Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection.

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

Chemical ligation of an entire DNA origami nanostructure.

Within the field of DNA nanotechnology, numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging applications. These structures typically suffer from a low tolerance against non-optimal environmental conditions including elevated temperatures. Here, we apply a chemical ligation method to covalently seal the nicks between adjacent 5' phosphorylated and 3' amine-modified strands within the DNA nanostructures. Using a cost-effective enzymatic strand modification procedure, we are able to batch-modify all DNA strands even of large DNA objects, such as origami nanostructures. The covalent strand linkage increases the temperature stability of the structures by ∼10 K. Generally, our method also allows a 'surgical' introduction of covalent strand linkages at preselected positions. It can also be used to map the strand ligation into chains throughout the whole nanostructure and identify assembly defects. We expect that our method can be applied to a large variety of DNA nanostructures, in particular when full control over the introduced covalent linkages and the absence of side adducts and DNA damages are required.

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron-Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the Cα-helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 µs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling.

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an 'off-switch' for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator.

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.

Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems.

Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.

Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids1-3. Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA4,5. Cyclic oligoadenylate, in turn, binds to and activates ribonucleases and other factors-via a CRISPR-associated Rossman-fold domain-and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the 'off-switch' that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This 'ring nuclease' is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight to the CRISPR system.

Control of cyclic oligoadenylate synthesis in a type III CRISPR system.

The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.

Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro.

In type I CRISPR-Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade-crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade-crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade-crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA.

Prespacer processing and specific integration in a Type I-A CRISPR system.

The CRISPR-Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3' ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3' ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR-Cas elements and host proteins across CRISPR types.

Real versus sham proximal biofield therapy in the treatment of warts of the hands and feet in adults: study protocol for a randomized controlled trial (MAGNETIK study).

BACKGROUND: Despite the lack of scientific studies on biofield therapies, they are widely acclaimed by patients. The mechanisms of action are not explained by current allopathic medical approaches. Warts are common and contagious viral lesions that may be refractory to standard dermatologic treatments such as cryotherapy, laser therapy, and keratolytic ointments. Biofield therapies are efficient in various pathologies. Their ability to treat warts has never been demonstrated in a scientific study with a robust methodology. Patients with refractory warts often place their trust in these alternative therapies because of the poor results obtained from traditional medicine. We propose a prospective, randomized, single-blind, assessor-blind trial to evaluate the efficacy of treatment of warts by biofield therapy. METHODS/DESIGN: Subjects with warts on their feet or hands will be randomized into two groups: real biofield therapy versus sham therapy. The diagnosis will be made at the time of inclusion, and follow-up will take place in week 3. Comparison of pictures of the warts at baseline and after 3 weeks will be used as the primary outcome measure. The hypothesis is that the extent of the disappearance of the original wart in the group treated by real biofield therapy will be 70% and that it will be 30% in the group treated by sham therapy. Using 90% power and an alpha risk of 5%, 31 subjects are required in each group for a two-tailed proportion comparison test. DISCUSSION: To our knowledge, this is the first study to evaluate the efficacy of biofield therapy on warts. Therefore, the aim of this study is to extend knowledge of biofield therapy to another area of medicine such as dermatology and to propose complementary or alternative practices to improve patient well-being. The main strength of the study is that it is a randomized, single-blind, assessor-blind, placebo-controlled study. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02773719 . Registered on 22 April 2016.

Structure of the CRISPR interference complex CSM reveals key similarities with cascade.

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I-III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family.

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR.

Site-directed spin labeling and pulsed electron-electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.

Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity.

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease.

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5'→3' polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron­sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3'→5' exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

The evolution and mechanisms of nucleotide excision repair proteins.

Nucleotide excision repair (NER) pathways remove a wide variety of bulky and helix-distorting lesions from DNA, and involve the coordinated action of damage detection, helicase and nuclease proteins. Most archaeal genomes encode eucaryal-type NER proteins, including the helicases XPB and XPD and nuclease XPF. These have been a valuable resource, yielding important mechanistic and structural insights relevant to human health. However, the nature of archaeal NER remains very uncertain. Here we review recent studies of archaeal NER proteins relevant to both eucaryal and archaeal NER systems and the evolution of repair pathways.

