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A cryo-electron microscopy structure of the DNA damage repair protein 53BP1 bound to a nucleosome illuminates the way 53BP1 recognizes two types of histone modifications (a methyl group and a ubiquitin moiety), and provides insight into the highly specified recognition and recruitment of 53BP1 to modified chromatin. Dual recognition of histone marks by 53BP1 Tudor domain proteins — containing a characteristic repeated structure that recognizes methylated arginine residues — mediate various protein–protein interactions, and Tudor domains are also employed as motifs to recognize different types of histone methylation marks. 53BP1, a protein involved early in the DNA damage response that determines the pathway of repair, has tandem Tudor domains. Daniel Durocher and colleagues have determined the cryo-electron microscopy structure of a 53BP1 dimer bound to a nucleosome core particle containing two types of histone modifications: a methyl group and a ubiquitin moiety. The structure illustrates how dual recognition of both marks as well as elements on the histones themselves mediates highly specified recognition and recruitment of 53BP1. DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair 1 , 2 , 3 . This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively 4 , 5 , 6 , 7 , 8 . RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7 , 8 ) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs 9 , 10 . 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain 10 , 11 . How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions 1 . However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop ‘loop-seq’—a high-throughput assay to measure the propensity for DNA looping—and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors 2 . Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a ‘mechanical code’ with broad functional implications. A high-throughput, chromosome-wide analysis of DNA looping reveals its contribution to the organization of chromatin, and provides insight into how nucleosomes are deposited and organised de novo.

The BRCA1–BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination 1 – 10 . The BRCA1–BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets 11 – 14 . The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1–BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks 15 , 16 . We further show that RING domains 17 in BRCA1–BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1–BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1–BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1–BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin 18 – 22 . These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer. The authors elucidate the mechanisms for the ubiquitylation specificity and recruitment of the ubiquitin ligase complex BRCA1–BARD1 to damaged DNA within chromatin to facilitate homologous recombination.

The DNA sensor cyclic GMP–AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer 1 . Upon activation by double-stranded DNA, cytosolic cGAS produces 2′3′ cGMP–AMP, which triggers the induction of inflammatory cytokines and type I interferons  2 – 7 . cGAS is also present inside the cell nucleus, which is replete with genomic DNA 8 , where chromatin has been implicated in restricting its enzymatic activity 9 . However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A–H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans . Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS–acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self–non-self discrimination of genomic DNA by cGAS. Using cryo-electron microscopy, the authors determine the structure of cGAS bound to nucleosomes and present evidence for the mechanism by which nucleosome binding to cGAS prevents cGAS dimerization and its binding to free double-stranded DNA.

Recent data suggest that histone modifications have a direct effect on nucleosomal architecture. Acetylation, methylation, phosphorylation and citrullination of the histone core may influence chromatin structure by affecting histone–histone and histone–DNA interactions, as well as the binding of histones to chaperones. Post-translational modifications of histones regulate all DNA-templated processes, including replication, transcription and repair. These modifications function as platforms for the recruitment of specific effector proteins, such as transcriptional regulators or chromatin remodellers. Recent data suggest that histone modifications also have a direct effect on nucleosomal architecture. Acetylation, methylation, phosphorylation and citrullination of the histone core may influence chromatin structure by affecting histone–histone and histone–DNA interactions, as well as the binding of histones to chaperones.

The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (γH2AX), which act as seeds for the formation of DNA-damage response foci 1 . It is unclear how these foci are rapidly assembled to establish a ‘repair-prone’ environment within the nucleus. Topologically associating domains are a key feature of 3D genome organization that compartmentalize transcription and replication, but little is known about their contribution to DNA repair processes 2 , 3 . Here we show that topologically associating domains are functional units of the DNA damage response, and are instrumental for the correct establishment of γH2AX–53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model in which H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the importance of chromosome conformation in the maintenance of genome integrity and demonstrates the establishment of a chromatin modification by loop extrusion. During the repair of double-stranded DNA breaks, cohesin mediates the extrusion of loops of DNA along which phosphorylated H2AX spreads to establish a repair zone.

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5′-GAT-3′ sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation. GATA 3 functions as a pioneer factor during cellular reprogramming. Here the authors delineate nucleosome positioning relative to GATA3 binding motifs and describe the structure of a GATA3–nucleosome complex; providing insight into how a pioneer factor interacts with nucleosomes and catalyze their local remodelling to produce an accessible enhancer.

Nucleic acids derived from pathogens induce potent innate immune responses 1 – 6 . Cyclic GMP–AMP synthase (cGAS) is a double-stranded DNA sensor that catalyses the synthesis of the cyclic dinucleotide cyclic GMP–AMP, which mediates the induction of type I interferons through the STING–TBK1–IRF3 signalling axis 7 – 11 . cGAS was previously thought to not react with self DNA owing to its cytosolic localization 2 , 12 , 13 ; however, recent studies have shown that cGAS is localized mostly in the nucleus and has low activity as a result of tight nuclear tethering 14 – 18 . Here we show that cGAS binds to nucleosomes with nanomolar affinity and that nucleosome binding potently inhibits its catalytic activity. To elucidate the molecular basis of cGAS inactivation by nuclear tethering, we determined the structure of mouse cGAS bound to human nucleosome by cryo-electron microscopy. The structure shows that cGAS binds to a negatively charged acidic patch formed by histones H2A and H2B via its second DNA-binding site 19 . High-affinity nucleosome binding blocks double-stranded DNA binding and maintains cGAS in an inactive conformation. Mutations of cGAS that disrupt nucleosome binding alter cGAS-mediated signalling in cells. Structural studies show that cyclic GMP–AMP synthase binds to nucleosomes through its DNA-binding site, which maintains it in an inactive conformation and prevents self-DNA binding.

Cyclic GMP–AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA 1 . After binding DNA, cGAS synthesizes the messenger 2′3′-cyclic GMP–AMP (cGAMP) 2 – 4 , which triggers cell-autonomous defence and the production of type I interferons and pro-inflammatory cytokines via the activation of STING 5 . In addition to responding to cytosolic microbial DNA, cGAS also recognizes mislocalized cytosolic self-DNA and has been implicated in autoimmunity and sterile inflammation 6 , 7 . Specificity towards pathogen- or damage-associated DNA was thought to be caused by cytosolic confinement. However, recent findings place cGAS robustly in the nucleus 8 – 10 , where tight tethering of chromatin is important to prevent autoreactivity to self-DNA 8 . Here we show how cGAS is sequestered and inhibited by chromatin. We provide a cryo-electron microscopy structure of the cGAS catalytic domain bound to a nucleosome, which shows that cGAS does not interact with the nucleosomal DNA, but instead interacts with histone 2A–histone 2B, and is tightly anchored to the ‘acidic patch’. The interaction buries the cGAS DNA-binding site B, and blocks the formation of active cGAS dimers. The acidic patch robustly outcompetes agonistic DNA for binding to cGAS, which suggests that nucleosome sequestration can efficiently inhibit cGAS, even when accessible DNA is nearby, such as in actively transcribed genomic regions. Our results show how nuclear cGAS is sequestered by chromatin and provides a mechanism for preventing autoreactivity to nuclear self-DNA. Biochemical and structural analyses show how tethering of the nucleotidyltransferase cGAS to chromatin prevents autoimmune recognition of nuclear DNA.

Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6–4 pyrimidine–pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA. Cryo-electron microscopy structures reveal that the DNA-repair factor UV-DDB exposes inaccessible nucleosome lesions for binding by inducing a translational shift in the nucleosome position.