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1.
Nat Rev Mol Cell Biol ; 22(12): 796-814, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34429537

RESUMO

The protein kinase ataxia telangiectasia mutated (ATM) is a master regulator of double-strand DNA break (DSB) signalling and stress responses. For three decades, ATM has been investigated extensively to elucidate its roles in the DNA damage response (DDR) and in the pathogenesis of ataxia telangiectasia (A-T), a human neurodegenerative disease caused by loss of ATM. Although hundreds of proteins have been identified as ATM phosphorylation targets and many important roles for this kinase have been identified, it is still unclear how ATM deficiency leads to the early-onset cerebellar degeneration that is common in all individuals with A-T. Recent studies suggest the existence of links between ATM deficiency and other cerebellum-specific neurological disorders, as well as the existence of broader similarities with more common neurodegenerative disorders. In this Review, we discuss recent structural insights into ATM regulation, and possible aetiologies of A-T phenotypes, including reactive oxygen species, mitochondrial dysfunction, alterations in transcription, R-loop metabolism and alternative splicing, defects in cellular proteostasis and metabolism, and potential pathogenic roles for hyper-poly(ADP-ribosyl)ation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/metabolismo , Doenças Neurodegenerativas/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Reparo do DNA , Homeostase , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Oxirredução , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , RNA/metabolismo
2.
Annu Rev Biochem ; 84: 711-38, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25580527

RESUMO

The ataxia-telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response, and it coordinates checkpoint activation, DNA repair, and metabolic changes in eukaryotic cells in response to DNA double-strand breaks and oxidative stress. Loss of ATM activity in humans results in the pleiotropic neurodegeneration disorder ataxia-telangiectasia. ATM exists in an inactive state in resting cells but can be activated by the Mre11-Rad50-Nbs1 (MRN) complex and other factors at sites of DNA breaks. In addition, oxidation of ATM activates the kinase independently of the MRN complex. This review discusses these mechanisms of activation, as well as the posttranslational modifications that affect this process and the cellular factors that affect the efficiency and specificity of ATM activation and substrate phosphorylation. I highlight functional similarities between the activation mechanisms of ATM, phosphatidylinositol 3-kinases (PI3Ks), and the other PI3K-like kinases, as well as recent structural insights into their regulation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Estresse Oxidativo , Fosfatidilinositol 3-Quinase/metabolismo , Processamento de Proteína Pós-Traducional
3.
Mol Cell ; 81(7): 1515-1533.e5, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33571423

RESUMO

Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Quebras de DNA de Cadeia Simples , Proteína Homóloga a MRE11/deficiência , Neocórtex/metabolismo , Poli ADP Ribosilação , Proteostase , Ataxias Espinocerebelares/metabolismo , Adulto , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Neocórtex/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia
4.
Mol Cell ; 75(1): 145-153.e5, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31153714

RESUMO

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.


Assuntos
DNA de Cadeia Simples/genética , Retroalimentação Fisiológica , RecQ Helicases/genética , Proteínas Recombinantes de Fusão/genética , Proteína de Replicação A/genética , Sítios de Ligação , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Recombinação Homóloga , Humanos , Microscopia de Fluorescência , Nucleossomos/química , Nucleossomos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação , Ligação Proteica , RecQ Helicases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula
5.
Genes Dev ; 33(23-24): 1751-1774, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31753913

RESUMO

Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.


Assuntos
Instabilidade Genômica , Estruturas R-Loop , Reparo de DNA por Recombinação/genética , Fatores de Transcrição/genética , Acetilação , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Células HEK293 , Células HeLa , Humanos , Transativadores/metabolismo , Fatores de Transcrição/análise , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
6.
Mol Cell ; 71(3): 419-427, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30057197

RESUMO

The Mre11 nuclease has been the subject of intensive investigation for the past 20 years because of the central role that Mre11/Rad50 complexes play in genome maintenance. The last two decades of work on this complex has led to a much deeper understanding of the structure, biochemical activities, and regulation of Mre11/Rad50 complexes from archaea, bacteria, and eukaryotic cells. This review will discuss some of the important findings over recent years that have illuminated roles for the Mre11 nuclease in these different contexts as well as the insights from structural biology that have helped us to understand its mechanisms of action.