The XBP-Bax1 helicase-nuclease complex unwinds and cleaves DNA: implications for eukaryal and archaeal nucleotide excision repair.

XPB helicase is an integral part of transcription factor TFIIH, required for both transcription initiation and nucleotide excision repair (NER). Along with the XPD helicase, XPB plays a crucial but only partly understood role in defining and extending the DNA repair bubble around lesions in NER. Archaea encode clear homologues of XPB and XPD, and structural studies of these proteins have yielded key insights relevant to the eukaryal system. Here we show that archaeal XPB functions with a structure-specific nuclease, Bax1, as a helicase-nuclease machine that unwinds and cleaves model NER substrates. DNA bubbles are extended by XPB and cleaved by Bax1 at a position equivalent to that cut by the XPG nuclease in eukaryal NER. The helicase activity of archaeal XPB is dependent on the conserved Thumb domain, which may act as the helix breaker. The N-terminal damage recognition domain of XPB is shown to be crucial for XPB-Bax1 activity and may be unique to the archaea. These findings have implications for the role of XPB in both archaeal and eukaryal NER and for the evolution of the NER pathway. XPB is shown to be a very limited helicase that can act on small DNA bubbles and open a defined region of the DNA duplex. The specialized functions of the accessory domains of XPB are now more clearly delineated. This is also the first direct demonstration of a repair function for archaeal XPB and suggests strongly that the role of XPB in transcription occurred later in evolution than that in repair.

The helicase XPD unwinds bubble structures and is not stalled by DNA lesions removed by the nucleotide excision repair pathway.

Xeroderma pigmentosum factor D (XPD) is a 5'-3' superfamily 2 helicase and the founding member of a family of DNA helicases with iron-sulphur cluster domains. As a component of transcription factor II H (TFIIH), XPD is involved in DNA unwinding during nucleotide excision repair (NER). Archaeal XPD is closely related in sequence to the eukaryal enzyme and the crystal structure of the archaeal enzyme has provided a molecular understanding of mutations causing xeroderma pigmentosum and trichothiodystrophy in humans. Consistent with a role in NER, we show that archaeal XPD can initiate unwinding from a DNA bubble structure, differentiating it from the related helicases FancJ and DinG. XPD was not stalled by substrates containing extrahelical fluorescein adducts, abasic sites nor a cyclobutane pyrimidine dimer, regardless of whether these modifications were placed on either the displaced or translocated strands. This suggests that DNA lesions repaired by NER may not present a barrier to XPD translocation in vivo, in contrast to some predictions. Preferential binding of a fluorescein-adducted oligonucleotide was observed, and XPD helicase activity was readily inhibited by both single- and double-stranded DNA binding proteins. These observations have several implications for the current understanding of the NER pathway.

Binding to PCNA in Euryarchaeal DNA Replication requires two PIP motifs for DNA polymerase D and one PIP motif for DNA polymerase B.

Replicative DNA polymerases possess a canonical C-terminal proliferating cell nuclear antigen (PCNA)-binding motif termed the PCNA-interacting protein (PIP) box. We investigated the role of the PIP box on the functional interactions of the two DNA polymerases, PabPol B (family B) and PabPol D (family D), from the hyperthermophilic euryarchaeon Pyrococcus abyssi, with its cognate PCNA. The PIP box was essential for interactions of PabPol B with PCNA, as shown by surface plasmon resonance and primer extension studies. In contrast, binding of PabPol D to PCNA was affected only partially by removing the PIP motif. We identified a second palindromic PIP box motif at the N-terminus of the large subunit of PabPol D that was required for the interactions of PabPol D with PCNA. Thus, two PIP motifs were needed for PabPol D for binding to PabPCNA. Moreover, the C-terminus of PabPCNA was essential for stimulation of PabPol D activity but not for stimulation of PabPol B activity. Neither DNA polymerase interacted with the PabPCNA interdomain connecting loop. Our data suggest that distinct processes are involved in PabPol D and PabPol B binding to PCNA, raising the possibility that Archaea require two mechanisms for recruiting replicative DNA polymerases at the replication fork.