Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3Tools Gong, Bei, Shin, Minsang, Sun, Jiali, Jung, Che-Hun, Bolt, Edward L., van der oost, John and Kim, Jeong-Sun (2014) Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3. Proceedings of the National Academy of Sciences, 111 (46). pp. 16359-16364. ISSN 1091-6490 Full text not available from this repository.
Official URL: http://www.pnas.org/content/111/46/16359
AbstractMobile 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.
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