title: Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity.
abstract: The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: James K
authors.initials: JK
authors.lastname: Nuñez
authors.affiliation: 1] Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. [2] Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Kranzusch
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Jonas
authors.initials: J
authors.lastname: Noeske
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Addison V
authors.initials: AV
authors.lastname: Wright
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Davies
authors.affiliation: 1] Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. [2] Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA. [3] Department of Chemistry, University of California, Berkeley, Berkeley, California, USA. [4] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D056704:Adaptive Immunity; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D018360:Crystallography, X-Ray; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000276:immunology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; C000591054:Cas2 protein, E coli; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C557650:YgbT protein, E coli