title: CRISPR Immunological Memory Requires a Host Factor for Specificity.
abstract: Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: James K
authors.initials: JK
authors.lastname: Nuñez
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Bai
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Harrington
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Tracey L
authors.initials: TL
authors.lastname: Hinder
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D056704:Adaptive Immunity; D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000276:immunology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000276:immunology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007156:Immunologic Memory; D034982:Integration Host Factors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D064130:CRISPR-Associated Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; C440502:integration host factor, E coli; C000591054:Cas2 protein, E coli; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C557650:YgbT protein, E coli