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

Description

DNA 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 endjoining (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 or hampering it. Among all these CtIP interactors, we focused on PRMT5 and CCAR2 and show that they play opposite roles in regulating DNA end resection. PRMT5 promotes initiation and stimulates processivity of DNA end resection, and it is required for survival to DSB-inducing agents. Otherwise, CCAR2 acts as an antagonist of CtIP by impairing resection initiation and limiting the extent of DNA that is resected, becoming an important 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.

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