A genome-wide screening uncovers the role of CCAR2 as an antagonist of DNA end resection

Abstract

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.

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

“…We reasoned that, as for almost every single DNA transaction, helicases would be in charge to reshape such unusual DNA configurations to facilitate the process. To find those helicases, we used an indirect approach and took advantage of the SeeSaw Reporter (SSR) (see Figure 1A, left) and the genome-wide screening we recently published (Gomez-Cabello et al, 2013;Ló pez-Saavedra et al, 2016) to look for different helicases in the choice between DSB repair pathways. This reporter analyses the choice between HR and NHEJ at very early stages; thus, it is particularly sensitive to changes in DNA resection velocity and/or processivity.…”

“…Briefly, the SSR measures the balance between NHEJ and HR based on the accumulation of distinct fluorescent proteins (GFP for NHEJ events and red fluorescent protein (RFP) for HR events; in this case, a specific subpathway termed single-strand annealing [SSA]; Ceccaldi et al, 2016). Alterations of the normal balance toward a relative increase of HR or NHEJ can be detected using this reporter (Gomez-Cabello et al, 2013;Jimeno et al, 2015;Ló pez-Saavedra et al, 2016). As expected (see Figure 1A, right), depletion of either BLM or RTEL1, proteins with known roles in HR at the level of DNA resection, skewed the balance toward an increase in NHEJ (Gomez-Cabello et al, 2013;Gravel et al, 2008;Youds et al, 2010).…”

Heterocyclic Analogs of Sulforaphane Trigger DNA Damage and Impede DNA Repair in Colon Cancer Cells: Interplay of HATs and HDACs

“…Since CtIP is a key player in the HR pathway, we employed the SSR assay, which is highly sensitive to DNA end resection . With the SSR 2.0 construct, DSBs induced by the meganuclease I‐SceI can be monitored as homology‐driven and homology‐independent repair events based on distinct fluorescent signals of red for HR and green for NHEJ ( Figure A,B).…”

“…Using a unique reporter assay that is sensitive to DNA end resection, we examined the early steps shared by the various HR pathways, and observed that the potent SFN analogs inhibited both HR and NHEJ pathways (Figure ). Effects of SFN analogs on CtIP turnover point to roles in HR, whereas inhibition of NHEJ might implicate other proteins, such as cell cycle and apoptosis regulator 2 (CCAR2), which is evolving as a “master regulator” of DNA repair . In order to further characterize the role of HR/NHEJ, other important players in the HR/NHEJ pathway, such as Ku70/80, BRCA1, RAD51 need to be tested in future experiments.…”

UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors

“…DSB repair pathway assays U2-OS cells bearing a single-copy integration of the reporters DR-GFP (Gene conversion), or SSR (NHEJ:HR) were used to analyze the different DSB repair pathways (Ló pez-Saavedra et al, 2016). In both cases, 4.000 cells were plated in 96-well plates.…”