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

Abstract

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.

Citation

Gong, B., Shin, M., Sun, J., Jung, C., Bolt, E. L., van der oost, J., & Kim, J. (2014). Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3. Proceedings of the National Academy of Sciences, 111(46), doi:10.1073/pnas.1410806111

• 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
• Electronic ISSN: 1091-6490
• Publisher: National Academy of Sciences
• Peer Reviewed: Peer Reviewed
• Volume: 111
• Issue: 46
• DOI: https://doi.org/10.1073/pnas.1410806111
• Keywords: Cas3, CRISPR, Cascade, Bacterial Immunity, Cas Proteins
• Public URL: http://eprints.nottingham.ac.uk/id/eprint/35113
• Publisher URL: http://www.pnas.org/content/111/46/16359
• Copyright Statement: Copyright information regarding this work can be found at the following address: http://eprints.nottingham.ac.uk/end_user_agreement.pdf