Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)

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Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)

2011; Elsevier BV; Volume: 286; Issue: 24 Linguagem: Inglês

10.1074/jbc.m111.238485

ISSN

1083-351X

Autores

Nathanael G. Lintner, Melina Kerou, Susan K. Brumfield, Shirley Graham, Huanting Liu, James H. Naismith, Matthew A Sdano, Nan Peng, Qunxin She, Valérie Copié, Mark Young, Malcolm F. White, C. Martin Lawrence,

Tópico(s)

Cytomegalovirus and herpesvirus research

Resumo

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea. In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea. IntroductionAdaptive and heritable immune systems have recently been recognized in Bacteria and Archaea. These systems consist of loci termed clustered regularly interspaced short palindromic repeats (CRISPRs) 6The abbreviations used are: CRISPRclustered regularly interspaced short palindromic (prokaryotic) repeatsCASCADECRIPSR-associated complex for antiviral defenseaCASCADEarchaeal (Apern) CASCADECasCRISPR-associatedcrRNACRISPR-RNARRMRNA-recognition motifCsaCRISPR-subtype ApernCseCRISPR-subtype E. coliCsyCRISPR-subtype YpestCstCRISPR-subtype TneapCsdCRISPR-subtype DvulgCshCRISPR-subtype HmariPAMprotospacer-adjacent motifTEMtransmission electron microscopyntnucleotide(s)dsdouble-strandedsssingle-strandedNi-NTAnickel-nitrilotriacetic acid. and their associated protein encoding (cas) genes (1van der Oost J. Jore M.M. Westra E.R. Lundgren M. Brouns S.J. Trends Biochem. Sci. 2009; 34: 401-407Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 2Horvath P. Barrangou R. Science. 2010; 327: 167-170Crossref PubMed Scopus (1532) Google Scholar, 3Sorek R. Kunin V. Hugenholtz P. 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Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 9Hale C.R. Zhao P. Olson S. Duff M.O. Graveley B.R. Wells L. Terns R.M. Terns M.P. Cell. 2009; 139: 945-956Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar) in which the variable sequences, also known as spacers, serve as guide sequences for the recognition and neutralization of invading DNA (7Barrangou R. Fremaux C. Deveau H. Richards M. Boyaval P. Moineau S. Romero D.A. Horvath P. Science. 2007; 315: 1709-1712Crossref PubMed Scopus (3704) Google Scholar, 8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 10Marraffini L.A. Sontheimer E.J. Science. 2008; 322: 1843-1845Crossref PubMed Scopus (1153) Google Scholar, 11Garneau J.E. Dupuis M.È. Villion M. Romero D.A. Barrangou R. Boyaval P. Fremaux C. Horvath P. Magadán A.H. Moineau S. Nature. 2010; 468: 67-71Crossref PubMed Scopus (1442) Google Scholar, 12Jore M.J. Lundgren M. van Duijn E. Butema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. Beijer M.R. Barendregt A. Zhou K. Snijders A.P. Dickman M.J. Doudna J.A. Boekema E.J. Heck A.J. van der Oost J. Brouns S.J.J. Nat. Struct. Mol. Biol. 2011; (in press)PubMed Google Scholar) or RNA (9Hale C.R. Zhao P. Olson S. Duff M.O. Graveley B.R. Wells L. Terns R.M. Terns M.P. Cell. 2009; 139: 945-956Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar).Co-occurrence patterns for cas genes within genomes and gene clusters suggest the Cas machinery takes on several different forms, referred to as subtypes (13Haft D. Selengut J. Mongodin E. Nelson K. Plos Comp. Biol. 2005; 1: 474-483Crossref Scopus (721) Google Scholar) or CRISPR-associated systems (14Makarova K.S. Grishin N.V. Shabalina S.A. Wolf Y.I. Koonin E.V. Biol. Direct. 2006; 1: 7Crossref PubMed Scopus (814) Google Scholar). These systems share a set of core cas genes, which include cas1–6, and eight sets of subtype specific genes (cse, csa, cst, csm, csy, csh, csn, and csd). A given CRISPR/Cas system will thus encode several of the core Cas proteins plus at least one of these eight subtypes. However, distant relationships across subtypes for several gene families have been recognized, such that some of the cas subtype gene families can be unified into superfamilies loosely based on clusters of orthologous groups (14Makarova K.S. Grishin N.V. Shabalina S.A. Wolf Y.I. Koonin E.V. Biol. Direct. 