RESUMEN
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 carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its role in this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5' flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks.
Asunto(s)
Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Endonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Reparación del ADN por Recombinación/genética , Sitios de Unión/genética , Proteínas Portadoras/genética , Catálisis , Línea Celular , Supervivencia Celular/genética , ADN/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas , Endonucleasas/genética , Humanos , Proteínas Nucleares/genética , Fosforilación/genética , Procesamiento Proteico-Postraduccional/genética , Radiación Ionizante , Recombinación GenéticaRESUMEN
The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells-a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Complejos Multiproteicos/metabolismo , Línea Celular , Humanos , Imagen Individual de MoléculaRESUMEN
The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5' flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.
Asunto(s)
Endonucleasas/genética , Células Eucariotas/metabolismo , Recombinación Homóloga/genética , ARN Helicasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Catálisis , Roturas del ADN de Doble Cadena , Daño del ADN/genética , ADN Helicasas , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo I/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Humanos , Enzimas Multifuncionales , Saccharomyces cerevisiae/genéticaRESUMEN
Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries. Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery. RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination. Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties. The RecO structure is totally different from the Rad52 structure. DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain. Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair.
Asunto(s)
Proteínas Bacterianas/química , Reparación del ADN , Proteínas de Unión al ADN/química , Deinococcus/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteína Recombinante y Reparadora de ADN Rad52 , Alineación de SecuenciaRESUMEN
The mammalian CtIP protein and its orthologs in other eukaryotes promote the resection of DNA double-strand breaks and are essential for meiotic recombination. Here we review the current literature supporting the role of CtIP in DNA end processing and the importance of CtIP endonuclease activity in DNA repair. We also examine the regulation of CtIP function by post-translational modifications, and its involvement in transcription- and replication-dependent functions through association with other protein complexes. The tumor suppressor function of CtIP likely is dependent on a combination of these roles in many aspects of DNA metabolism.
Asunto(s)
Proteínas Portadoras/química , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN/metabolismo , Recombinación Homóloga , Proteínas Nucleares/química , Procesamiento Proteico-Postraduccional , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN/química , Roturas del ADN de Cadena Simple , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.
Asunto(s)
Reparación del ADN/genética , Endonucleasas/genética , Fosforilación/genética , Proteínas de Saccharomyces cerevisiae/genética , Autofagia/genética , Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Saccharomyces cerevisiae/genéticaRESUMEN
DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.
Asunto(s)
Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Puntos de Control de la Fase G1 del Ciclo Celular , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Endodesoxirribonucleasas , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Ratones , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteína de Replicación A/metabolismo , Translocación Genética , Proteínas Supresoras de TumorRESUMEN
RecF pathway proteins play an important role in the restart of stalled replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR, and RecO initiate homologous recombination (HR) by loading of the RecA recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein. The specific role of RecF in this process is not well understood. Previous studies have proposed that RecF directs the RecOR complex to boundaries of damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA junctions. RecF belongs to ABC-type ATPases, which function through an ATP-dependent dimerization. Here, we demonstrate that the RecF of Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer, and that the DNA binding and ATPase activity of RecF depend on both the structure of DNA substrate, and the presence of RecR. We found that RecR interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to ssDNA and dimerization, likely due to increasing the ATPase rate. The DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific protein-protein interactions without significant contributions from RecR-DNA interactions. Finally, RecF neither alone nor in complex with RecR preferentially binds to the ss/dsDNA junction. Our data suggest that the specificity of the RecFOR complex toward the boundaries of DNA damaged regions may result from a network of protein-protein and DNA-protein interactions, rather than a simple recognition of the ss/dsDNA junction by RecF.
Asunto(s)
Proteínas Bacterianas/metabolismo , Daño del ADN/fisiología , Reparación del ADN/fisiología , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Deinococcus/metabolismo , Adenosina Trifosfatasas/metabolismo , Replicación del ADN/fisiología , Dimerización , Complejos Multiproteicos/metabolismo , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína , Rec A Recombinasas/metabolismoRESUMEN
RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double-stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP-dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.