53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone ‘code’ produced by DSB signalling.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini^1,2. This function of 53BP1 requires interactions with PTIP³ and RIF1^4,5,6,7,8,9, the latter of which recruits REV7 (also known as MAD2L2) to break sites^10,11. 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^12,13, 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.

Poly-(ADP)-ribose polymerase (PARP) inhibition is synthetic lethal with deficiency for homologous recombination (HR), a pathway essential for DNA double-strand break repair. PARP inhibitors (PARPi) therefore hold great promise for the treatment of tumors with disruptive mutations in BRCA1/2 or other HR factors. Unfortunately, PARPi resistance has proved to be a major problem in the clinic. Knowledge about PARPi resistance is expanding quickly, revealing four main mechanisms that alter drug availability, affect (de)PARylation enzymes, restore HR, or restore replication fork stability. We discuss how studies on resistance mechanisms have yielded important insights into the regulation of DNA double-strand break (DSB) repair and replication fork protection, and how these studies could pave the way for novel treatment options to target resistance mechanisms or acquired vulnerabilities.

Shutting Down Repair to Protect: Cells repair DNA double-strand breaks (DSBs) by halting the cell cycle and activating the machinery involved in mending the breaks. However, during mitosis neither the DNA damage checkpoint nor DSB repair occur, apparently leaving the cell extremely vulnerable to DSBs. Orthwein et al. (p. 189 , published online 20 March) found that the DSB response was blocked by the phosphorylation of two crucial repair factors, RNF8 and PB531, preventing their recruitment to the site of damage. Restoring DSB repair during mitosis caused end-to-end chromosome fusions, which are catastrophic for chromosome segregation and normal cell division, explaining why the repair machinery is shut down during cell division.

Modulation of DNA repair pathway choice by a potent inhibitor of 53BP1 improves the efficiency of homology-dependent genome editing in human and mouse cells. Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR)1,2,3. However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ)4. We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells4,5 and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants6 for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.