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2017 ; 170
(1
): 48-60.e11
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Structure Basis for Directional R-loop Formation and Substrate Handover
Mechanisms in Type I CRISPR-Cas System
#MMPMID28666122
Xiao Y
; Luo M
; Hayes RP
; Kim J
; Ng S
; Ding F
; Liao M
; Ke A
Cell
2017[Jun]; 170
(1
): 48-60.e11
PMID28666122
show ga
Type I CRISPR systems feature a sequential dsDNA target searching and degradation
process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3,
respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca
type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to
scout for sequence complementarity, and (2) further unwinding of the entire
protospacer to form a full R-loop. These structures provide the much-needed
temporal and spatial resolution to resolve key mechanistic steps leading to Cas3
recruitment. In the early steps, PAM recognition causes severe DNA bending,
leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop
formation triggers conformational changes in Cascade, licensing Cas3 to bind. The
same process also generates a bulge in the non-target DNA strand, enabling its
handover to Cas3 for cleavage. The combination of both negative and positive
checkpoints ensures stringent yet efficient target degradation in type I
CRISPR-Cas systems.