2006; 1: 7Crossref PubMed Scopus (814) Google Scholar). In addition, many CRISPR/Cas systems include a third cluster of genes that belong to the repeat associated mysterious protein superfamily and are named cmr1–6 (13Haft D. Selengut J. Mongodin E. Nelson K. Plos Comp. Biol. 2005; 1: 474-483Crossref Scopus (721) Google Scholar).Several activities of the CRISPR-associated protein machinery are now recognized. One function is the acquisition and insertion of new spacers into the CRISPR loci. Whereas little is known about this process, it is thought to involve Cas1 and Cas2. A second function is processing the CRISPR transcript to produce crRNA, and several endoribonucleases that process crRNA have now been identified, including Pyrococcus furiosus Cas6. A third function is the use of crRNA to guide neutralization of non-host RNA with the RNAi-like activity of the CMR complex, which has been demonstrated in P. furiosus. Interestingly, however, Cas6 and the CMR complex lack apparent homology to eukaryotic RNAi protein machinery in both primary sequence and three-dimensional structure (9Hale C.R. Zhao P. Olson S. Duff M.O. Graveley B.R. Wells L. Terns R.M. Terns M.P. Cell. 2009; 139: 945-956Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar, 15Beloglazova N. Brown G. Zimmerman M.D. Proudfoot M. Makarova K.S. Kudritska M. Kochinyan S. Wang S. Chruszcz M. Minor W. Koonin E.V. Edwards A.M. Savchenko A. Yakunin A.F. J. Biol. Chem. 2008; 280: 20361-20371Abstract Full Text Full Text PDF Scopus (158) Google Scholar, 16Carte J. Wang R. Li H. Terns R.M. Terns M.P. Genes Dev. 2008; 22: 3489-3496Crossref PubMed Scopus (408) Google Scholar, 17Wang R. Preamplume G. Terns M.P. Terns R.M. Li H. Structure. 2011; 19: 257-264Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Last, and most relevant, a fourth function of CRISPR/Cas is the use of crRNA to guide neutralization of invading DNA (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar). This activity is mediated by the CRISPR-associated complex for antiviral defense (CASCADE) (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar).The characterization of Escherichia coli CASCADE revealed a complex composed of the Cse1–4 subtype proteins and Cas5e. Collectively, these 5 proteins are also known as CasA–CasE. Together, they form a 405-kDa complex with crRNA that allows recognition of single- and double-stranded target DNAs complementary to the bound crRNA (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 12Jore M.J. Lundgren M. van Duijn E. Butema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. Beijer M.R. Barendregt A. Zhou K. Snijders A.P. Dickman M.J. Doudna J.A. Boekema E.J. Heck A.J. van der Oost J. Brouns S.J.J. Nat. Struct. Mol. Biol. 2011; (in press)PubMed Google Scholar). Following recognition by CASCADE, the nuclease/helicase activity of Cas3 is then recruited to degrade the invading DNA (18Sinkunas T. Gasiunas G. Fremaux C. Barrangou R. Horvath P. Siksnys V. EMBO J. 2011; 30: 1335-1342Crossref PubMed Scopus (291) Google Scholar). Each component of E. coli CASCADE, along with Cas3, is required for viral resistance in vivo (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar).Transmission electron microscopy, small angle x-ray scattering, and non-covalent mass spectrometry reveal that E. coli CASCADE has an unusual quaternary structure, consisting of six copies of CasC(Cse4), which form the core or backbone of the CASCADE complex (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 12Jore M.J. Lundgren M. van Duijn E. Butema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. Beijer M.R. Barendregt A. Zhou K. Snijders A.P. Dickman M.J. Doudna J.A. Boekema E.J. Heck A.J. van der Oost J. Brouns S.J.J. Nat. Struct. Mol. Biol. 2011; (in press)PubMed Google Scholar). This core is complemented by single copies of CasA, CasD(Cas5e) and CasE(Cse3), with CasE exhibiting an endoribonuclease activity that specifically cleaves the CRISPR transcript (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 19Perez-Rodriguez R. Haitjema C. Huang Q. Nam K.H. Bernardis S. Ke A. Delisa M.P. Mol. 