Phosphorylation-mediated conformational change regulates human SLFN11.

Human Schlafen 11 (SLFN11) is sensitizing cells to DNA damaging agents by irreversibly blocking stalled replication forks, making it a potential predictive biomarker in chemotherapy. Furthermore, SLFN11 acts as a pattern recognition receptor for single-stranded DNA (ssDNA) and functions as an antiviral restriction factor, targeting translation in a codon-usage-dependent manner through its endoribonuclease activity. However, the regulation of the various SLFN11 functions and enzymatic activities remains enigmatic.

Structural basis for human OGG1 processing 8-oxodGuo within nucleosome core particles.

Base excision repair (BER) is initialized by DNA glycosylases, which recognize and flip damaged bases out of the DNA duplex into the enzymes active site, followed by cleavage of the glycosidic bond. Recent studies have revealed that all types of DNA glycosylases repair base lesions less efficiently within nucleosomes, and their repair activity is highly depended on the lesion's location within the nucleosome. To reveal the underlying molecular mechanism of this phenomenon, we determine the 3.

Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation.

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7.

Energy-driven genome regulation by ATP-dependent chromatin remodellers.

The packaging of DNA into chromatin in eukaryotes regulates gene transcription, DNA replication and DNA repair. ATP-dependent chromatin remodelling enzymes (re)arrange nucleosomes at the first level of chromatin organization. Their Snf2-type motor ATPases alter histone-DNA interactions through a common DNA translocation mechanism.

Hexasome-INO80 complex reveals structural basis of noncanonical nucleosome remodeling.

Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B.

Structural basis for TBP displacement from TATA box DNA by the Swi2/Snf2 ATPase Mot1.

The Swi2/Snf2 family transcription regulator Modifier of Transcription 1 (Mot1) uses adenosine triphosphate (ATP) to dissociate and reallocate the TATA box-binding protein (TBP) from and between promoters. To reveal how Mot1 removes TBP from TATA box DNA, we determined cryogenic electron microscopy structures that capture different states of the remodeling reaction. The resulting molecular video reveals how Mot1 dissociates TBP in a process that, intriguingly, does not require DNA groove tracking.

Mre11-Rad50: the DNA end game.

The Mre11-Rad50-(Nbs1/Xrs2) complex is an evolutionarily conserved factor for the repair of DNA double-strand breaks and other DNA termini in all kingdoms of life. It is an intricate DNA associated molecular machine that cuts, among other functions, a large variety of free and obstructed DNA termini for DNA repair by end joining or homologous recombination, yet leaves undamaged DNA intact. Recent years have brought progress in both the structural and functional analyses of Mre11-Rad50 orthologs, revealing mechanisms of DNA end recognition, endo/exonuclease activities, nuclease regulation and DNA scaffolding.

Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions.

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer.

Structural mechanism of extranucleosomal DNA readout by the INO80 complex.

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo-electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA.

Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations.

Mechanistic understanding of human SLFN11.

Schlafen 11 (SLFN11) is an interferon-inducible antiviral restriction factor with tRNA endoribonuclease and DNA binding functions. It is recruited to stalled replication forks in response to replication stress and inhibits replication of certain viruses such as the human immunodeficiency virus 1 (HIV-1) by modulating the tRNA pool. SLFN11 has been identified as a predictive biomarker in cancer, as its expression correlates with a beneficial response to DNA damage inducing anticancer drugs.

Ice thickness monitoring for cryo-EM grids by interferometry imaging.

While recent technological developments contributed to breakthrough advances in single particle cryo-electron microscopy (cryo-EM), sample preparation remains a significant bottleneck for the structure determination of macromolecular complexes. A critical time factor is sample optimization that requires the use of an electron microscope to screen grids prepared under different conditions to achieve the ideal vitreous ice thickness containing the particles. Evaluating sample quality requires access to cryo-electron microscopes and a strong expertise in EM.

Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing.

Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased levels of cGAMP and enhanced IFN-I signature. To identify disease mechanisms and potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients.

Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50.

DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin.

Novel Poxin Stable cGAMP-Derivatives Are Remarkable STING Agonists.

2',3'-cGAMP is a cyclic A- and G-containing dinucleotide second messenger, which is formed upon cellular recognition of foreign cytosolic DNA as part of the innate immune response. The molecule binds to the adaptor protein STING, which induces an immune response characterized by the production of type I interferons and cytokines. The development of STING-binding molecules with both agonistic as well as antagonistic properties is currently of tremendous interest to induce or enhance antitumor or antiviral immunity on the one hand, or to treat autoimmune diseases on the other hand.

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides c GAMP.

The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines.

Structural and biochemical characterization of human Schlafen 5.

The Schlafen family belongs to the interferon-stimulated genes and its members are involved in cell cycle regulation, T cell quiescence, inhibition of viral replication, DNA-repair and tRNA processing. Here, we present the cryo-EM structure of full-length human Schlafen 5 (SLFN5) and the high-resolution crystal structure of the highly conserved N-terminal core domain. We show that the core domain does not resemble an ATPase-like fold and neither binds nor hydrolyzes ATP.

SIRPα-αCD123 fusion antibodies targeting CD123 in conjunction with CD47 blockade enhance the clearance of AML-initiating cells.

Background: Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 "don't eat me signal" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies.

BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.

The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae.

OAS1/RNase L executes RIG-I ligand-dependent tumor cell apoptosis.

Cytoplasmic double-stranded RNA is sensed by RIG-I-like receptors (RLRs), leading to induction of type I interferons (IFN-Is), proinflammatory cytokines, and apoptosis. Here, we elucidate signaling mechanisms that lead to cytokine secretion and cell death induction upon stimulation with the bona fide RIG-I ligand 5'-triphosphate RNA (3p-RNA) in tumor cells. We show that both outcomes are mediated by dsRNA-receptor families with RLR being essential for cytokine production and IFN-I-mediated priming of effector pathways but not for apoptosis.

Nuclear cGAS: guard or prisoner?

cGAS, an innate immune sensor of cellular stress, recognizes double-stranded DNA mislocalized in the cytosol upon infection, mitochondrial stress, DNA damage, or malignancy. Early models suggested that cytosolic localization of cGAS prevents autoreactivity to nuclear and mitochondrial self-DNA, but this paradigm has shifted in light of recent findings of cGAS as a predominantly nuclear protein tightly bound to chromatin. This has raised the question how nuclear cGAS is kept inactive while being surrounded by chromatin, and what function nuclear localization of cGAS may serve in the first place? Cryo-EM structures have revealed that cGAS interacts with nucleosomes, the minimal units of chromatin, mainly via histones H2A/H2B, and that these protein-protein interactions block cGAS from DNA binding and thus prevent autoreactivity.

Genome information processing by the INO80 chromatin remodeler positions nucleosomes.

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones.