Genetic and proteomic study of ercc4/ xpf in dna repair and human diseases
- Autores: Maria Marín Vilar
- Directores de la Tesis: Jordi Surrallés Calonge (dir. tes.), Massimo Bogliolo (codir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2018
- Idioma: español
- Tribunal Calificador de la Tesis: Valeria Naim (presid.), Laia Castells Roca (secret.), Pau Castillo Bosch (voc.)
- Programa de doctorado: Programa de Doctorado en Genética por la Universidad Autónoma de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
A change in an organism’s DNA can affect all the aspects of its life, until the point of compromising it. To overcome this, cells have evolved sophisticated machineries of DNA damage repair. Thus, our DNA contains the necessary information to produce proteins that participate in damage recognition, binding, excision and in the reestablishment of correct genetic information. One of these proteins is the XPF endonuclease, which is the catalytic subunit of the stable heterodimer XPF-ERCC1, able to incise at the 5’ side at different DNA damages. This essential protein is encoded by ERCC4 gene and participates in multiple genome maintenance pathways including nucleotide excision repair (NER), interstrand crosslink (ICL) repair, double strand break (DSB) repair pathways such as microhomology-mediated end joining (MMEJ) and single strand annealing (SSA). XPF has also been suggested to have possible backup roles in repairing oxidative damage and in telomere maintenance besides to have a role in the response of cancer cells to chemotherapy. Considering its wide involvement in multiple DNA repair pathways, it is not surprising that ERCC4 mutations are associated to a range of human diseases including Xeroderma Pigmentosum (XP), Segmental Progeria (XFE), Fanconi Anemia (FA), Cockayne Syndrome (CS) and several cases combined diseases of Xeroderma and Cockayne syndromes (XPCSCD). A better understanding of (i) the correlation between the pathogenic mutations and patients’ phenotype and (ii) the essential DNA repair mechanisms is expected to promote a faster development of possible treatments. Here we report a detailed overview of functional studies performed with a set of cells containing pathogenic XPF mutations in a genetically homogeneous background. The selected XPF mutants, located in different domains of the protein, and the resulting human syndromes, were the following: XPFR153P (XFE), XPFI225M (XP), XPFL230P (FA), XPFC236R (CS), XPFR589W (XP/XPCSCD), XPFR689S (FA), XPFR799W (XP/XFE-CS). Detailed functional studies include the analyses of NER pathway (UVC sensitivity, UDS and RRS) and ICLR pathway (ICL sensitivity, ICL-induced G2/M arrest and ICL-induced chromosome fragility). Our results emphasize the importance of other factors, beyond protein position of the variant, such as protein levels, cell localization and the molecular interactions, in order to associate any XPF mutant to a clinical phenotype. In this framework, and to increase our knowledge about XPF interactions that can regulate XPF functions in the distinct DNA repair pathways, we investigated the XPF interactome. The implementation of the most advanced proteomic techniques including tandem affinity purification, co-immunoprecipitation and SILAC, coupled to mass spectrometry led us to identify a new XPF interactor involved in genome maintenance: USP11, a deubiquitinase that is known to regulate the activity of functionally related proteins such as BRCA2 or XPC. Here we prove XPF-USP11 interaction occurs irrespective of DNA damage and is DNA–independent. We also demonstrate that USP11 regulates DSB repair by SSA and possibly NER but is not involved in HR or ICLR.
