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Cell. 2015 Nov 5;163(4):854-65. doi: 10.1016/j.cell.2015.10.003.

Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

Author information

1
Department of Chemistry, Columbia University, New York, NY 10027, USA.
2
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
3
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
4
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
5
Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
6
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.
7
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address: ecg2108@cumc.columbia.edu.

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.

PMID:
26522594
PMCID:
PMC4636941
DOI:
10.1016/j.cell.2015.10.003
[Indexed for MEDLINE]
Free PMC Article

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