The shieldin complex mediates 53BP1-dependent DNA repair

Abstract

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini. This function of 53BP1 requires interactions with PTIP and RIF1, the latter of which recruits REV7 (also known as MAD2L2) to break sites. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

SHLD 2/ FAM 35A co‐operates with REV 7 to coordinate DNA double‐strand break repair pathway choice

“…Through its N‐terminal domain, SHLD2 is mobilized to and accumulates at sites of DNA damage in a 53BP1‐, RIF1‐, and REV7‐dependent manner, in accordance with several recent studies describing the role of SHLD2 in the DNA damage response (Barazas et al , ; Dev et al , ; Ghezraoui et al , ; Gupta et al , ; Mirman et al , ; Noordermeer et al , ; Tomida et al , ). Importantly, we show that the N‐terminus of SHLD2 has very limited DNA‐binding capacity, which support a model where the recruitment of SHDL2 to DSBs is promoted by protein–protein interactions (Noordermeer et al , ). This finding corroborates the initial observation made by Gupta et al (), showing that the N‐terminal domain of SHLD2 is critical for its association with REV7.…”

“…By performing structure prediction analyses on SHLD2 protein sequence using Motif Scan (MyHits, SIB, Switzerland) and InterProScan5 (Jones et al , ), we identified a putative N‐terminal DNA‐binding domain (NUMOD3 domain) and a structural motif in its C‐terminus that we labeled PFAM (Fig A). Recently, structural prediction analyses of SHLD2 also defined a N‐terminal motif promoting protein–protein as well as three putative OB‐fold like domains at its C‐terminus (Dev et al , ; Ghezraoui et al , ; Gupta et al , ; Noordermeer et al , ; Tomida et al , ). Finally, previous phospho‐proteomic analysis identified a S/Q substrate of ATM/ATR following DNA damage at position 339 (Matsuoka et al , ).…”

BRCA1 Mutation-Specific Responses to 53BP1 Loss-Induced Homologous Recombination and PARP Inhibitor Resistance

“…Such cell line variations may stem from the activity of additional factors that contribute to PALB2 recruitment to DNA breaks such as RNF168, ATR or RPA (Luijsterburg et al, 2017; Murphy et al, 2014; Yazinski et al, 2017). Moreover, KO of DNA end resection inhibitory proteins involved in the shieldin complex were recently shown to promote PARPi resistance in a range of human and mouse BRCA1 mutant cell lines (Barazas et al, 2018; Dev et al, 2018; Mirman et al, 2018; Noorder-meer et al, 2018; Tomida et al, 2018). We anticipate that similar to 53BP1 KO, the presence of BRCA1 hypomorphs in combination with shieldin KO may facilitate a greater level of PARPi resistance compared with shieldin KO and the absence of mutant BRCA1 protein expression.…”

Phase separation of 53 BP 1 determines liquid‐like behavior of DNA repair compartments

“…The DDR is initiated at DNA break sites by the ATM kinase, which phosphorylates histone variant H2AX to generate cH2AX (Shiloh & Ziv, 2013;Blackford & Jackson, 2017). Restrained resection is achieved by 53BP1dependent recruitment of RIF1, REV7, and the Shieldin complex (Dev et al, 2018;Findlay et al, 2018;Ghezraoui et al, 2018;Gupta et al, 2018;Mirman et al, 2018;Noordermeer et al, 2018;Setiaputra & Durocher, 2019). 53BP1 generates sizeable chromatin domains, which scaffold the assembly of downstream effectors and shield DNA lesions against excessive nucleolytic digestion.…”