Activation of Thoeris antiviral system via SIR2 effector filament assembly.

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems1-7. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components8-12. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD+ (refs. 12-14). Although the structure of ThsA has been solved15, the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD+ depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.

The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.

Oriented Soft DNA Curtains for Single-Molecule Imaging.

Over the past 20 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 a 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 a 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 by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end 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 the 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 the binding location and position of individual fluorescently labeled proteins on DNA.

The H-subunit of the restriction endonuclease CglI contains a prototype DEAD-Z1 helicase-like motor.

CglI is a restriction endonuclease from Corynebacterium glutamicum that forms a complex between: two R-subunits that have site specific-recognition and nuclease domains; and two H-subunits, with Superfamily 2 helicase-like DEAD domains, and uncharacterized Z1 and C-terminal domains. ATP hydrolysis by the H-subunits catalyses dsDNA translocation that is necessary for long-range movement along DNA that activates nuclease activity. Here, we provide biochemical and molecular modelling evidence that shows that Z1 has a fold distantly-related to RecA, and that the DEAD-Z1 domains together form an ATP binding interface and are the prototype of a previously undescribed monomeric helicase-like motor. The DEAD-Z1 motor has unusual Walker A and Motif VI sequences those nonetheless have their expected functions. Additionally, it contains DEAD-Z1-specific features: an H/H motif and a loop (aa 163-aa 172), that both play a role in the coupling of ATP hydrolysis to DNA cleavage. We also solved the crystal structure of the C-terminal domain which has a unique fold, and demonstrate that the Z1-C domains are the principal DNA binding interface of the H-subunit. Finally, we use small angle X-ray scattering to provide a model for how the H-subunit domains are arranged in a dimeric complex.

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases.

The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5'-GCCGC-3' site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes.

Restriction endonuclease AgeI is a monomer which dimerizes to cleave DNA.

Although all Type II restriction endonucleases catalyze phosphodiester bond hydrolysis within or close to their DNA target sites, they form different oligomeric assemblies ranging from monomers, dimers, tetramers to higher order oligomers to generate a double strand break in DNA. Type IIP restriction endonuclease AgeI recognizes a palindromic sequence 5΄-A/CCGGT-3΄ and cuts it ('/' denotes the cleavage site) producing staggered DNA ends. Here, we present crystal structures of AgeI in apo and DNA-bound forms. The structure of AgeI is similar to the restriction enzymes that share in their target sites a conserved CCGG tetranucleotide and a cleavage pattern. Structure analysis and biochemical data indicate, that AgeI is a monomer in the apo-form both in the crystal and in solution, however, it binds and cleaves the palindromic target site as a dimer. DNA cleavage mechanism of AgeI is novel among Type IIP restriction endonucleases.

Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI.

Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5'-W/CCGGW-3' (W stands for A or T, '/' denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an 'open' configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes.

Crystal structure of the R-protein of the multisubunit ATP-dependent restriction endonuclease NgoAVII.

The restriction endonuclease (REase) NgoAVII is composed of two proteins, R.NgoAVII and N.NgoAVII, and shares features of both Type II restriction enzymes and Type I/III ATP-dependent restriction enzymes (see accompanying paper Zaremba et al., 2014). Here we present crystal structures of the R.NgoAVII apo-protein and the R.NgoAVII C-terminal domain bound to a specific DNA. R.NgoAVII is composed of two domains: an N-terminal nucleolytic PLD domain; and a C-terminal B3-like DNA-binding domain identified previously in BfiI and EcoRII REases, and in plant transcription factors. Structural comparison of the B3-like domains of R.NgoAVII, EcoRII, BfiI and the plant transcription factors revealed a conserved DNA-binding surface comprised of N- and C-arms that together grip the DNA. The C-arms of R.NgoAVII, EcoRII, BfiI and plant B3 domains are similar in size, but the R.NgoAVII N-arm which makes the majority of the contacts to the target site is much longer. The overall structures of R.NgoAVII and BfiI are similar; however, whilst BfiI has stand-alone catalytic activity, R.NgoAVII requires an auxiliary cognate N.NgoAVII protein and ATP hydrolysis in order to cleave DNA at the target site. The structures we present will help formulate future experiments to explore the molecular mechanisms of intersubunit crosstalk that control DNA cleavage by R.NgoAVII and related endonucleases.

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme.

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5'-CCTGG-3'). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5'-ACTGGG-3') complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C-DNA and EcoRII-N-DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C-DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs.

DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis.

The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6-7 nucleotides) downstream of the asymmetric recognition sequence 5'-GCCGC-3'. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.

An Engineered SS Bridge Blocks the Conformational Change Required for the Nuclease Activity of BfiI.

The type IIS restriction endonuclease BfiI is a homodimer, and each monomer is composed of the N-terminal catalytic and C-terminal DNA recognition domains connected by a 28-residue linker segment. In the crystal in the absence of cognate DNA, BfiI exists in a "closed" conformation, in which an interdomain linker occludes a putative DNA binding surface at the catalytic domain and sterically hinders access to the active site. Cognate DNA binding presumably triggers a conformational change from the inactive "closed" state to the catalytically competent "open" state. Here we show that the disulfide SS bridge engineered at the domain interface locks the enzyme in the "closed" state. In the "closed" SS-linked state, BfiI binds cognate DNA with the same affinity as the wild-type enzyme but does not cut it, indicating that cross-linking introduces a restraint on the conformational transition, which couples DNA recognition and cleavage. Disruption of the interdomain SS bridge by the reducing agent restores the DNA cleavage ability of BfiI.

Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease.

Restriction endonuclease Bse634I recognizes and cleaves the degenerate DNA sequence 5'-R/CCGGY-3' (R stands for A or G; Y for T or C, '/' indicates a cleavage position). Here, we report the crystal structures of the Bse634I R226A mutant complexed with cognate oligoduplexes containing ACCGGT and GCCGGC sites, respectively. In the crystal, all potential H-bond donor and acceptor atoms on the base edges of the conserved CCGG core are engaged in the interactions with Bse634I amino acid residues located on the α6 helix. In contrast, direct contacts between the protein and outer base pairs are limited to van der Waals contact between the purine nucleobase and Pro203 residue in the major groove and a single H-bond between the O2 atom of the outer pyrimidine and the side chain of the Asn73 residue in the minor groove. Structural data coupled with biochemical experiments suggest that both van der Waals interactions and indirect readout contribute to the discrimination of the degenerate base pair by Bse634I. Structure comparison between related enzymes Bse634I (R/CCGGY), NgoMIV (G/CCGGC) and SgrAI (CR/CCGGYG) reveals how different specificities are achieved within a conserved structural core.

Structural basis for sequence-specific recognition of guide and target strands by the Archaeoglobus fulgidus Argonaute protein.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like 'long' and truncated 'short' pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a truncated long-B Argonaute protein encoded by an archaeon Archaeoglobus fulgidus. We demonstrate that AfAgo associates with small RNA molecules carrying 5'-terminal AUU nucleotides in vivo, and characterize its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.

smFRET Detection of Cis and Trans DNA Interactions by the BfiI Restriction Endonuclease.

Protein-DNA interactions are fundamental to many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and mechanisms for target search differ across proteins. Especially challenging phenomena to monitor and understand are transient binding events that occur across two DNA target sites, whether occurring in cis or trans. Type IIS restriction endonucleases rely on such interactions. They play a crucial role in safeguarding bacteria against foreign DNA, including viral genetic material. BfiI, a type IIS restriction endonuclease, acts upon a specific asymmetric sequence, 5-ACTGGG-3, and precisely cuts both upper and lower DNA strands at fixed locations downstream of this sequence. Here, we present two single-molecule Förster resonance energy-transfer-based assays to study such interactions in a BfiI-DNA system. The first assay focuses on DNA looping, detecting both "Phi"- and "U"-shaped DNA looping events. The second assay only allows in trans BfiI-target DNA interactions, improving the specificity and reducing the limits on observation time. With total internal reflection fluorescence microscopy, we directly observe on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binds longer to target sites and that BfiI rarely changes conformations during binding. This newly developed assay could be employed for other DNA-interacting proteins that bind two targets and for the dsDNA substrate BfiI-PAINT, a useful strategy for DNA stretch assays and other super-resolution fluorescence microscopy studies.

Catalytic activity control of restriction endonuclease--triplex forming oligonucleotide conjugates.

Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both in vivo and in vitro for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. In this study we present a proof-of-concept demonstration of two strategies to generate restriction endonuclease-TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI-TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease-TFO conjugate technology one step closer to potential in vivo applications.

A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3'-5' and 5'-3' strands by rotating a single active site.

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.

DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme.

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer-dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.

Disarming of type I-F CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9.

CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection.

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

Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour 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 defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.