Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation

Huo, YW; Nam, Ki Hyun; Ding, Fang; Hj, Lee; Wu, Lijun; Xiao, YB; Farchione,; Zhou, Shi-Sheng; Rajashankar, Kanagalaghatta R.; Kurinov, Igor; Rg, Zhang; Ke, AL

doi:10.1038/nsmb.2875

Cited by 165 publications

(225 citation statements)

References 40 publications

(84 reference statements)

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“…1A) will cofunction with the Cst complex as the effector nuclease/helicase. Based on expectations from other Type I systems, the Cst crRNP may function as a surveillance complex that recruits Cas3 nuclease to cleave invader DNA (Brouns et al 2008;Cady and O'Toole 2011;Jore et al 2011;Wiedenheft et al 2011a,b;Nam et al 2012;Sashital et al 2012;van Duijn et al 2012;Westra et al 2012b;Mulepati and Bailey 2013;Sinkunas et al 2013;Hochstrasser et al 2014;Huo et al 2014). We likely did not detect Cas3 in our isolated Cst crRNP complexes (Fig.…”

Section: Cas Protein Composition and Functional Roles Of Csa And Cst mentioning

confidence: 69%

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Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Majumdar

Zhao

Pfister

et al. 2015

RNA

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CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, and III) each target destruction of foreign nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr (Type III-B) Cas proteins associated with one of two size classes of crRNAs and cleaves complementary target RNAs. Here, we have isolated and characterized two additional native Pfu crRNPs containing either Csa (Type I-A) or Cst (Type I-G) Cas proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by mass spectrometry and immunoblotting and the crRNAs by RNA sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from all seven Pfu CRISPR loci and contain identical 5 ′ ends (8-nt repeat-derived 5 ′ tag sequences) but heterogeneous 3 ′ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3 ′ end processing pathways following primary cleavage of common pre-crRNAs. Like other previously characterized Type I CRISPR-Cas effector complexes, we predict that the newly identified Pfu Csa and Cst crRNPs each function to target invading DNA, adding an additional layer of protection beyond that afforded by the previously characterized RNA targeting Cmr complex.

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Section: Cas Protein Composition and Functional Roles Of Csa And Cst mentioning

confidence: 69%

“…We likely did not detect Cas3 in our isolated Cst crRNP complexes (Fig. 2) because the Cas3 protein may not associate with Cst complexes in the absence of (invader) DNA targets (Hochstrasser et al 2014;Huo et al 2014).…”

Section: Cas Protein Composition and Functional Roles Of Csa And Cst mentioning

confidence: 99%

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Majumdar

Zhao

Pfister

et al. 2015

RNA

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“…While this paper was in revision, the crystal structure of an irondependent Cas3 from Thermobifida fusca (TfusCas3) was published in complex with ssDNA derived from the expression host (46). In TfusCas3, ssDNA binding causes differences in relative orientation of RD2 and CTD compared with the structure we observe in TteCas3 (Fig.…”

Section: Discussionmentioning

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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“…[18][19][20] In the systems targeting foreign DNA, Cas3 is known to be involved in DNA unwinding and degradation as a single-stranded DNA (ssDNA) nuclease. 13,21 However, the Type III-B system exclusively targets RNA and little is known about the degradation mechanism of RNA after being cleaved by the CMR complex. 22,23 The hyperthermophilic archaea Archaeoglobus fulgidus DSM 4304 possesses 3 CRISPR loci and at least 20 putative cas genes.…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease

2015

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In prokaryotes, the CRISPR/Cas system is known to target and degrade invading phages and foreign genetic elements upon subsequent infection. However, the structure and function of many Cas proteins remain largely unknown, due to the high diversity of Cas proteins. Here we report 3 crystal structures of Archaeoglobus fulgidus Csx3 (AfCsx3) in free form, in complex with manganese ions and in complex with a single-stranded RNA (ssRNA) fragment, respectively. AfCsx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structurebased biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at 2 separate surfaces within the AfCsx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, AfCsx3 displays robust 3 0 -deadenylase activity in the presence of manganese ions, which strongly suggests that AfCsx3 functions as a deadenylation exonuclease. Taken together, our results indicate that AfCsx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of AfCsx3 in the Type III-B CRISPR/ Cas system.

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Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation

Cited by 165 publications

References 40 publications

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

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

Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease

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