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Science. 2010; 329: 1355-1358Crossref PubMed Scopus (485) Google Scholar).Of the E. coli CASCADE protein components, only the CasC(Cse4) and CasD(Cas5e) protein families are observed in the other Cas subtypes or Cas systems with recognizable CASCADE components (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 13Haft D. Selengut J. Mongodin E. Nelson K. Plos Comp. Biol. 2005; 1: 474-483Crossref Scopus (721) Google Scholar, 14Makarova K.S. Grishin N.V. Shabalina S.A. Wolf Y.I. Koonin E.V. Biol. Direct. 2006; 1: 7Crossref PubMed Scopus (814) Google Scholar), where their distant homologues are known under a variety of different names, and the nomenclature can be quite confusing. However, increased recognition of the central role of these protein families in CRISPR/Cas is leading to the adoption of unifying nomenclature, and the superfamilies that contain CasD and CasC are now increasingly referred to as Cas5 and Cas7, respectively. Interestingly, these superfamilies display conserved gene synteny, and thus, are likely to represent evolutionarily conserved elements that lie at the core of CASCADE structure and function.Sulfolobus species represent an important model system for Archaea in general, and the Crenarchaeota in particular, and have been quite useful for investigating the function of CRISPR/Cas (15Beloglazova N. Brown G. Zimmerman M.D. Proudfoot M. Makarova K.S. Kudritska M. Kochinyan S. Wang S. Chruszcz M. Minor W. Koonin E.V. Edwards A.M. Savchenko A. Yakunin A.F. J. Biol. Chem. 2008; 280: 20361-20371Abstract Full Text Full Text PDF Scopus (158) Google Scholar, 22Mojica F.J. Díez-Villaseñor C. García-Martínez J. Soria E. J. Mol. 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Tsutakawa S.E. Alsbury D.L. Copié V. Young M.J. Tainer J.A. Lawrence C.M. J. Mol. Biol. 2011; 405: 939-955Crossref PubMed Scopus (62) Google Scholar, 31Manica A. Zebec Z. Teichmann D. Schleper C. Mol. Microbiol. 2011; 80: 481-491Crossref PubMed Scopus (79) Google Scholar). The Sulfolobus solfataricus P2 genome encodes six CRISPR loci designated CRISPRs A-F (32Lillestøl R. Shah S.A. Brügger K. Redder P. Phan H. Christiansen J. Garret R.A. Mol. Microbiol. 2009; 72: 259-272Crossref PubMed Scopus (183) Google Scholar), and multiple paralogs of cas, csa (CRISPR subtype Apern), and cmr gene products, including three paralogs of the CASCADE-like cas5/cas7 gene cassette. In S. solfataricus the Cas5 and Cas7 proteins have been generally referred to as Cas5a and Csa2, respectively. Here we report the identification of an archaeal CASCADE (aCASCADE), and confirm the central roles of archaeal Csa2 and Cas5a in this complex. We also report the functional characterization of Csa2 and Cas5a in the recognition of crRNA and invading DNA by aCASCADE, and the crystallographic structure of Csa2, the first structure for a member of the Cas7 superfamily.DISCUSSIONHere we report the first structure of a Cas7 protein, and the isolation and characterization of a complex that bears many of the hallmarks expected of an archaeal CASCADE. Similar to E. coli CASCADE (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 12Jore M.J. Lundgren M. van Duijn E. Butema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. Beijer M.R. Barendregt