An OB-fold complex controls the repair pathways for DNA double-strand breaks

Abstract

53BP1 with its downstream proteins, RIF1, PTIP and REV7, antagonizes BRCA1-dependent homologous recombination (HR) and promotes non-homologous end joining (NHEJ) in an unclear manner. Here we show that REV7 forms a complex with two proteins, FAM35A and C20ORF196. We demonstrate that FAM35A preferentially binds single-strand DNA (ssDNA) in vitro, and is recruited to DSBs as a complex with C20ORF196 and REV7 downstream of RIF1 in vivo. Epistasis analysis shows that both proteins act in the same pathway as RIF1 in NHEJ. The defects in HR pathway to repair DSBs and the reduction in resection of broken DNA ends in BRCA1-mutant cells can be largely suppressed by inactivating FAM35A or C20ORF196, indicating that FAM35A and C20ORF196 prevent end resection in these cells. Together, our data identified a REV7–FAM35A–C20ORF196 complex that binds and protects broken DNA ends to promote the NHEJ pathway for DSB repair.

Shieldin – the protector of DNA ends

“…Although structural information on the SHLD2 OB‐folds is currently unavailable, the purified SHLD2 C‐terminus binds to DNA, with a strong preference for ssDNA that is consistent with the binding properties of other OB‐fold proteins . ssDNA binding in vitro is abolished by the same aromatic residue mutations that disable the ability of SHLD2 to suppress HR, underlining ssDNA binding as a key function of SHLD2 .…”

“…The SHLD2 C‐terminus purified in complex with SHLD1 from HEK293T cells binds ssDNA with a dissociation constant of approximately 10 nM , an intermediate affinity between RPA (< 1 nM) and RAD51 (> 100 nM) . However, the SHLD2 C‐terminus expressed in Escherichia coli has 1–2 orders of magnitude lower affinity for ssDNA than the protein complex purified from human cells . Co‐expression with SHLD1 increases the stability of the SHLD2 C‐terminus in mammalian cells , but whether SHLD1 stimulates the affinity of SHLD2 for ssDNA remains an open question.…”

53BP1: a DSB escort

“…The second mechanism by which 53BP1 promotes PARPi sensitivity involves the Rif1/ Shieldin/CST/Polα/Primase axis. When this function of 53BP1 is disabled, BRCA1-deficient cells regain their ability to repair PARPi-induced DSBs by HDR and survive (Chapman et al 2013;Escribano-Diaz et al 2013;Feng et al 2013;Zimmermann et al 2013;Xu et al 2015;Barazas et al 2018;Dev et al 2018;Gao et al 2018;Ghezraoui et al 2018;Gupta et al 2018;Mirman et al 2018;Noordermeer et al 2018). The proposed mechanism for this reactivation of HDR is discussed below.…”

“…Concerning the mechanism by which 53BP1 limits the formation of ssDNA at DNA breaks, there are two main models ( Fig. 7; Barazas et al 2018;Dev et al 2018;Findlay et al 2018;Gao et al 2018;Ghezraoui et al 2018;Gupta et al 2018;Mirman et al 2018;Noordermeer et al 2018). In the first model, 53BP1 uses the loading of Shieldin onto the ssDNA to protect the 5 ′ end from resection (Fig.…”

“…7A). The finding that Shld2 alone or in complex with Shld1 can bind to ssDNA is promising in this regard (Dev et al 2018;Findlay et al 2018;Gao et al 2018;Noordermeer et al 2018), but it remains to be seen whether Shieldin binds sufficiently close to the 5 ′ end to form a barrier to nucleolytic attack. For Shieldin to block resection upon engaging the DNA end, at least some ssDNA must B A Figure 7.…”