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Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Gong, Bei; Shin, Minsang; Sun, Jiali; Jung, Che-Hun; Bolt, Edward L.; van der oost, John; Kim, Jeong-Sun

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3 Thumbnail


Bei Gong

Minsang Shin

Jiali Sun

Che-Hun Jung

Professor of Molecular Biology

John van der oost

Jeong-Sun Kim


Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a “Cascade” ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called “interference.” After target recogni- tion by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mecha- nism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD- nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mecha- nism for 3′ to 5′ nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3′ to 5′ trans- location of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

Journal Article Type Article
Acceptance Date Sep 26, 2014
Online Publication Date Nov 3, 2014
Publication Date Nov 18, 2014
Deposit Date Jul 19, 2016
Publicly Available Date Jul 19, 2016
Journal Proceedings of the National Academy of Sciences
Print ISSN 0027-8424
Electronic ISSN 1091-6490
Publisher National Academy of Sciences
Peer Reviewed Peer Reviewed
Volume 111
Issue 46
Keywords Cas3, CRISPR, Cascade, Bacterial Immunity, Cas Proteins
Public URL
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