Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus

Staals, Raymond H.J.; Agari, Yoshihiro; Maki-Yonekura, Saori; Zhu, Yifan; Taylor, David W.; Duijn, Esther van; Barendregt, Arjan; Vlot, Marnix; Koehorst, Jasper J.; Sakamoto, Keiko; Masuda, Akira; Dohmae, Naoshi; Schaap, Peter J.; Doudna, Jennifer A.; Heck, Albert J. R.; Yonekura, Koji; Oost, John van der; Shinkai, Akeo

doi:10.1016/j.molcel.2013.09.013

Cited by 219 publications

(405 citation statements)

References 41 publications

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“…Type II CRISPR systems are characterized by Cas9, a multidomain protein with a bilobed architecture harboring two nuclease sites that together catalyze cleavage of invading DNA (12, 21-24). The Csm system in CRISPR type IIIA and a related Cmr complex in type IIIB cleave DNA and RNA, respectively, forming a helical structure around crRNA that is structurally similar to Cascade (14,17,18,25,26).Whereas Cas9 and Csm/Cmr effector complexes directly recognize and nucleolytically degrade invading genes (12, 14, 16, 17, 21), Cascade complex by itself is not a nuclease for degradation of invader DNA (13). In Escherichia coli K-12 (type IE), Cascade recognizes and binds crRNA to complimentary sequence in target DNA, generating an RNA mediated displacement loop (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid within double-stranded (ds) target DNA (13).…”

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confidence: 99%

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Gong

Shin

Sun

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HDnucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3′ to 5′ nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3′ to 5′ translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.M any bacteria and archaea can eliminate invading phages and plasmids by activating a defense system that is based on processing of clustered regularly interspaced short palindromic repeats (CRISPRs) by CRISPR-associated (Cas) proteins and various additional proteins. These CRISPR-Cas systems are diverse in structure and function, but common principles of action allow their classification into three major types (I, II, and III) with some further divisions into subtypes (e.g., type IA-F) (1-6).Common CRISPR-mediated immunity processes are usually defined into three stages: (i) acquisition of a short DNA segment (protospacer) from an invading virus or plasmid, and its insertion at the leader-proximal end of a CRISPR locus (7, 8), (ii) generation of small mature CRISPR RNAs (crRNAs) from a longer transcript of a CRISPR locus (9-11), and (iii) interference of foreign nucleic acid invaders by a crRNA-containing ribonucleoprotein effector complex (12-18).Different interference effector complexes characterize the three major CRISPR types. Cascade (Cas complex for antiviral defense) complexes are synonymous with interference in type I CRISPRs, comprising multiple proteins in a helical structure around crRNA that targets invader DNA (13,19, 20). Type II CRISPR systems are characterized by Cas9, a multidomain protein with a bilobed architecture harboring two nuclease sites that together catalyze cleavage of invading DNA (12, 21-24). The Csm system in CRISPR type IIIA and a related Cmr ...

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confidence: 99%

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Gong

Shin

Sun

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Most type III-B effector complexes comprise six proteins (Cmr1-6) (Gasiunas et al, 2014); however, in Sulfolobus solfataricus, these effector complexes contain an additional protein (Cmr7) (Zhang et al, 2012b). Recognized target RNA is cleaved at regular intervals in the 3' to 5' direction (Staals et al, 2013). This digestion pattern indicates the presence of multiple active sites within the backbone of the Cmr complex (Staals et al, 2013), which is composed of multiple subunits of Cmr4 and Cmr5.…”

Section: Target Recognition and Degradationmentioning

confidence: 99%

“…Cascade Thermofilum pendens (Hrle et al, 2014) I-E Cse1, repeat group 2 Cas6 homologues Escherichia coli (Westra et al, 2010) II-C lack of Csn2 and Cas4 Neisseria meningitides III-A Csm2 Cas6 homologues, Csm Staphylococcus epidermidis (Marraffini and III Cas10 Csm Sontheimer, 2008) III-B Cmr5 Cas6 homologues, Cmr Thermus thermophilus (Staals et al, 2013), Cmr RNA Sulfolubus solfataricus (Zhang et al, 2012b) is PAM-independent and is based on the proximity of a repeat sequence to a spacer sequence in the CRISPR locus, which hinders its cleavage (Marraffini and Sontheimer, 2010). The current classification of CRISPR-Cas systems is based on the sequences of the cas genes, the sequences of the repeats within the CRISPR arrays, and the organization of the cas operons (Makarova et al, 2011).…”

