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1.
Nucleic Acids Res ; 52(2): 525-547, 2024 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-38084926

RESUMEN

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.


Asunto(s)
Daño del ADN , Proteínas , Animales , Proteínas/genética , ADN/genética , ADN/metabolismo , Replicación del ADN , Reparación del ADN , Mamíferos/genética
2.
Nat Cell Biol ; 24(1): 62-73, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-35013556

RESUMEN

Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.


Asunto(s)
Cromatina/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteína que Contiene Valosina/metabolismo , Línea Celular Tumoral , Disulfiram/análogos & derivados , Disulfiram/farmacología , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Sumoilación , Factores de Transcripción/metabolismo , Ubiquitinación
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