Assuntos
Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/fisiologia , Hidrolases Anidrido Ácido , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/genética
7.
Mol Cell ; 71(2): 332-342.e8, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30017584

RESUMO

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA Ligase Dependente de ATP/genética , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku/genética , Camundongos
8.
Mol Cell ; 65(1): 91-104, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-27939942

RESUMO

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/metabolismo , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/enzimologia , Genótipo , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/genética , Estresse Oxidativo , Fenótipo , Fosforilação , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção
9.
Mol Cell ; 67(5): 891-898.e4, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28867292

RESUMO

DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/enzimologia , Reparo de DNA por Recombinação , Imagem Individual de Molécula , Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular/genética , Adutos de DNA/genética , Adutos de DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Difusão , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Proteína Homóloga a MRE11 , Microscopia de Fluorescência , Proteínas Nucleares/genética , Nucleossomos/genética , Fatores de Tempo
10.
Genes Dev ; 31(3): 260-274, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28242625

RESUMO

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.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias Ósseas/metabolismo , Cromatina/metabolismo , Reparo do DNA , Proteínas Nucleares/metabolismo , Osteossarcoma/metabolismo , Sequência de Aminoácidos , Proteína BRCA1/genética , Neoplasias Ósseas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Instabilidade Genômica , Células HEK293 , Chaperonas de Histonas , Histonas/genética , Histonas/metabolismo , Recombinação Homóloga , Humanos , Proteínas Nucleares/genética , Osteossarcoma/genética , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas
11.
Mol Cell ; 64(3): 593-606, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27814491

RESUMO

The human Mre11/Rad50/Nbs1 (hMRN) complex is critical for the sensing, processing, and signaling of DNA double-strand breaks. The nuclease activity of Mre11 is essential for mammalian development and cell viability, although the regulation and substrate specificity of Mre11 have been difficult to define. Here we show that hMRN catalyzes sequential endonucleolytic and exonucleolytic activities on both 5' and 3' strands of DNA ends containing protein adducts, and that Nbs1, ATP, and adducts are essential for this function. In contrast, Nbs1 inhibits Mre11/Rad50-catalyzed 3'-to-5' exonucleolytic degradation of clean DNA ends. The hMRN endonucleolytic cleavage events are further stimulated by the phosphorylated form of the human C-terminal binding protein-interacting protein (CtIP) DNA repair enzyme, establishing a role for CtIP in regulating hMRN activity. These results illuminate the important role of Nbs1 and CtIP in determining the substrates and consequences of human Mre11/Rad50 nuclease activities on protein-DNA lesions.


Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Adutos de DNA/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Hidrolases Anidrido Ácido , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adutos de DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteína Homóloga a MRE11 , Mutação , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Transdução de Sinais , Spodoptera , Especificidade por Substrato
12.
Mol Cell ; 64(3): 580-592, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27814490

RESUMO

The Mre11/Rad50/Nbs1 complex initiates double-strand break repair by homologous recombination (HR). Loss of Mre11 or its nuclease activity in mouse cells is known to cause genome aberrations and cellular senescence, although the molecular basis for this phenotype is not clear. To identify the origin of these defects, we characterized Mre11-deficient (MRE11-/-) and nuclease-deficient Mre11 (MRE11-/H129N) chicken DT40 and human lymphoblast cell lines. These cells exhibit increased spontaneous chromosomal DSBs and extreme sensitivity to topoisomerase 2 poisons. The defects in Mre11 compromise the repair of etoposide-induced Top2-DNA covalent complexes, and MRE11-/- and MRE11-/H129N cells accumulate high levels of Top2 covalent conjugates even in the absence of exogenous damage. We demonstrate that both the genome instability and mortality of MRE11-/- and MRE11-/H129N cells are significantly reversed by overexpression of Tdp2, an enzyme that eliminates covalent Top2 conjugates; thus, the essential role of Mre11 nuclease activity is likely to remove these lesions.


Assuntos
Antígenos de Neoplasias/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas Nucleares/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Fatores de Transcrição/genética , Hidrolases Anidrido Ácido , Animais , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Galinhas , DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Regulação da Expressão Gênica , Instabilidade Genômica/efeitos dos fármacos , Humanos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Proteína Homóloga a MRE11 , Mutação , Proteínas Nucleares/metabolismo , Diester Fosfórico Hidrolases , Proteínas de Ligação a Poli-ADP-Ribose , Transdução de Sinais , Inibidores da Topoisomerase II/farmacologia , Fatores de Transcrição/metabolismo
13.
PLoS Biol ; 18(7): e3000606, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687490

RESUMO

The 70 kDa heat shock protein (HSP70) family of chaperones are the front line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here, we demonstrate quantitative identification of HSP70 and 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides, but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (SOD1) mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-activated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteoma/metabolismo , Linhagem Celular , Humanos , Mutação/genética , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , Especificidade por Substrato , Ubiquitina/metabolismo
14.
EMBO Rep ; 22(1): e50500, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33245190

RESUMO

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function.


