title: Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.
abstract: CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027, USA.
authors.forename: Sy
authors.initials: S
authors.lastname: Redding
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Sternberg
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Myles
authors.initials: M
authors.lastname: Marshall
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Gibb
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Prashant
authors.initials: P
authors.lastname: Bhat
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Chantal K
authors.initials: CK
authors.lastname: Guegler
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Blake
authors.initials: B
authors.lastname: Wiedenheft
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; Innovative Genomics Initiative, University of California, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA; 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
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address: ecg2108@cumc.columbia.edu.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Greene
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics*; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D004265:DNA Helicases / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008954:Models, Biological; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D064130:CRISPR-Associated Proteins; D004279:DNA, Viral; D029968:Escherichia coli Proteins; C000591054:Cas2 protein, E coli; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C557650:YgbT protein, E coli; D004265:DNA Helicases; C574631:ygcB protein, E coli