RESUMO
DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.
Assuntos
Dano ao DNA , Proteínas , Animais , Proteínas/genética , DNA/genética , DNA/metabolismo , Replicação do DNA , Reparo do DNA , Mamíferos/genéticaRESUMO
The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.
Assuntos
Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/química , Instabilidade Genômica , Sequência de Aminoácidos , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Etoposídeo/química , Formaldeído/química , Expressão Gênica , Humanos , Cinética , Mutação , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Síndrome , Raios UltravioletaRESUMO
The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.
Assuntos
Adenosina Trifosfatases/metabolismo , Ataxina-3/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Homeostase , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatases/genética , Ataxina-3/genética , Sobrevivência Celular , Cromatina/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
We demonstrate a role for protein kinase casein kinase 2 (CK2) in the phosphorylation and regulation of the M3-muscarinic receptor in transfected cells and cerebellar granule neurons. On agonist occupation, specific subsets of receptor phosphoacceptor sites (which include the SASSDEED motif in the third intracellular loop) are phosphorylated by CK2. Receptor phosphorylation mediated by CK2 specifically regulates receptor coupling to the Jun-kinase pathway. Importantly, other phosphorylation-dependent receptor processes are regulated by kinases distinct from CK2. We conclude that G protein-coupled receptors (GPCRs) can be phosphorylated in an agonist-dependent fashion by protein kinases from a diverse range of kinase families, not just the GPCR kinases, and that receptor phosphorylation by a defined kinase determines a specific signalling outcome. Furthermore, we demonstrate that the M3-muscarinic receptor can be differentially phosphorylated in different cell types, indicating that phosphorylation is a flexible regulatory process where the sites that are phosphorylated, and hence the signalling outcome, are dependent on the cell type in which the receptor is expressed.
Assuntos
Caseína Quinase II/fisiologia , Receptor Muscarínico M3/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Caseína Quinase II/antagonistas & inibidores , Células Cultivadas , Sequência Consenso , Cricetinae , Cricetulus , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptor Muscarínico M3/química , Transdução de Sinais , Quinases de Receptores Adrenérgicos beta/metabolismoRESUMO
The ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5'-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.
Assuntos
Adenosina Trifosfatases/metabolismo , Morte Celular/genética , DNA/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Radiação Ionizante , HumanosRESUMO
The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/metabolismo , Tolerância a Radiação , Neoplasias da Bexiga Urinária/radioterapia , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Proteínas de Ligação a DNA/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Células HEK293 , Humanos , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Especificidade por Substrato , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
Changes in synaptic strength mediated by ionotropic glutamate N-methyl-D-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the G(q/11)-coupled M(3)-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M(3)-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity.
Assuntos
Cerebelo/metabolismo , Receptor Muscarínico M3/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Cerebelo/citologia , Retroalimentação Fisiológica , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , N-Metilaspartato/farmacologia , Plasticidade Neuronal , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositóis/metabolismo , Fosforilação , Receptor Muscarínico M3/química , Receptor Muscarínico M3/deficiência , Receptor Muscarínico M3/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate-a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)-is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3' phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.
Assuntos
Adenosina Trifosfatases/metabolismo , Adutos de DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Reparo do DNA , Replicação do DNA , Epistasia Genética , Humanos , Proteínas de Membrana/química , Diester Fosfórico Hidrolases/metabolismo , Proteína SUMO-1/metabolismo , SumoilaçãoRESUMO
The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop.
Assuntos
Quinase 1 do Ponto de Checagem/fisiologia , Proteínas de Ligação a DNA/fisiologia , Modelos Genéticos , Animais , Quinase 1 do Ponto de Checagem/metabolismo , Quebras de DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Fosforilação , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
DNA double strand breaks (DSBs) are the most cytotoxic DNA lesions and, if not repaired, lead to chromosomal rearrangement, genomic instability and cell death. Cells have evolved a complex network of DNA repair and signalling molecules which promptly detect and repair DSBs, commonly known as the DNA damage response (DDR). The DDR is orchestrated by various post-translational modifications such as phosphorylation, methylation, ubiquitination or SUMOylation. As DSBs are located in complex chromatin structures, the repair of DSBs is engineered at two levels: (i) at sites of broken DNA and (ii) at chromatin structures that surround DNA lesions. Thus, DNA repair and chromatin remodelling machineries must work together to efficiently repair DSBs. Here, we summarize the current knowledge of the ubiquitin-dependent molecular unfoldase/segregase p97 (VCP in vertebrates and Cdc48 in worms and lower eukaryotes) in DSB repair. We identify p97 as an essential factor that regulates DSB repair. p97-dependent extraction of ubiquitinated substrates mediates spatio-temporal protein turnover at and around the sites of DSBs, thus orchestrating chromatin remodelling and DSB repair. As p97 is a druggable target, p97 inhibition in the context of DDR has great potential for cancer therapy, as shown for other DDR components such as PARP, ATR and CHK1.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Proteína com Valosina/metabolismo , Animais , HumanosRESUMO
The G-protein coupled receptor (GPCR) gene family represents one of the largest families in the mammalian genome. The flexibility of signalling and widespread tissue distribution of these receptors has allowed GPCRs to be employed in the physiological regulation of nearly all biological functions. This, coupled with the fact that it is possible to chemically produce highly specific ligands to these receptors have made GPCRs attractive targets for pharmacological intervention in a wide variety of disease states. When targeting GPCRs in therapeutic drug design it is traditional, and eminently sensible, to focus on ligands that will provide agonism, antagonism or allosteric modulation. However, as more is understood of the mechanisms that regulate GPCRs, and in particular the dynamic covalent modifications that might endow tissue specific functions, then these regulatory processes may provide alternative targets for GPCR drug discovery. In this review we consider three of the covalent modifications which are considered to regulate the function of GPCRs namely; receptor phosphorylation, palmitoylation and ubiquitination. In particular, we will describe the mechanisms of modification, the functional consequences and the relationship between these three covalent modification events.