There are two major and alternative pathways to repair DNA double-strand breaks: non-homologous end-joining and homologous recombination. Here we identify and characterize novel factors involved in choosing between these pathways; in this study we took advantage of the SeeSaw Reporter, in which the repair of double-strand breaks by homology-independent or -dependent mechanisms is distinguished by the accumulation of green or red fluorescence, respectively. Using a genome-wide human esiRNA (endoribonuclease-prepared siRNA) library, we isolate genes that control the recombination/end-joining ratio. Here we report that two distinct sets of genes are involved in the control of the balance between NHEJ and HR: those that are required to facilitate recombination and those that favour NHEJ. This last category includes CCAR2/DBC1, which we show inhibits recombination by limiting the initiation and the extent of DNA end resection, thereby acting as an antagonist of CtIP.

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 overexpression.

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The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

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 0 UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP-at the level of mRNA stability through ALC1 and eIF4A1.

No abstract

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

RAD51 paralogs are a group of conserved proteins in eukaryotes that are involved in the repair of DNA breaks at several levels. On one hand, they help the strand invasion step catalyzed by RAD51. Also, they play late roles in Holliday Junction metabolism.Here we uncover a new role of the RAD51 paralogs at an earlier event in the repair of broken chromosomes. All five RAD51 paralogs affect the balance between double strand break repair pathways. Specifically, they favor homology-mediated repair over non-homologous end-joining. Such role is independent of RAD51 or the checkpoint activity of these proteins. Moreover, it defines a novel control point of double strand break repair independent and subsequent to DNA-end resection initiation.Mechanistically, RAD51 paralogs limit the retention of Ku80 at the sites of DNA breaks. Thus, our data extend the role of this family of proteins to the earliest event of double strand break repair.