Assuntos
Aldeído Oxirredutases , Mitofagia , Aldeído Oxirredutases/metabolismo , Senescência Celular , Oxirredução , Estresse Oxidativo/genética
15.
Cell ; 135(2): 250-60, 2008 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-18957200

RESUMO

The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes.


Assuntos
Proteínas Arqueais/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Pyrococcus furiosus/metabolismo , Proteínas Arqueais/isolamento & purificação , DNA/metabolismo , Endodesoxirribonucleases/isolamento & purificação , Exodesoxirribonucleases/isolamento & purificação , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Mapeamento de Interação de Proteínas , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética
16.
Mol Cell ; 54(6): 1022-1033, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24837676

RESUMO

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.


Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação/genética , Sítios de Ligação/genética , Proteínas de Transporte/genética , Catálise , Linhagem Celular , Sobrevivência Celular/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases , Endonucleases/genética , Humanos , Proteínas Nucleares/genética , Fosforilação/genética , Processamento de Proteína Pós-Traducional/genética , Radiação Ionizante , Recombinação Genética
17.
Crit Rev Biochem Mol Biol ; 54(4): 371-384, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31577154

RESUMO

The repair of DNA double-strand breaks occurs through a series of defined steps that are evolutionarily conserved and well-understood in most experimental organisms. However, it is becoming increasingly clear that repair does not occur in isolation from other DNA transactions. Transcription of DNA produces topological changes, RNA species, and RNA-dependent protein complexes that can dramatically influence the efficiency and outcomes of DNA double-strand break repair. The transcription-associated history of several double-strand break repair factors is reviewed here, with an emphasis on their roles in regulating R-loops and the emerging role of R-loops in coordination of repair events. Evidence for nucleolytic processing of R-loops is also discussed, as well as the molecular tools commonly used to measure RNA-DNA hybrids in cells.


Assuntos
Reparo do DNA/genética , DNA/genética , Estruturas R-Loop/genética , RNA/genética , Transcrição Gênica , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Helicases/genética , Endodesoxirribonucleases/genética , Humanos , RNA Helicases/genética , Recombinação Genética
19.
Nucleic Acids Res ; 45(9): 5255-5268, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28369545

RESUMO

The Mre11-Rad50-Nbs1(Xrs2) (MRN/X) complex is critical for the repair and signaling of DNA double strand breaks. The catalytic core of MRN/X comprised of the Mre11 nuclease and Rad50 adenosine triphosphatase (ATPase) active sites dimerizes through association between the Rad50 ATPase catalytic domains and undergoes extensive conformational changes upon ATP binding. This ATP-bound 'closed' state promotes binding to DNA, tethering DNA ends and ATM activation, but prevents nucleolytic processing of DNA ends, while ATP hydrolysis is essential for Mre11 endonuclease activity at blocked DNA ends. Here we investigate the regulation of ATP hydrolysis as well as the interdependence of the two functional active sites. We find that double-stranded DNA stimulates ATP hydrolysis by hMRN over ∼20-fold in an end-dependent manner. Using catalytic site mutants to create Rad50 dimers with only one functional ATPase site, we find that both ATPase sites are required for the stimulation by DNA. MRN-mediated endonucleolytic cleavage of DNA at sites of protein adducts requires ATP hydrolysis at both sites, as does the stimulation of ATM kinase activity. These observations suggest that symmetrical engagement of the Rad50 catalytic head domains with ATP bound at both sites is important for MRN functions in eukaryotic cells.


Assuntos
Domínio Catalítico , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Hidrolases Anidrido Ácido , Trifosfato de Adenosina/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Humanos , Hidrólise , Complexos Multiproteicos/metabolismo , Ligação Proteica , Multimerização Proteica
20.
Proc Natl Acad Sci U S A ; 113(9): E1170-9, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26884156

RESUMO

Exonuclease 1 (Exo1) is a 5'→3' exonuclease and 5'-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.


Assuntos
Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Exodesoxirribonucleases/fisiologia , Biocatálise , Dano ao DNA , Humanos , Saccharomyces cerevisiae/metabolismo
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