Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway.

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing.

Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis.

The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.

Structural Mechanism of EMRE-Dependent Gating of the Human Mitochondrial Calcium Uniporter.

Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.

SnapShot: S-Phase Entry and Exit.

S-phase entry and exit are regulated by hundreds of protein complexes that assemble "just in time," orchestrated by a multitude of distinct events. To help understand their interplay, we have created a tailored visualization based on the Minardo layout, highlighting over 80 essential events. This complements our earlier visualization of M-phase, and both can be displayed together, giving a comprehensive overview of the events regulating the cell division cycle. To view this SnapShot, open or download the PDF.

Mechanisms for Zinc and Proton Inhibition of the GluN1/GluN2A NMDA Receptor.

N-methyl-D-aspartate receptors (NMDARs) play essential roles in memory formation, neuronal plasticity, and brain development, with their dysfunction linked to a range of disorders from ischemia to schizophrenia. Zinc and pH are physiological allosteric modulators of NMDARs, with GluN2A-containing receptors inhibited by nanomolar concentrations of divalent zinc and by excursions to low pH. Despite the widespread importance of zinc and proton modulation of NMDARs, the molecular mechanism by which these ions modulate receptor activity has proven elusive. Here, we use cryoelectron microscopy to elucidate the structure of the GluN1/GluN2A NMDAR in a large ensemble of conformations under a range of physiologically relevant zinc and proton concentrations. We show how zinc binding to the amino terminal domain elicits structural changes that are transduced though the ligand-binding domain and result in constriction of the ion channel gate.

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins.

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes.

The endosomal sorting complexes required for transport (ESCRT) pathway mediates cellular membrane remodeling and fission reactions. The pathway comprises five core complexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. These soluble complexes are typically recruited to target membranes by site-specific adaptors that bind one or both of the early-acting ESCRT factors: ALIX and ESCRT-I/ESCRT-II. These factors, in turn, nucleate assembly of ESCRT-III subunits into membrane-bound filaments that recruit the AAA ATPase Vps4. Together, ESCRT-III filaments and Vps4 remodel and sever membranes. Here, we review recent advances in our understanding of the structures, activities, and mechanisms of the ESCRT-III and Vps4 machinery, including the first high-resolution structures of ESCRT-III filaments, the assembled Vps4 enzyme in complex with an ESCRT-III substrate, the discovery that ESCRT-III/Vps4 complexes can promote both inside-out and outside-in membrane fission reactions, and emerging mechanistic models for ESCRT-mediated membrane fission.

The picky mTORC1 in metabolic enzyme degradation.

In this issue of Molecular Cell, Yi et al.1 demonstrate that reduced mTORC1 activity induces the CTLH E3 ligase-dependent degradation of HMGCS1, an enzyme in the mevalonate pathway, thus revealing a unique connection between mTORC1 signaling and the degradation of a specific metabolic enzyme via the ubiquitin-proteasome system.

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein-coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of Sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC⋅BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, the multipass translocon, and Sec61 for the biogenesis of diverse membrane proteins in human cells.

mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.

GATOR2 rings GATOR1 to speak to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) senses cellular leucine levels through the GATOR1/2-Rag axis. Jiang et al. show that the Ring domains of GATOR2 subunits maintain the integrity of the complex and promote ubiquitination and inhibition of GATOR1, thereby leading to mTORC1 activation.

Establishment of dsDNA-dsDNA interactions by the condensin complex.

Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a "diffusion capture" model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture.

Ring domains are essential for GATOR2-dependent mTORC1 activation.

The GATOR2-GATOR1 signaling axis is essential for amino-acid-dependent mTORC1 activation. However, the molecular function of the GATOR2 complex remains unknown. Here, we report that disruption of the Ring domains of Mios, WDR24, or WDR59 completely impedes amino-acid-mediated mTORC1 activation. Mechanistically, via interacting with Ring domains of WDR59 and WDR24, the Ring domain of Mios acts as a hub to maintain GATOR2 integrity, disruption of which leads to self-ubiquitination of WDR24. Physiologically, leucine stimulation dissociates Sestrin2 from the Ring domain of WDR24 and confers its availability to UBE2D3 and subsequent ubiquitination of NPRL2, contributing to GATOR2-mediated GATOR1 inactivation. As such, WDR24 ablation or Ring deletion prevents mTORC1 activation, leading to severe growth defects and embryonic lethality at E10.5 in mice. Hence, our findings demonstrate that Ring domains are essential for GATOR2 to transmit amino acid availability to mTORC1 and further reveal the essentiality of nutrient sensing during embryonic development.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.

Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.

Topoisomerases I and II facilitate condensin DC translocation to organize and repress X chromosomes in C. elegans.

Condensins are evolutionarily conserved molecular motors that translocate along DNA and form loops. To address how DNA topology affects condensin translocation, we applied auxin-inducible degradation of topoisomerases I and II and analyzed the binding and function of an interphase condensin that mediates X chromosome dosage compensation in C. elegans. TOP-2 depletion reduced long-range spreading of condensin-DC (dosage compensation) from its recruitment sites and shortened 3D DNA contacts measured by Hi-C. TOP-1 depletion did not affect long-range spreading but resulted in condensin-DC accumulation within expressed gene bodies. Both TOP-1 and TOP-2 depletion resulted in X chromosome derepression, indicating that condensin-DC translocation at both scales is required for its function. Together, the distinct effects of TOP-1 and TOP-2 suggest two distinct modes of condensin-DC association with chromatin: long-range DNA loop extrusion that requires decatenation/unknotting of DNA and short-range translocation across genes that requires resolution of transcription-induced supercoiling.

m6A RNA methylation regulates promoter- proximal pausing of RNA polymerase II.

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.

WNK1 is an assembly factor for the human ER membrane protein complex.

The assembly of nascent proteins into multi-subunit complexes is a tightly regulated process that must occur at high fidelity to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble cytosolic subunits to function. Here, we show that the kinase with no lysine 1 (WNK1), known for its role in hypertension and neuropathy, functions as an assembly factor for the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and directly competes for binding of E3 ubiquitin ligases, permitting assembly. Depletion of WNK1 thus destabilizes both the EMC and its membrane protein clients. This work describes an unexpected role for WNK1 in protein biogenesis and defines the general requirements of an assembly factor that will apply across the proteome.

Reconstitution of the destruction complex defines roles of AXIN polymers and APC in ß-catenin capture, phosphorylation, and ubiquitylation.

The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.

Structure of an activated DNA-PK and its implications for NHEJ.

DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKINs) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 Å at best. Here, we report the cryoelectron microscopy (cryo-EM) structures of DNA-PKcs (DNA-PK catalytic subunit) bound to a DNA end or complexed with Ku70/80 and DNA in both inactive and activated forms at resolutions of 3.7 Å overall and 3.2 Å for FATKINs. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.