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Identificación de nuevas proteínas que interaccionan con CtlP: Implicación de PRMT5 y CCAR2 en la resección del ADN

AuthorsLópez-Saavedra, Ana
AdvisorHuertas Sánchez, Pablo
Issue Date2017
PublisherUniversidad de Sevilla
AbstractDNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesion that can arise from either endogenous and exogenous genotoxic stresses. There are two major and alternative pathways to repair DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). The choice between these repair mechanisms is highly regulated in order to ensure an accurate repair and maintenance of genomic stability. The best-known regulatory step is DNA end resection, which leads to repair by HR whereas inhibits NHEJ. DNA end resection is an evolutionary conserved process that consists in 5’-3’ degradation of each DNA end, giving rise to 3’ ssDNA tails. CtIP is a key factor involved in initiation of end resection in humans, acting as a hub that integrates several cellular and environmental cues through different post-translational modifications and protein-protein interactions. Thus, in order to identify additional factors that regulate CtIP resection role, we performed Tandem Affinity Purification assay using U2OS cells expressing a double-tagged (GFP and FLAG) CtIP fusion. We isolated 10 new CtIP constitutive binding proteins, most of them with an effect in DNA end resection, either by stimulating it (CCAR2) or hampering it (PRMT5, SF3B3, DHX15, DHX9 and KIF11). Among all these novel CtIP interactors, we focused on PRMT5 and CCAR2, whose depletion show opposite phenotypes in regulating DNA end resection, and analysed their roles in DSB repair through the association with CtIP. PRMT5, like CtIP, promotes initiation and stimulates processivity of DNA end resection, thus favouring the repair by homologous recombination. In addition, PRMT5 is required for survival to DSB-inducing agents, such as ionizing radiation and camptothecin, but not etoposide. Otherwise, CCAR2 acts as an antagonist of CtIP by impairing resection initiation and limiting the extent of DNA that is resected. Although CCAR2 can localize at DSBs, the interaction between CCAR2 and CtIP occurs mainly outside of damaged chromatin and in a damage-independent manner. CCAR2 recruitment is mediated by ATM activity and phosphorylation of threonine-454 of CCAR2 by ATM is required for its function as regulator of the length of resection tracts. Despite affecting DNA end resection in different ways, depletion of either PRMT5 or CCAR2 disturbs the balance between NHEJ and HR for the repair of DSBs, suggesting that both proteins are essentials for preserving genomic integrity.
DescriptionPrograma Oficial de Doctorado en Biología Molecular y Biomedicina.
Appears in Collections:(CABIMER) Tesis
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