Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli

van Erp, Paul  B. G.; Jackson, Ryan N.; Carter, Joshua; Golden, Sarah M.; Bailey, Scott; Wiedenheft, Blake

doi:10.1093/nar/gkv793

Title: Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli

Journal Article · Mon Aug 03 00:00:00 EDT 2015 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkv793· OSTI ID:1252758

van Erp, Paul B. G. ^[1]; Jackson, Ryan N. ^[1]; Carter, Joshua ^[1]; Golden, Sarah M. ^[1]; Bailey, Scott ^[2]; Wiedenheft, Blake ^[1]

Montana State Univ., Bozeman, MT (United States)
Johns Hopkins School of Public Health, Baltimore, MD (United States)

In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: USDOE; National Science Foundation (NSF); National Inst. of Health

Grant/Contract Number:: AC02-06CH11357; AC02-76SF00515; P41GM103393; F32 GM108436; P20GM10347; R01GM097330; P20GM103500; R01GM108888; EPS-110134

OSTI ID:: 1252758

Journal Information:: Nucleic Acids Research, Vol. 43, Issue 17; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 38 works

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