Section: Structure Of the Crispr-cas Systemmentioning

confidence: 99%

“…The backbone of the type III crRNP complex is composed of multiple Csm3 (type III-A) or Cmr4 (type III-B) proteins (Rouillon et al, 2013;Spilman et al, 2013;Staals et al, 2013). Like type I CRISPR-Cas effector complexes, the crRNA is positioned along the backbone in type III complexes (Rouillon et al, 2013).…”

Section: Target Recognition and Degradationmentioning

confidence: 99%

“…Recognized target RNA is cleaved at regular intervals in the 3' to 5' direction (Staals et al, 2013). This digestion pattern indicates the presence of multiple active sites within the backbone of the Cmr complex (Staals et al, 2013), which is composed of multiple subunits of Cmr4 and Cmr5. …”

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CRISPR-Cas Systems in Prokaryotes

Burmistrz

Pyrć

2015

Pol J Microbiol

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A b s t r a c tProkaryotic organisms possess numerous strategies that enable survival in hostile conditions. Among others, these conditions include the invasion of foreign nucleic acids such as bacteriophages and plasmids. The clustered regularly interspaced palindromic repeats-CRISPRassociated proteins (CRISPR-Cas) system provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat. This mechanism of immunity is based on short fragments of foreign DNA incorporated within the hosts genome. After transcription, these fragments guide protein complexes that target foreign nucleic acids and promote their degradation. The aim of this review is to summarize the current status of CRISPR-Cas research, including the mechanisms of action, the classification of different types and subtypes of these systems, and the development of new CRISPR-Cas-based molecular biology tools. K e y w o r d s: CRISPR-Cas, prokaryotesBurmistrz M. and Pyrć K. 3 194 spacer sequences can be of either foreign or self-origin (Stern et al., 2010;Vercoe et al., 2013). The proteomic component is responsible for the incorporation of new template sequences, processing them into a form that enables base pairing with target nucleic acids, as well as for scanning and cleavage of target DNA or RNA. Of note, not all CRISPR-Cas systems discovered to date are active (Haft et al., 2005;van der Ploeg, 2009). Structure of the CRISPR-Cas systemThe genomic component of the CRISPR-Cas system is formed by a series of variable spacers, which in some cases share sequence similarity with viruses, plasmids, or bacteria. These regions are interspaced with repeat sequences that are identical or almost identical within a single CRISPR cassette. The length of the repeat sequences varies between 25 and 40 nt, whereas the length of the spacer sequences varies between 21 and 72 nt. As mentioned above, some spacers show high homology with foreign nucleic acids, but the origin of a significant percentage of spacers remains unknown. The sequence homology between the spacer and the target is the major determinant of nucleic acid degradation of the target. Some bacterial species contain more than one CRISPR locus within their genome (Louwen et al., 2014). Depending on the specific bacterial species or strain, a CRISPR locus may contain from a few to several hundred repeat spacer units; however, most commonly, a single CRISPR locus contains approximately 50 units. The CRISPR repeat sequences play an important role during both the acquisition of new spacers and the transcription and maturation of CRISPR RNA (crRNA). Based on the sequence similarity, the CRISPR repeats are assigned into groups (Kunin et al., 2007). These groups were taken into account in the current classification of CRISPR-Cas systems (Makarova et al., 2011). Although most CRISPR arrays are located on chromosomal DNA, there are examples of CRISPRs located on plasmids (Godde and Bickerton, 2006). In general, CRISPR loci are flanked by A/T rich leader sequences containing...

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Structure of Csm2 elucidates the relationship between small subunits of CRISPR‐Cas effector complexes

Venclovas

2016

FEBS Letters

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Type I and type III CRISPR-Cas effector complexes share similar architecture and have homologous key subunits. However, the relationship between the so-called small subunits of these complexes remains a contentious issue. Here, it is shown that the recently solved structure of Thermotoga maritima Csm2 represents a dimer with the extensive structure swapping between monomers. Unswapping the structure generates a compact globular monomer which shares similar structure and surface properties with Cmr5, the small subunit of a related Cmr complex. Detailed analysis of available structures of small subunits reveals that they all have a common fold suggesting their common origin.

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Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus

Cited by 219 publications

References 41 publications

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

CRISPR-Cas Systems in Prokaryotes

Structure of Csm2 elucidates the relationship between small subunits of CRISPR‐Cas effector complexes

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