Biology subjects

Sasnauskas, G.

Publications and source records attributed to Sasnauskas, G..

8 recordsLinked to original sources

bioRxiv2026

Csx15 is a broad specificity ring nuclease bridging CRISPR-Cas and CBASS cyclic nucleotide signaling systems

Prokaryotic antiviral type III CRISPR-Cas and CBASS systems use cyclic nucleotides as second messengers to activate diverse effector proteins. Whereas CBASS signaling typically triggers abortive infection, type III CRISPR-Cas signaling is tightly regulated through controlled signal production and degradation. Here, we define the molecular mechanism of the CRISPR-associated Csx15 protein family and demonstrate that Csx15 functions as a broad specificity ring nuclease capable of degrading cyclic nucleotide signals from both CRISPR-Cas and CBASS pathways. Structural and biochemical analyses reveal that Csx15 adopts diverse oligomeric states and undergoes ligand-induced conformational transitions that promote filament assembly required for catalytic activity, analogous to filament-forming SAVED-domain effectors. Finally, we provide evidence that Csx15 is a close relative of SAVED domains suggesting that Csx15-like proteins may have also contributed to the evolution of CBASS cyclic nucleotide signaling.

biochemistry↗

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

Off-target detection of CRISPR-Cas9 nuclease in vitro with CROFT-Seq

Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases could lead to unintended changes in the edited genome. Detection of off-target sites is critical to make genome editing technology safe and predictable. Although current in vitro methods for off-target detection can identify these sites, they are time-consuming, complex, and relatively costly. Here, we present CROFT-Seq (CRISPR nuclease off-target detection by sequencing), a sensitive, rapid, and cost-effective assay for the genome-wide detection of Cas9 off-target sites in vitro. CROFT-Seq performs comparably to the common currently used in vitro methods and serves as a valuable and efficient tool for the rapid assessment of genome-editing nuclease specificity. Notably, a high proportion of the top-ranked off-targets identified by CROFT-Seq were validated in cells, highlighting its effectiveness as a predictor of off-target sites.

biochemistry↗

bioRxiv2024

Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated SAVED-Lon protease CalpL, which forms a tripartite effector system together with an anti-{sigma} factor, CalpT, and an ECF-like {sigma} factor, CalpS. Here we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.

biochemistry↗

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↗

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↗