53BP1 cooperation with the REV7–shieldin complex underpins DNA structure-specific NHEJ

Abstract

53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination, and reduced fidelity of long-range V(D)J recombination. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive, unlike 53BP1-deficient cells, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.

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

“…Here, we provide further insight into the role of SHLD2 in DNA repair and show that SHLD2 acts as a downstream effector of REV7 in the NHEJ pathway. 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 , ).…”

“…Using a mass spectrometry‐based approach, we identified SHLD2 as a high‐confidence interactor of REV7. While this association has been previously reported (Hein et al , ), it is only recently that its biological relevance has been further investigated (Dev et al , ; Ghezraoui et al , ; Gupta et al , ; Mirman et al , ; Noordermeer et al , ; Tomida et al , ). Up to now, SHLD2 remained an enigma in regard to its physiological functions.…”

“…However, it remains largely unclear how they modulate the proper balance between NHEJ and HR. Several recent studies have recently tackled to decipher the effectors of the 53BP1‐RIF1‐REV7 axis (Barazas et al , ; Dev et al , ; Ghezraoui et al , ; Gupta et al , ; Mirman et al , ; Noordermeer et al , ; Tomida et al , ), and our work contributes to this effort by providing further insight into the role of REV7 in DNA repair pathway choice.…”

“…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 , ).…”

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.…”