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1.
Mol Cell ; 83(23): 4290-4303.e9, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37951216

RESUMO

Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.


Assuntos
RNA , Ubiquitina , Humanos , RNA/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Aldeídos , Biossíntese de Proteínas
2.
Mol Cell ; 82(5): 889-890, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-35245455

RESUMO

Krastev et al. (2022) identify a cellular mechanism that counteracts cytotoxic trapping of PARP1 induced by clinical PARP inhibitors. SUMO-targeted ubiquitylation of trapped PARP1 is shown to trigger the enzymes' extraction from chromatin by the p97 ATPase.


Assuntos
Cromatina , Inibidores de Poli(ADP-Ribose) Polimerases , Cromatina/genética , Extratos Vegetais , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ubiquitinação/efeitos dos fármacos
3.
EMBO J ; 43(12): 2397-2423, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38760575

RESUMO

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Decitabina , Ubiquitina-Proteína Ligases , Decitabina/farmacologia , Humanos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Metilação de DNA/efeitos dos fármacos , Antimetabólitos Antineoplásicos/farmacologia , Animais , Sumoilação/efeitos dos fármacos
4.
Mol Cell ; 80(1): 102-113.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853547

RESUMO

Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.


Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , Linhagem Celular , Proteínas de Ligação a DNA/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Domínios Proteicos , Relação Estrutura-Atividade
5.
Nucleic Acids Res ; 49(2): 902-915, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33348378

RESUMO

Repair of covalent DNA-protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/fisiologia , Ubiquitinação , Catálise , Linhagem Celular , Cromatina/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Enzimas Desubiquitinantes/metabolismo , Técnicas de Inativação de Genes , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Peptidase 7 Específica de Ubiquitina/fisiologia
6.
Nat Protoc ; 19(10): 2891-2914, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38890499

RESUMO

Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples.


Assuntos
DNA , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Reagentes de Ligações Cruzadas/química , Humanos , Animais , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteínas/química , Eletroforese em Gel de Poliacrilamida/métodos
7.
Nat Cell Biol ; 26(5): 797-810, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38600235

RESUMO

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.


Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Adutos de DNA/metabolismo , Adutos de DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo por Excisão , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição , Transcrição Gênica , Ubiquitinação , Raios Ultravioleta
8.
Nat Commun ; 14(1): 352, 2023 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-36681662

RESUMO

DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA , Animais , Humanos , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mamíferos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo
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