Biology subjects

Zaremba, M.

Publications and source records attributed to Zaremba, M..

8 recordsLinked to original sources

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↗

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

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

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion

Defense-associated sirtuins (DSR) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbor an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defense systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

microbiology↗

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

Oriented Soft DNA Curtains for Single Molecule Imaging

Over the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single- or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

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↗