Abstract
DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair.
Highlights
Due to their highly recombinogenic and pro-apoptotic potentials, DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions
FAM35A accumulates at DSBs in a 53BP1, RIF1- and REV7-dependent manner
To get better insight into the interactome of REV7, we performed a standard affinity purification (AP) followed by mass spectrometry (MS) (AP-MS) (Fig EV1.A), where REV7 was tagged with the Flag epitope and stably expressed in the human embryonic kidney 293 (HEK293) cell line using the Flp-In/T-REX system (Fig EV1.B)
Summary
Due to their highly recombinogenic and pro-apoptotic potentials, DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions Their inaccurate resolution can result in point mutations, small deletions/insertions, chromosomal rearrangements or loss of gross genetic information that drive genomic instability, carcinogenesis and cell death (reviewed in (Tubbs and Nussenzweig, 2017)). To avoid these deleterious outcomes, cells have deployed a complex network of proteins to signal and repair DSBs. One critical step during the DSB response consists in the choice between two mutually exclusive DNA repair pathways: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) (reviewed in (Ceccaldi et al, 2016)). DNA repair pathway choice is under the control of two antagonizing factors, 53BP1 and BRCA1 (reviewed in (Hustedt and Durocher, 2016))
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