RESUMEN
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.
Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/química , Línea Celular , Proteínas de Unión al ADN/química , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina/fisiología , Ubiquitinación , Catálisis , Línea Celular , Cromatina/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/química , Enzimas Desubicuitinizantes/metabolismo , Técnicas de Inactivación de Genes , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Peptidasa Específica de Ubiquitina 7/fisiologíaRESUMEN
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.
Asunto(s)
Síndrome de Cockayne , ADN Helicasas , Enzimas Reparadoras del ADN , Reparación del ADN , Proteínas de Unión a Poli-ADP-Ribosa , ARN Polimerasa II , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patología , Aductos de ADN/metabolismo , Aductos de ADN/genética , Daño del ADN , ADN Helicasas/metabolismo , ADN Helicasas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Reparación por Escisión , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Receptores de Interleucina-17 , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Factores de Transcripción , Transcripción Genética , Ubiquitinación , Rayos UltravioletaRESUMEN
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.