Biology subjects

Silanskas, A.

Publications and source records attributed to Silanskas, A..

9 recordsLinked to original sources

bioRxiv2026

Viral SSB-bound ssDNA activates the bacterial anti-phage defense system DARNA

To protect themselves against phage infection, bacteria employ diverse defense systems that are typically activated specifically upon infection. However, the mechanisms of activation and self versus non-self discrimination for most systems remain poorly understood. Here, we show that the bacterial immunity protein DARNA, once activated, cleaves a subset of host tRNAs, thereby inhibiting phage propagation. Although phages escape DARNA-mediated defense through mutations in the gene encoding single-stranded DNA-binding protein (SSB), we find that phage SSBs do not directly stimulate DARNA. Instead, DARNA is activated by single-stranded DNA presented by phage SSB, but not by the host SSB. The recognition of an endogenous nucleic acid signal promoted by a viral protein ensures that DARNA can detect and respond to a broad range of viruses while avoiding auto-immunity.

microbiology↗

bioRxiv2026

Deoxydinucleotides activate the bacterial anti-phage defense system ApeA

Bacteria and archaea encode diverse antiviral defense systems, many of which rely on toxic effector proteins that are activated specifically upon bacteriophage infection. However, the mechanisms by which infection is recognized and coupled to effector activation remain poorly understood for most antiviral systems. Here, we focus on ApeA, a HEPN-domain antiviral protein that confers immunity through cleavage of host tRNAs within their anticodon loops. We show that ApeA proteins form large doughnut-shaped oligomers that are activated upon ligand binding in a conserved protein pocket distinct from the catalytic center. In the Ec2ApeA variant, this pocket specifically recognizes 5'-phosphorylated deoxydinucleotides that likely arise as intermediates of host genome degradation by viral nucleases, thereby enabling Ec2ApeA to achieve a broad protection profile. Together, our results reveal how small-molecule products of virus-induced host cell destruction function as signals that activate bacterial immune defenses.

molecular biology↗

bioRxiv2025

Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation

During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins like Cas4, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a supercomplex with the Cas1-Cas2 integrase and the Csn2 protein. However, the supercomplex structure and the role of the ring-like Csn2 protein remain unknown. Here, we present cryo-electron microscopy structures of the type II-A prespacer selection supercomplex in the DNA-scanning and two different PAM-bound configurations. Our study uncovers the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems, and reveals the role of the accessory protein Csn2, which serves as a platform for the assembly of Cas9 and Cas1-Cas2 integrase on prespacer DNA, reminiscent of the sliding clamp in DNA replication. Repurposing of Cas9 by the CRISPR adaptation machinery for prespacer selection characterized here demonstrates Cas9 plasticity and expands our knowledge of the Cas9 biology.

microbiology↗

bioRxiv2025

Activation of SPARDA defense system by filament assembly reveals beta-relay signaling mechanism widespread in prokaryotic Argonautes

Present in all three domains of life, Argonaute (Ago) proteins use short oligonucleotides as guides to recognize complementary nucleic acid targets and are involved in RNA silencing (eukaryotes) or host defense against invading DNA (prokaryotes). Here, we show that the SPARDA (short prokaryotic Argonaute, DNase associated) system functions in anti-plasmid defense. Upon detection of invading plasmid DNA, it degrades both the invader and host DNA, inducing cell death and preventing further spread of the invader. Upon activation, the recognition signal of the bound guide/target duplex is relayed to other functional SPARDA sites through a structural region, which we termed beta-relay. The associated dramatic conformational changes trigger the formation of a filament, in which the DREN nuclease domains form tetramers poised to cleave dsDNA. Furthermore, we identified the presence of beta-relay in all pAgo clades, providing new insights into the structural mechanisms of pAgo proteins.

biochemistry↗

bioRxiv2024

TIR domains produce histidine-ADPR conjugates as immune signaling molecules in bacteria

TIR domains are central components of pattern recognition immune proteins across all domains of life. In both bacteria and plants, TIR-domain proteins were shown to recognize pathogen invasion and then produce immune signaling molecules exclusively comprising nucleotide moieties. Here we show that the TIR domain protein of the type II Thoeris defense system in bacteria produces a unique signaling molecule comprising the amino acid histidine conjugated to ADP-ribose (His-ADPR). His-ADPR is generated in response to phage infection and activates the cognate Thoeris effector by binding a Macro domain located at the C-terminus of the effector protein. By determining the crystal structure of a ligand-bound Macro domain, we describe the structural basis for His-ADPR recognition. Our analyses furthermore reveal a family of phage proteins that bind and sequester His-ADPR signaling molecules, allowing phages to evade TIR- mediated immunity. These data demonstrate diversity in bacterial TIR signaling and reveal a new class of TIR-derived immune signaling molecules combining nucleotide and amino acid moieties.

microbiology↗

bioRxiv2023

Advancing Understanding of DNA-BfiI Restriction Endonuclease Cis and Trans Interactions through smFRET Technology

Monitoring of DNA-protein interactions is essential in understanding many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and the mechanisms for target search differ across proteins. Revealing temporal interactions with two target sites, both in Cis and in Trans, is crucial in target search mechanisms studies. Here, we present two single-molecule Forster resonance energy transfer (smFRET)-based assays to study BfiI-DNA interactions. The first assay, smFRET-based DNA looping assay, detects both "Phi" and "U"-shaped DNA looping events. We modified it to only allow in Trans BfiI-target DNA interactions to improve specificity and reduce limitations in the observation time. Our TIRF microscopy measurements directly observe the on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binding events last longer on target sites and that the BfiI rarely changes conformations during binding. This newly developed assay could be useful for other two-targets-binding DNA-interacting proteins and could be employed for dsDNA substrate BfiI-PAINT, which is useful for DNA stretch-assays and other super-resolution fluorescence microscopy studies.

biophysics↗

bioRxiv2021

Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2-protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

molecular biology↗

bioRxiv2020

Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

BackgroundArgonaute (Ago) proteins are found in all three domains of life. The best characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are monomeric proteins, all composed of four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. It is believed that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. ResultsIn this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal {beta}-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including single-molecule FRET, SAXS and AFM, to test the possible dimerization of AfAgo. SAXS results confirm that WT AfAgo, but not the dimerization surface mutant AfAgo{Delta}, forms a homodimer both in the apo-form and when bound to a nucleic acid. Single molecule FRET and AFM studies demonstrate that the dimeric WT AfAgo binds two ends of a linear DNA fragment, forming a relatively stable DNA loop. ConclusionOur results show that contrary to other characterized Ago proteins, AfAgo is a stable homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

biochemistry↗

bioRxiv2019

PAM recognition by miniature CRISPR-Cas14 triggers programmable double-stranded DNA cleavage

In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas nucleases that recognize and cleave dsDNA in PAM dependent manner. Categorized as class 2 type V-F they come from the Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5 T- or C-rich PAM sequence triggers double stranded (ds) DNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in E. coli and find that some but not all can. Altogether, our findings show that miniature Cas nucleases are functional CRISPR-Cas defense systems and have the potential to be harnessed as programmable nucleases for genome editing.

biochemistry↗