Characterisation of DNA-protein crosslink repair with the Purification of x-linked Proteins technique

Weickert Vivancos, Pedro (2024): Characterisation of DNA-protein crosslink repair with the Purification of x-linked Proteins technique. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

DNA-Protein Crosslinks (DPCs) have emerged as an important source of endogenous and exogenous DNA damage. DPCs arise when proteins get covalently trapped on DNA, what can happen during the action of enzymes that naturally establish covalent intermediates with DNA -such as topoisomerases- but also by the action of reactive compounds. One of these compounds, formaldehyde, is an environmental toxin and a metabolite produced during one-carbon metabolism and methanol detoxification. Formaldehyde is extremely reactive and generates protein-protein crosslinks, RNA-protein crosslinks and DPCs. DPCs are toxic because they can block essential chromatin transactions such as replication or transcription. Their toxicity has been exploited in chemotherapy with the use of different drugs that induce these genomic lesions either specifically, camptothecin (CPT) traps TOP1 or etoposide traps TOP2, or unspecifically, with compounds like cisplatin commonly used in breast cancer treatment. DPC-repair generally involves specialized proteases which mediate the destruction of the protein adduct. In higher eukaryotes, the metalloprotease SPRTN is recruited to the lesion in a process that involves the collision of the replication fork with the adduct. SPRTN is activated by the DNA structure generated by polymerase stalling, cleaves the protein and allows peptide bypass by translesion synthesis (TLS). The study of DPC-repair and the identification of DPCs generated by non-specific crosslinkers has remained elusive due to limited methodology to study these lesions. Previous methods to isolate DPCs, rely on DNA precipitation and are prone to technical biases due to the presence of protein aggregates, giving false positives, or changes in DNA precipitation behaviour due to the variable crosslinked protein nature, giving false negatives. Therefore, I established during my master thesis, and optimized during my PhD, a method for the isolation, identification and monitoring of DPCs, the Purification of x-linked Proteins (PxP). Using PxP in combination with mass spectrometry, I uncovered the identity of DPCs generated by physiologically relevant levels of formaldehyde. Strikingly, formaldehyde-induced DPCs are less complex than anticipated, they mostly consist of crosslinked nucleosomes. Then, we decided to apply PxP for the study of 5-azadC-induced DPCs, which consists mostly of the DNA Methyl Transferase 1 (DNMT1). We observed DNMT1-DPC formation in a dose-dependent manner and could monitor their repair kinetics. We discovered that repair of this crosslinks involved the SUMOylation of the adduct and SUMO-targeted ubiquitylation by the StUbL RNF4, which triggered proteasomal degradation of crosslinked DNMT1. In agreement with biochemical data and previous work, RNF4 knock-out cells displayed 5-azadC sensitivity. Interestingly, we observed a cleaved DNMT1-DPC fragment whose appearance was dependent on the same modifications -SUMO and ubiquitin- but not generated by the proteasome. We identified SPRTN as the enzyme responsible for this cleavage and, through structure function analysis, conclude that the UBZ domain is responsible for the recruitment of SPRTN to DNMT1-DPCs. Strikingly, only the loss of the UBZ domain completely phenocopied the SPRTN-ΔC allele, which is causative for Ruijs-Aalfs syndrome. Our data support a replication-independent role for the metalloprotease SPRTN during DPC-repair and highlight the importance of the UBZ domain during lesion recognition. Given the multiple applications of the PxP for the study of DPC biology, we decided to write an article in which we described the method step by step to facilitate its implementation in other labs. We show that the PxP can be used to isolate also other types of DPCs such as those generated by non-competitive inhibitors (e.g etoposide) and that it can successfully isolate relevant physiological DPCs, like those generated by HMCES after short wavelength ultraviolet light (UVC) exposure. In conclusion, we believe that this manuscript will be a great resource for laboratories which do research on the DNA damage field.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 10. Mai 2024
• 1. Bericht­erstatter:in: Stingele, Julian