title: CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.
abstract: In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.
authors.affiliation: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, California Institute for Quantitative Biosciences, and Department of Chemistry, University of California, Berkeley, CA 94720.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Hochstrasser
authors.forename: David W
authors.initials: DW
authors.lastname: Taylor
authors.forename: Prashant
authors.initials: P
authors.lastname: Bhat
authors.forename: Chantal K
authors.initials: CK
authors.lastname: Guegler
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Sternberg
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020285:Cryoelectron Microscopy; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021621:Imaging, Three-Dimensional; D046911:Macromolecular Substances / Q000737:chemistry / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D021122:Protein Subunits; D034622:RNA Interference; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D012329:RNA, Bacterial; D004265:DNA Helicases; C574631:ygcB protein, E coli
references.citation: J Biol Chem. 2013 Aug 2;288(31):22184-92
references.pmid: 23760266
references.citation: J Comput Chem. 2004 Oct;25(13):1605-12
references.pmid: 15264254
references.citation: Mol Cell. 2013 Oct 10;52(1):135-145
references.pmid: 24119403
references.citation: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10098-103
references.pmid: 21646539
references.citation: Science. 2007 Mar 23;315(5819):1709-12
references.pmid: 17379808
references.citation: Nat Struct Mol Biol. 2011 May;18(5):529-36
references.pmid: 21460843
references.citation: Nature. 2011 Sep 21;477(7365):486-489
references.pmid: 21938068
references.citation: Science. 2008 Aug 15;321(5891):960-4
references.pmid: 18703739
references.citation: J Struct Biol. 2005 Jul;151(1):41-60
references.pmid: 15890530
references.citation: Nat Rev Microbiol. 2011 Jun;9(6):467-77
references.pmid: 21552286
references.citation: Mol Cell. 2013 Oct 10;52(1):146-52
references.pmid: 24119404
references.citation: Mol Cell. 2013 Oct 10;52(1):124-34
references.pmid: 24119402
references.citation: Nat Protoc. 2009;4(3):363-71
references.pmid: 19247286
references.citation: Mol Cell. 2012 Feb 10;45(3):292-302
references.pmid: 22227116
references.citation: J Struct Biol. 2004 Nov;148(2):194-204
references.pmid: 15477099
references.citation: J Struct Biol. 2007 Jan;157(1):38-46
references.pmid: 16859925
references.citation: Microbiology (Reading). 2009 Mar;155(Pt 3):733-740
references.pmid: 19246744
references.citation: J Struct Biol. 2009 Apr;166(1):95-102
references.pmid: 19263523
references.citation: J Struct Biol. 1999 Dec 1;128(1):82-97
references.pmid: 10600563
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86
references.pmid: 22949671
references.citation: Nature. 2012 Feb 15;482(7385):331-8
references.pmid: 22337052
references.citation: J Struct Biol. 1996 Jan-Feb;116(1):17-24
references.pmid: 8742718
references.citation: Mol Cell. 2012 Jun 8;46(5):606-15
references.pmid: 22521690
references.citation: Trends Genet. 2014 Mar;30(3):111-8
references.pmid: 24555991
references.citation: J Struct Biol. 2004 Jan-Feb;145(1-2):91-9
references.pmid: 15065677
references.citation: EMBO J. 2013 Feb 6;32(3):385-94
references.pmid: 23334296
references.citation: Cell. 2014 Feb 27;156(5):935-49
references.pmid: 24529477
references.citation: Mol Cell. 2012 Jun 8;46(5):595-605
references.pmid: 22521689
references.citation: Science. 2014 Mar 14;343(6176):1247997
references.pmid: 24505130
references.citation: J Struct Biol. 2010 Jun;170(3):427-38
references.pmid: 20338243
references.citation: J Struct Biol. 2007 Jan;157(1):47-55
references.pmid: 16931051
references.citation: J Mol Evol. 2005 Feb;60(2):174-82
references.pmid: 15791728
references.citation: Nature. 2010 Nov 4;468(7320):67-71
references.pmid: 21048762
references.citation: Science. 2012 Aug 17;337(6096):816-21
references.pmid: 22745249
references.citation: Cell. 2009 Nov 25;139(5):945-56
references.pmid: 19945378
references.citation: Ultramicroscopy. 2005 Aug;104(1):8-29
references.pmid: 15935913