Christophe Rouillon

University of St Andrews · BioMedical Sciences Research Complex

CRISPR defense systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition3–5. Among a large set of proteins that were linked to type...

CRISPR antiviral defense systems such as the well-known DNA-targeting Cas9- and the more complex RNA-targeting type III systems are widespread in bacteria and archea. The type III systems can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition. These second messen...

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 met...

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 free...

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 abort...

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 abort...

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 abort...

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 abort...

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...

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, bu...

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 oligoadenyl...

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 oligoadenylat...

The CRISPR-Cas system provides adaptive immunity against mobile genetic elements in prokaryotes, utilising small CRISPR RNAs which direct effector complexes to degrade invading entities. Type III effector complexes were recently demonstrated to synthesise a novel second messenger, cyclic oligoadenylate (cOA), on binding target RNA. cOA in turn bind...

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 orchestr...

Raw data for the kinetic analysis and cOA production presented in Figure 4B.

Raw data for the kinetic analysis presented in Figure 6B.

Raw data for the kinetic analysis presented in Figure 7A.

Raw data for the cOA production data, quantified in triplicate.

Raw data for the kinetic analysis presented in Figure 9B.

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 in...

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,...

Ribonucleoprotein (RNP) complexes from CRISPR–Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base...

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)....

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....

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 compl...

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 (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fus...

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, yi...

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 s...

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 Pyro...

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 enzy...

DNA replication in Archaea, as in other organisms, involves large protein complexes called replisomes. In the Euryarchaeota subdomain, only two putative replicases have been identified, and their roles in leading and lagging strand DNA synthesis are still poorly understood. In this study, we focused on the coupling of proliferating cell nuclear ant...

DNA replication occurs through a large protein complex assembly called replisome. An understanding of its structure and function requires its in vitro reassembly from individual subunits. In all living organisms, the elongation of a primed DNA template by DNA polymerase, with high processivity, requires the accessory protein DNA sliding clamp (PCNA...