A. Zhou K. Snijders A.P. Dickman M.J. Doudna J.A. Boekema E.J. Heck A.J. van der Oost J. Brouns S.J.J. Nat. Struct. Mol. Biol. 2011; (in press)PubMed Google Scholar), aCASCADE includes a Cas7/CasC protein (Csa2), a CasD/Cas5e ortholog (Cas5a), and the complex co-purifies from S. solfataricus with processed crRNA. Furthermore, the recombinant Csa2-Cas5a complex produced in E. coli specifically binds crRNA, which in turn, recognizes single-stranded "target" DNA in vitro. Finally, the complex isolated from S. solfataricus also co-purifies with the more weakly interacting or lower abundance components, Cas6, Csa5, and perhaps Csa4. In S. solfataricus aCASCADE, Cas6 appears to serve a function analogous to that of E. coli CasE/Cse3 (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar). Thus, there are clear orthologs in aCASCADE for each component of E. coli CASCADE except CasA and CasB, components that appear to be limited to the E. coli CRISPR/Cas subtype (13Haft D. Selengut J. Mongodin E. Nelson K. Plos Comp. Biol. 2005; 1: 474-483Crossref Scopus (721) Google Scholar, 14Makarova K.S. Grishin N.V. Shabalina S.A. Wolf Y.I. Koonin E.V. Biol. Direct. 2006; 1: 7Crossref PubMed Scopus (814) Google Scholar), although the possibility that Csa4 (Sso1401/Cas8a2) is functionally similar to CasA might be considered. Importantly, the presence of core CASCADE components in S. solfataricus aCASCADE (Csa2/Cas5a), as well as Cas6, suggests that structural and functional studies of aCASCADE are relevant not only to the Apern subtype (Csa) CASCADE, but are also generally relevant to orthologs in other CRISPR/Cas subtypes, especially the Cst, Csh, and Csm subtypes that associate with Cas6 (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 13Haft D. Selengut J. Mongodin E. Nelson K. Plos Comp. Biol. 2005; 1: 474-483Crossref Scopus (721) Google Scholar, 14Makarova K.S. Grishin N.V. Shabalina S.A. Wolf Y.I. Koonin E.V. Biol. Direct. 2006; 1: 7Crossref PubMed Scopus (814) Google Scholar, 61Lawrence C.M. White M.F. Structure. 2011; 19: 142-144Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar).S. solfataricus Cas6Although annotated as Cas6 orthologs, the crenarchaeal Cas6 proteins are highly diverged from the well characterized euryarchaeal Cas6 protein (16Carte J. Wang R. Li H. Terns R.M. Terns M.P. Genes Dev. 2008; 22: 3489-3496Crossref PubMed Scopus (408) Google Scholar). The two protein families share little sequence similarity beyond the glycine-rich region that is the hallmark of these proteins. Most significantly, the proposed Tyr-His-Lys catalytic triad of Pyrococcus Cas6 (16Carte J. Wang R. Li H. Terns R.M. Terns M.P. Genes Dev. 2008; 22: 3489-3496Crossref PubMed Scopus (408) Google Scholar) does not appear to be conserved. It is therefore significant to see that S. solfataricus Cas6 functions like its Pyrococcus counterpart in vitro, processing the pre-crRNA transcript to generate crRNA, and that like Pyrococcus Cas6, Csy4, and CasE(Cse3) (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 16Carte J. Wang R. Li H. Terns R.M. Terns M.P. Genes Dev. 2008; 22: 3489-3496Crossref PubMed Scopus (408) Google Scholar, 21Haurwitz R.E. Jinek M. Wiedenheft B. Zhou K. Doudna J.A. Science. 2010; 329: 1355-1358Crossref PubMed Scopus (485) Google Scholar), S. solfataricus Cas6 generates crRNA with an 8-base 5′ handle that contains the conserved GAAA(C/G) motif (62Kunin V. Sorek R. Hugenholtz P. Genome Biol. 2007; 8: R61Crossref PubMed Scopus (343) Google Scholar). Our data also suggest that Cas6 may show only moderate affinity for aCASCADE. Like many organisms utilizing Cas6, S. solfataricus contains both the CMR and CASCADE systems. Indeed, it appears that Cas6 is associated with all CRISPR subtypes (Cst, Csh, Csm, and Csa) predicted or shown to contain unstructured CRISPR repeats and both the CMR and CASCADE systems (13Haft D. Selengut J. Mongodin E. Nelson K. Plos Comp. Biol. 2005; 1: 474-483Crossref Scopus (721) Google Scholar, 17Wang R. Preamplume G. Terns M.P. Terns R.M. Li H. Structure. 