RESUMO
SUMO conjugation is known to occur in response to double-stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3-specific peptidase, SENP7, interacts with the chromatin repressive KRAB-associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA-damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.
Assuntos
Cromatina/metabolismo , Endopeptidases/metabolismo , Reparo de DNA por Recombinação , Motivos de Aminoácidos , Sequência de Aminoácidos , Montagem e Desmontagem da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Helicases/metabolismo , Endopeptidases/química , Células HEK293 , Células HeLa , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteína SUMO-1/metabolismoRESUMO
The opposing activities of 53BP1 and BRCA1 influence pathway choice in DNA double-strand-break repair. How BRCA1 counteracts the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2â¼ubiquitin and demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitination by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1-deficient cells. BRCA1-BARD1's function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin and optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning, and the need for SMARCAD1 in olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus, BRCA1-BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair.
Assuntos
Proteína BRCA1/genética , Cromatina/metabolismo , DNA Helicases/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteína BRCA1/metabolismo , Sítios de Ligação , Camptotecina/farmacologia , Cromatina/química , Cromatina/efeitos dos fármacos , Clonagem Molecular , Quebras de DNA de Cadeia Dupla , Clivagem do DNA/efeitos dos fármacos , DNA Helicases/metabolismo , DNA de Neoplasias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Ftalazinas/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
AIMS: In cancer therapy, research has focused on the development of nanocarriers that can aid diagnosis, deliver therapeutic agents and monitor treatment progress. This study introduces high-resolution synchrotron x-ray fluorescence microscopy (SR-XFM) to investigate intracellular localization of novel lanthanide-coated nanoparticles in human cells and their genotoxicity screening after internalization. MATERIALS & METHODS: Noble metal nanoparticles coated with cerium and luminescent europium complexes have been developed as platforms for bioimaging and potential biodelivery applications. The intracellular distribution after internalization has been analyzed by ultrasensitive SR-XFM and genotoxicity evaluated using γ-H2AX DNA damage foci phosphorylation assay. RESULTS: We demonstrate the unprecedented capability of SR-XFM for extremely sensitive nanoimaging and intracellular elemental distribution analysis of noble metal nanoparticles in cells. Furthermore, we show that, depending on the charge of the coating complex and the presence of the DNA cargo, the internalization of functionalized nanoparticles by human fibroblasts can cause elevated levels of DNA damage detected by histone H2AX phosphorylation. CONCLUSION: The variable genotoxic impact of newly designed nanovectors emphasizes the need for careful and comprehensive testing of biological responses of all new nanoconstructs intended for future clinical applications. This can be greatly facilitated by SR-XFM nanoimaging of nanoparticles in cells at very low concentrations.
Assuntos
Dano ao DNA/fisiologia , Diagnóstico por Imagem/métodos , Elementos da Série dos Lantanídeos/química , Nanopartículas Metálicas/química , Microscopia de Fluorescência/métodos , Síncrotrons , Linhagem Celular , Dano ao DNA/genética , Histonas/metabolismo , Humanos , FosforilaçãoRESUMO
Meckel-Gruber syndrome (MKS) is an autosomal recessive lethal malformation syndrome characterized by renal cystic dysplasia, central nervous system malformations (typically, posterior occipital encephalocele), and hepatic developmental defects. Two MKS genes, MKS1 and MKS3, have been identified recently. The present study describes the cellular, sub-cellular and functional characterization of the novel proteins, MKS1 and meckelin, encoded by these genes. In situ hybridization studies for MKS3 in early human embryos showed transcript localizations in agreement with the tissue phenotype of MKS patients. Both MKS proteins predominantly localized to epithelial cells, including proximal renal tubules and biliary epithelial cells. MKS1 localized to basal bodies, while meckelin localized both to the primary cilium and to the plasma membrane in ciliated cell-lines and primary cells. Meckelin protein with the Q376P missense mutation was unable to localize at the cell membrane. siRNA-mediated reduction of Mks1 and Mks3 expression in a ciliated epithelial cell-line blocked centriole migration to the apical membrane and consequent formation of the primary cilium. Co-immunoprecipitation experiments show that wild-type meckelin and MKS1 interact and, in three-dimensional tissue culture assays, epithelial branching morphogenesis was severely impaired. These results suggest that MKS proteins mediate a fundamental developmental stage of ciliary formation and epithelial morphogenesis.