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Science. 2018 Jul 6;361(6397). pii: eaat0839. doi: 10.1126/science.aat0839. Epub 2018 Jun 7.

Structure basis for RNA-guided DNA degradation by Cascade and Cas3.

Author information

1
Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA.
2
Department of Cell Biology, Harvard Medical School, 250 Longwood Avenue, SGM 509, Boston, MA 02115, USA.
3
Department of Cell Biology, Harvard Medical School, 250 Longwood Avenue, SGM 509, Boston, MA 02115, USA. ailong.ke@cornell.edu maofu_liao@hms.harvard.edu.
4
Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA. ailong.ke@cornell.edu maofu_liao@hms.harvard.edu.

Abstract

Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo-electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop-forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5'-triphosphate-dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.

PMID:
29880725
DOI:
10.1126/science.aat0839

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