2011; 19: 257-264Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 61Lawrence C.M. White M.F. Structure. 2011; 19: 142-144Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 62Kunin V. Sorek R. Hugenholtz P. Genome Biol. 2007; 8: R61Crossref PubMed Scopus (343) Google Scholar). The potentially loose association of Cas6 in the S. solfataricus aCASCADE may allow it to function in the initial processing of the CRISPR transcript for both CRISPR systems.Structural Models for aCASCADEThe overexpression of Csa2 in S. solfataricus results in the production of extended right-handed helical assemblies of variable length. The ability of the extended Csa2 assembly to bind crRNA and to protect the crRNA from RNase digestion suggests that the crRNA is bound by protein along its entire length. That the assembly copurifies with Cas5a, Csa5, and Cas6, in addition to the crRNA, also suggests that many aspects of the assembly are physiologically relevant. However, the extended helical filaments observed in preparations from S. solfataricus are longer than needed to accommodate a single crRNA; one turn of the Csa2 helix should be more than sufficient to accomplish this. Thus, if these helices are physiologically relevant, they are likely to harbor multiple crRNAs and could potentially be used in succession to screen potential target DNA for a match to the collection of bound crRNA.Alternatively, we note that the open symmetry of the Csa2 assembly coupled with high concentrations of Csa2 from overexpression in S. solfataricus and substoichiometric amounts of endogenous Cas5a, Csa5, or Cas6, might allow the assembly to grow to physiologically irrelevant lengths, particularly in the presence of stabilizing crRNA. Thus, we also consider a shorter model, in which native aCASCADE includes a single crRNA and a limited number of Csa2 subunits, resulting in an arch-shaped structure corresponding to less than one turn of helix. Indeed, this second model is consistent with the most recent model for E. coli CASCADE, which binds a single crRNA and is observed as a smaller arch-shaped particle (12Jore M.J. Lundgren M. van Duijn E. Butema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. Beijer M.R. Barendregt A. Zhou K. Snijders A.P. Dickman M.J. Doudna J.A. Boekema E.J. Heck A.J. van der Oost J. Brouns S.J.J. Nat. Struct. Mol. Biol. 2011; (in press)PubMed Google Scholar). We suggest that the arch-shaped backbone of E. coli CASCADE is explained by the presence of a half-turn or more of CasC helix (supplemental Fig. S5), and that the helical, open symmetry of aCASCADE is also present in the CasC backbone of E. coli CASCADE.In either model, the major function of Csa2 appears to be the construction of an extended assembly that functions to support the crRNA spacer sequence along its entire length (thus protecting it from RNase digestion). At the same time, we anticipate that the Watson-Crick edges of the bases must remain solvent exposed and available for interaction with target DNA, such that aCASCADE also effectively templates or presents the crRNA spacer sequence for DNA recognition. This suggests that some of the conserved surface features on Csa2, including His160, will tightly interact with RNA in a sequence independent manner. Collectively, the interactions with the Csa2 protomers might also serve to stabilize a hybrid RNA/target-DNA complex, and/or destabilize bound dsDNA, allowing DNA within the cell to be surveyed for homology to the bound crRNA spacer.How is crRNA specifically recruited to aCASCADE? The data are consistent with a cooperative CASCADE assembly process that is dependent on the presence of crRNA, as we do not