Mechanism of Foreign DNA Recognition and Degradation by the Type-I CRISPR System in Escherechia coli

CRISPR;RNA-based immune system;DNA targeting;Cascade;Cas3;DNA helicase;DNA-binding protein;Nuclease;RNA;RNA-binding protein;Biophysics

Mulepati, SabinLeahy, Daniel J. ;

Johns Hopkins University

关键词: CRISPR;    RNA-based immune system;    DNA targeting;    Cascade;    Cas3;    DNA helicase;    DNA-binding protein;    Nuclease;    RNA;    RNA-binding protein;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39299/Sabin%20Mulepati%20Thesis_Final.docx?sequence=2&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) immune system is used by bacteria and archaea to gain immunity from mobile genetic elements like phage DNA and plasmids. In Escherichia coli, small RNA derived from its CRISPR loci (crRNA) are integrated into a large ribonucleoprotein complex called Cascade, which is then used as a surveillance complex to find foreign DNA based on sequence complementary. Previous studies suggested that Cascade recruits an additional nuclease-helicase protein called Cas3 to silence foreign DNA in order to gain immunity. To understand the roles of Cascade and Cas3, we carried out structural and biochemical studies on both of these essential components of the CRISPR immune system.Here, we report the crystal structure of the Cascade complex from E. coli bound to its target DNA to 3.03 Å. The structure reveals a DNA-RNA hybrid at the core of the complex, forming a heavily distorted, discontinuous, arched-ladder that locally forms short A-form-like duplexes. Bases in both strands of the hybrid are flipped out at regular intervals due to the organization of the protein subunits in the complex. The structure presented here shows how Cascade-like complexes have evolved to form a distorted hybrid that is likely primed for recruitment of Cas3 for further degradation of the invasive DNA.We also report the crystal structure of the HD nuclease domain of Cas3 from T. thermophilus, and characterize its nuclease active site. Based on additional biochemical analysis, we show that the HD nuclease likely uses a two-metal-ion-dependent cleavage mechanism. Furthermore, using individually purified Cascade and Cas3 from E. coli, we reconstituted CRISPR-mediated plasmid degradation in vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking, and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.Taken together, the results of our study explain how foreign DNA is identified and degraded by the CRISPR immune system, and provide a solid framework for future studies.