. These aggregates are either directed towards autophagy for destruction or sequestered into an inclusion, termed the aggresome, at the centrosome. Utilizing high-resolution quantitative analysis, here, we define aggresome assembly at the centrosome in human cells. Centriolar satellites are proteinaceous granules implicated in the trafficking of proteins to the centrosome. During aggresome assembly, satellites were required for the growth of the aggresomal structure from an initial ring of phosphorylated HSP27 deposited around the centrioles. The seeding of this phosphorylated HSP27 ring depended on the centrosomal proteins CP110, CEP97 and CEP290. Owing to limiting amounts of CP110, senescent cells, which are characterized by the accumulation of protein aggregates, were defective in aggresome formation. Furthermore, satellites and CP110-CEP97-CEP290 were required for the aggregation of mutant huntingtin. Together, these data reveal roles for CP110-CEP97-CEP290 and satellites in the control of cellular proteostasis and the aggregation of disease-relevant proteins.
Assuntos
Centríolos , Agregados Proteicos , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1). PRDX1 reduces peroxides and coordinates the signaling actions of protein binding partners. Loss of PRDX1 inhibits irradiation-induced RAD51 foci formation and represses HR DNA repair. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. Following irradiation cells deficient in PRDX1 had increased incorporation of the sulfenylation probe DAz-2 in RAD51 Cys319, a functionally-significant, thiol that PRDX1 is critical for maintaining in a reduced state. Molecular dynamics (MD) simulations of dT-DNA bound to a non-oxidized RAD51 protein showed tight binding throughout the simulation, while dT-DNA dissociated from an oxidized Cys319 RAD51 filament. These novel data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Rad51 Recombinase , Difosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , DNA/metabolismo , Reparo do DNA , Fibroblastos/metabolismo , Recombinação Homóloga , Humanos , Camundongos , Oxirredução , Peróxidos , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RiboseRESUMO
The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.
Assuntos
Cromátides/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Animais , Antígenos Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Galinhas , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II , Histonas/análise , Histonas/metabolismo , Humanos , Autoantígeno Ku , Ovalbumina/genética , Proteínas Serina-Treonina Quinases/metabolismo , Rad51 Recombinase/análise , Proteínas de Saccharomyces cerevisiae , Proteínas Supressoras de Tumor/metabolismoRESUMO
Telomere repeat sequences are added to linear chromosome ends by telomerase, an enzyme comprising a reverse transcriptase (TERT) and an RNA template component (TR). We aimed to investigate TR in the DT40 B-cell tumour line using gene targeting, but were unable to generate TR nulls, suggesting a requirement for TR in DT40 proliferation. Disruption of one TR allele reduced telomerase activity and caused a progressive decline in telomere and G-strand overhang length. We then examined the interactions between TR and cellular DNA double-strand break (DSB) repair. Deletion in TR+/- cells of the gene encoding the non-homologous end-joining protein, Ku70, caused rapid loss of G-strand overhangs. Ku70-/-TR+/- cells proliferated more slowly than either single mutant and showed frequent mitotic aberrations. Activation of the DNA damage response was observed in TR-deficient cells and was exacerbated by Ku deficiency, although frequent telomeric DNA damage signals were not observed until late passages. This activation of the DNA damage response was suppressed by deletion of Rad54, a key homologous recombination gene. These findings suggest that Ku and telomerase cooperate to block homologous recombination from acting on telomeres.
Assuntos
Antígenos Nucleares/metabolismo , Galinhas/genética , Galinhas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , RNA/genética , Telomerase/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Aberrações Cromossômicas , Dano ao DNA , Heterozigoto , Autoantígeno Ku , RNA/metabolismo , Telomerase/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
The BRCA2 interactor, centrobin, is a centrosomal protein that has been implicated in centriole duplication and microtubule stability. We used genome editing to ablate CNTROB in hTERT-RPE1 cells and observed an increased frequency of monocentriolar and acentriolar cells. Using a novel monoclonal antibody, we found that centrobin primarily localizes to daughter centrioles but also associates with mother centrioles upon serum starvation. Strikingly, centrobin loss abrogated primary ciliation upon serum starvation. Ultrastructural analysis of centrobin nulls revealed defective axonemal extension after mother centriole docking. Ciliogenesis required a C-terminal portion of centrobin that interacts with CP110 and tubulin. We also depleted centrobin in zebrafish embryos to explore its roles in an entire organism. Centrobin-depleted embryos showed microcephaly, with curved and shorter bodies, along with marked defects in laterality control, morphological features that indicate ciliary dysfunction. Our data identify new roles for centrobin as a positive regulator of vertebrate ciliogenesis.