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.

DNA double‐strand breaks are a threat to genome integrity and cell viability. The nucleolytic processing of broken DNA ends plays a central role in dictating the repair processes that will mend these lesions. Usually, DNA end resection promotes repair by homologous recombination, whereas minimally processed ends are repaired by non‐homologous end joining. Important in this process is the chromatin‐binding protein 53BP1, which inhibits DNA end resection. How 53BP1 shields DNA ends from nucleases has been an enduring mystery. The recent discovery of shieldin, a four‐subunit protein complex with single‐stranded DNA‐binding activity, illuminated a strong candidate for the ultimate effector of 53BP1‐dependent end protection. Shieldin consists of REV7, a known 53BP1‐pathway component, and three hitherto uncharacterized proteins: C20orf196 (SHLD1), FAM35A (SHLD2), and CTC‐534A2.2 (SHLD3). Shieldin promotes many 53BP1‐associated activities, such as the protection of DNA ends, non‐homologous end joining, and immunoglobulin class switching. This review summarizes the identification of shieldin and the various models of shieldin action and highlights some outstanding questions requiring answers to gain a full molecular understanding of shieldin function.

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Graphical Abstract Highlights d The crystal structure of EspB reveals a fused PE/PPE homology domain d EspB has a stabilized bipartite secretion signal that targets the EccCb1 ATPase d EspB oligomerizes to form a ring-shaped heptamer d A model of the heptamer was fit to EM density and crosslinking data In Brief Mycobacterium tuberculosis exports virulence factors to its surface using the ESX-1 secretion system, progressing the infection of human macrophages. Solomonson et al. show that one of these factors, EspB, adopts a PE/PPE-like fold and oligomerizes to form a barrel-shaped structure with heptameric symmetry. Accession Numbers 4WJ1 4WJ2 3J83 Solomonson et al., 2015, Structure 23, 1-13 March 3, SUMMARYMycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors.

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8–MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB–MCM8–MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8–MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.

Highlights d Loss of APE2 is lethal in cells with mutations in BRCA1 or BRCA2 d The APE2 DNA repair nuclease removes endogenous DNA 3 0 blocks d 3 0 blocks arising from ribonucleotides cause APEX2-BRCA1/ 2 synthetic lethality d DNA 3 0 block-resolving pathways are vulnerabilities for HRdeficient tumor cells

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway–Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3’ CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

Background:The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) complex regulates transcription through chromatin modification and other mechanisms. Results: The overall structure of SAGA and the arrangement of all subunits within this complex were determined. Conclusion: SAGA is flexible and is composed of core modules that support peripheral catalytic modules. Significance: Understanding the structural mechanisms of SAGA multifunctionality improves the understanding of other chromatin-modifying complexes.