see extended helices of recombinant Csa2 or Csa2/Cas5a in the absence of crRNA. In addition, whereas the Csa2 backbone of aCASCADE is expected to bind the variable CRISPR spacer in a sequence-independent manner, the complex clearly distinguishes crRNA from other cellular RNAs, most likely through sequence-specific interactions with the 5′ and 3′ handles. For these reasons, it is attractive to consider roles for the additional aCASCADE components (Cas5a, Cas6, Csa4, and Csa5) in specific crRNA recognition. Such interactions might also serve to nucleate (crRNA induced oligomerization) or terminate growth of the Csa2 helix, and thus govern the length of the Csa2 backbone in aCASCADE. Termination of helix growth could, in turn, limit the length of the nucleoprotein filament to a single crRNA, giving rise to an E. coli-like aCASCADE assembly, rather than the extended nucleoprotein filaments seen when Csa2 alone is overexpressed in S. solfataricus. Thus, guided by our own data, and by the current model for E. coli CASCADE (8Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Science. 2008; 321: 960-964Crossref PubMed Scopus (1618) Google Scholar, 12Jore M.J. Lundgren M. van Duijn E. Butema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. Beijer M.R. Barendregt A. Zhou K. Snijders A.P. Dickman M.J. Doudna J.A. Boekema E.J. Heck A.J. van der Oost J. Brouns S.J.J. Nat. Struct. Mol. Biol. 2011; (in press)PubMed Google Scholar), we propose the model for aCASCADE presented in Fig. 6. The structural core of aCASCADE is modeled as a partial turn of Csa2 helix, with crRNA running along the length of the Csa2 assembly, and Cas5a, Cas6, Csa4, Csa5, or other unidentified proteins at the 5′- and 3′-ends, where they may serve to initiate and terminate growth of the complex.Once assembled, CASCADE must probe DNA within the cell for sequences complimentary to the bound crRNA spacers. Although we cannot predict with certainty which surface CASCADE might be utilized for this process, we note that the concave surface formed by a partial turn of the Csa2 helix is large enough to accommodate, or wrap around dsDNA, or a hybrid RNA-DNA complex. Furthermore, several of our TEM-based models indicate that the partial Csa2 helix can be docked to dsDNA in a coaxial arrangement, that is, with the Csa2 helical axis coincident with that of the DNA double helix, such that the Csa2 helix wraps around the dsDNA. In such an arrangement, the Csa2 protomers would be positioned along the dsDNA, each equidistant from the DNA, potentially facilitating DNA surveillance. For these reasons, we tentatively place the conserved surfaces of Csa2 and crRNA along the concave surface of the oligomeric Csa2 arch. However, we must emphasize that there is, as yet, no experimental evidence to support the position of the DNA or crRNA within the proposed model. On the other hand, this model is similar to the current model for RecA, where RecA binds ssDNA to form a helical nucleoprotein filament, which in turn catalyzes recognition of homologous dsDNA and strand exchange to produce the heteroduplex. In particular, crystallographic and EM studies indicate RecA and its eukaryotic homologs do indeed wrap around dsDNA such that the helical axes of the protein and nucleic acid are coincident with each other (63Chen Z. Yang H. Pavletich N.P. Nature. 2008; 453: 489-494Crossref PubMed Scopus (493) Google Scholar, 64Sheridan S.D. Yu X. Roth R. Heuser J.E. Sehorn M.G. Sung P. Egelman E.H. Bishop D.K. Nucleic Acids Res. 2008; 36: 4057-4066Crossref PubMed Scopus (79) Google Scholar), similar to the tentative model for CASCADE that we propose here.Upon reflection, additional advantages of the unusual open symmetry of the Csa2 oligomer become apparent, part

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Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)