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

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (REF: BB/M021017/1 to MFW).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.Publisher PDFPeer reviewe

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

En libre-accès sur Archimer : http://archimer.ifremer.fr/doc/2009/publication-7317.pdfInternational audienceReplicative 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

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

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (Grant REF BB/S000313/1 to MFW) and the Wellcome Trust (Grant 210486/Z/18/Z to CMC).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.Publisher PDFPeer reviewe

Contribution of vertical and horizontal components of ground reaction forces on global motor moment during a golf swing: a preliminary study

No abstract availabl

Control of cyclic oligoadenylate synthesis in a type III CRISPR system

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (REF: BB/M000400 /1 to MFW), and a Royal Society Challenge Grant (REF: CH160014 to MFW).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.Publisher PDFPeer reviewe

Determination of the intervertebral spinal axial rotation in a golf player population: a preliminary study

No abstract availabl

Biomechanical analysis of the golf swing: methodological effect of angular velocity component on the identification of the kinematic sequence

The golf swing is a complex whole-body motion for which a proximal-to-distal transfer of the segmental angular velocitiesfrom the pelvis to the club is believed to be optimal for maximizing the club head linear velocity. However, previous experimental resultsabout such timing (or kinematic sequence) are contradictory. Nevertheless, methods that were used in these studies differed significantly,in particular, those regarding the component of the angular velocity vector selected for the identification of the kinematic sequence.Hence, the aim of this study was to investigate the effect of angular velocity vector component selection on the identified kinematicsequence. Methods: Thirteen golfers participated in this study and performed driver swings in a motion capture laboratory. Seven meth-ods based on different component selection of segmental angular velocities (vector norm, component normal-to-sagittal, frontal, trans-versal and swing planes, segment longitudinal component and a method mixing longitudinal and swing plane components) were tested.Results: Results showed the critical influence of the component chosen to identify the kinematic sequence with almost as many kine-matic sequences as the number of tested methods for every golfer. Conclusion: One method seems to show the strongest correlation toperformance but none of them can be assessed as a reference method for the identification of the golf swing kinematic sequence. Re-garding the limited time lag between the different peak occurrences and the uncertainty sources of current materials, development ofsimulation studies would be more suitable to identify the optimal kinematic sequence for the golf swin

Effect of shoulder model complexity in upper-body kinematics analysis of the golf swing

The golf swing is a complex full body movement during which the spine and shoulders are highly involved. In order to determine shoulder kinematics during this movement, multibody kinematics optimization (MKO) can be recommended to limit the effect of the soft tissue artifact and to avoid joint dislocations or bone penetration in reconstructed kinematics. Classically, in golf biomechanics research, the shoulder is represented by a 3 degrees-of-freedom model representing the glenohumeral joint. More complex and physiological models are already provided in the scientific literature. Particularly, the model used in this study was a full body model and also described motions of clavicles and scapulae. This study aimed at quantifying the effect of utilizing a more complex and physiological shoulder model when studying the golf swing. Results obtained on 20 golfers showed that a more complex and physiologically-accurate model can more efficiently track experimental markers, which resulted in differences in joint kinematics. Hence, the model with 3 degrees-of-freedom between the humerus and the thorax may be inadequate when combined with MKO and a more physiological model would be beneficial. Finally, results would also be improved through a subject-specific approach for the determination of the segment lengths

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

Skoltech Ph.D. program in the Life Sciences (to A.K.); European Research Council consolidator grant [GA 724863 to R.S].; NIH [R01 GM10407] and Russian Science Foundation [14-14-00988] grants to K.S.; UMNIK grant 8115GU/2015 to O.M., and institutional support from Skoltech to K.S. Funding for open access charge: Skolkovo Institute of Science and Technology internal funding.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.Publisher PDFPeer reviewe

Influence of the projection plane and the markers choice on the X-factor computation of the golf swing X-factor: a case study

Study of variability induced by the methology choice on the X-factor computation of the golf swing. Based on optoelectronic measurements. Variability of the choice of upper limb, plane of projection and instant of computation