Cryo-EM structures of the Spo11 core complex bound to DNA.

DNA double-strand breaks that initiate meiotic recombination are formed by the topoisomerase-relative enzyme Spo11, supported by conserved auxiliary factors. Because high-resolution structural data have not been available, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-electron microscopy structures at up to 3.3-Å resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104 and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3'-OH and first 5' overhanging nucleotide, establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.

Structures, mechanisms and applications of RNA-centric CRISPR-Cas13.

Prokaryotes are equipped with a variety of resistance strategies to survive frequent viral attacks or invading mobile genetic elements. Among these, CRISPR-Cas surveillance systems are abundant and have been studied extensively. This Review focuses on CRISPR-Cas type VI Cas13 systems that use single-subunit RNA-guided Cas endonucleases for targeting and subsequent degradation of foreign RNA, thereby providing adaptive immunity. Notably, distinct from single-subunit DNA-cleaving Cas9 and Cas12 systems, Cas13 exhibits target RNA-activated substrate RNase activity. This Review outlines structural, biochemical and cell biological studies toward elucidation of the unique structural and mechanistic principles underlying surveillance effector complex formation, precursor CRISPR RNA (pre-crRNA) processing, self-discrimination and RNA degradation in Cas13 systems as well as insights into suppression by bacteriophage-encoded anti-CRISPR proteins and regulation by endogenous accessory proteins. Owing to its programmable ability for RNA recognition and cleavage, Cas13 provides powerful RNA targeting, editing, detection and imaging platforms with emerging biotechnological and therapeutic applications.

The CRISPR effector Cam1 mediates membrane depolarization for phage defence.

Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors1-5. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain6,7. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA.

While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 may modify the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.

Cryo-EM structure of the Spo11 core complex bound to DNA.

The DNA double-strand breaks that initiate meiotic recombination are formed by topoisomerase relative Spo11, supported by conserved auxiliary factors. Because high-resolution structural data are lacking, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-EM structures at up to 3.3 Å resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104, and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3'-OH and first 5' overhanging nucleotide, definitively establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.

Advances in structure-guided mechanisms impacting on the cGAS-STING innate immune pathway.

The metazoan cGAS-STING innate immunity pathway is triggered in response to cytoplasmic double-stranded DNA (dsDNA), thereby providing host defense against microbial pathogens. This pathway also impacts on autoimmune diseases, cellular senescence and anti-tumor immunity. The cGAS-STING pathway was also observed in the bacterial antiviral immune response, known as the cyclic oligonucleotide (CDN)-based anti-phage signaling system (CBASS). This review highlights a structure-based mechanistic perspective of recent advances in metazoan and bacterial cGAS-STING innate immune signaling by focusing on the cGAS sensor, cGAMP second messenger and STING adaptor components, thereby elucidating the specificity, activation, regulation and signal transduction features of the pathway.

Molecular basis for Nse5-6 mediated regulation of Smc5/6 functions.

The Smc5/6 complex (Smc5/6) is important for genome replication and repair in eukaryotes. Its cellular functions are closely linked to the ATPase activity of the Smc5 and Smc6 subunits. This activity requires the dimerization of the motor domains of the two SMC subunits and is regulated by the six non-SMC subunits (Nse1 to Nse6). Among the NSEs, Nse5 and Nse6 form a stable subcomplex (Nse5-6) that dampens the ATPase activity of the complex. However, the underlying mechanisms and biological significance of this regulation remain unclear. Here, we address these issues using structural and functional studies. We determined cryo-EM structures of the yeast Smc5/6 derived from complexes consisting of either all eight subunits or a subset of five subunits. Both structures reveal that Nse5-6 associates with Smc6's motor domain and the adjacent coiled-coil segment, termed the neck region. Our structural analyses reveal that this binding is compatible with motor domain dimerization but results in dislodging the Nse4 subunit from the Smc6 neck. As the Nse4-Smc6 neck interaction favors motor domain engagement and thus ATPase activity, Nse6's competition with Nse4 can explain how Nse5-6 disfavors ATPase activity. Such regulation could in principle differentially affect Smc5/6-mediated processes depending on their needs of the complex's ATPase activity. Indeed, mutagenesis data in cells provide evidence that the Nse6-Smc6 neck interaction is important for the resolution of DNA repair intermediates but not for replication termination. Our results thus provide a molecular basis for how Nse5-6 modulates the ATPase activity and cellular functions of Smc5/6.