ALC1/eIF4A1-mediated regulation of CtIP mRNA stability controls DNA end resection

During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 5' UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP-at the level of mRNA stability through ALC1 and eIF4A1

The Helicase PIF1 Facilitates Resection over Sequences Prone to Forming G4 Structures

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpressio

Non-canonical DNA structures in Double Strand Break repair

DNA double-strand breaks (DSBs) pose a serious threat to genome stability, so their proper repair is crucial for cell viability. To repair them, the cell has evolved two main alternative mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR). The correct choice between these two pathways is a key point to obtain a faithful restoration of the broken DNA sequence. One of best-known events regulating this decision is the generation of 3’ single-stranded DNA overhangs in a process known as DNA resection. Many different factors are involved in this process such as cell stemness, chromatin status or DNA conformation. In this Thesis, we investigated the role of G-quadruplexes, a noncanonical DNA structure, in DNA resection. First, we studied the impact of G-quadruplexes in this process and showed that DNA resection is impaired in the presence of these structures, both in vivo and in vitro. In addition, we demonstrated that PIF1α helicase activity unwinding G-quadruplexes promotes resection over these structures and that this process requires other factors such as BRCA1 and TOP2β. Furthermore, we took advantage of the key role of factors modulating DNA resection to regulate homologous recombination efficiency to search for new compounds that could affect CRISPR-Cas9 mediated homologous recombination efficiency.Los cortes que afectan a las dos cadenas del ADN suponen una seria amenaza a la estabilidad del genoma por lo que su reparación es crucial para la viabilidad celular. Para repararlos, la célula ha desarrollado principalmente dos mecanismos, la unión de extremos no homólogos (NHEJ) y la recombinación homóloga (HR). La correcta elección entre ambas rutas es un punto clave para conseguir restauran fielmente la secuencia de ADN dañada. Uno de los eventos más conocidos que regulan esta decisión es la generación de fibras de ADN de cadena sencilla colgantes en el extremo 3’ del corte de ADN en un proceso conocido como resección del ADN. Existen muchos factores involucrados en este proceso tal y como el grado de diferenciación de la célula, el estado de la cromatina o la conformación del ADN. En esta Tesis, se ha investigado el papel de los G-cuadruplexes, una estructura no canónica del ADN, en la resección del ADN. En primer lugar, se estudió el impacto de los G-cuadruplexes en este proceso y se ha mostrado que la resección del ADN se encuentra perjudicada en presencia de estas estructuras, tanto in vivo como in vitro. Además, se ha demostrado que la actividad helicasa de PIF1α en la resolución de los Gcuadruplexes promueve la resección a través de estas estructuras y que este proceso requiere de otros factores como BRCA1 y TOP2β. Asimismo, aprovechamos la importancia de la modulación de la resección del ADN por distintos factores en la regulación la eficiencia de la recombinación homóloga para buscar new compuestos que puedan influir en la eficiencia de la recombinación homóloga mediada por CRISPR-Cas9