In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kDa complex is called Cascade. Here we report the 3.03Å crystal structure of Cascade bound to a single-stranded DNA target. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This non-canonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

Background:The CRISPR-Cas immune system protects E. coli against invasive DNA. Results: We have reconstituted this immune system in vitro using recombinant proteins. Conclusion: Degradation of invasive DNA is tightly regulated and unidirectional. Significance: Reconstitution provides an invaluable tool for understanding the CRISPR-Cas immune system.

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPRassociated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the singlestranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system. Clustered regularly interspaced short palindromic repeat (CRISPR) 2 loci are composed of short DNA repeat sequences separated by stretches of variable spacer sequences (1) that are derived from viral and plasmid DNA (2-5). CRISPR loci are located near clusters of CRISPR-associated (cas) genes that, together with the RNA transcribed from the CRISPR loci, mediate the resistance pathway (6, 7). Although cas gene clusters are extremely diverse in both gene number, sequence, and organization, they can be grouped into three types of CRISPR/ Cas system (6). cas3 is the defining gene of the type I system, which is also the most widespread of the systems, being found in ϳ95% of all genomes containing cas genes (6).CRISPR/Cas systems operate in three stages: adaptation, expression, and interference. During adaptation, foreign DNA is recognized, processed, and integrated into CRISPR loci as new spacer sequences. In the expression stage, CRISPR loci are transcribed and processed into small RNAs (crRNA). In the final interference stage, crRNAs serve as the guide sequences in the silencing of target nucleic acid.In the type I system, the cascade (CRISPR-associated complex for antiviral defense) catalyzes the processing of CRISPR transcripts into crRNA (7). Cascade is a 405-kDa complex comprised of five functionally essential Cas proteins (Cse1 (CRISPR subtype Escherichia coli 1), Cse2, Cas5, Cas6, and Cas7) (6 -8). Following processing, the mature crRNA remains associated with cascade (7) and targets this complex to bind foreign dsDNA. Upon binding, cascade melts the dsDNA, allowing crRNA to form Watson-Crick base pairs with the complementary strand, whereas the noncomplementary strand is exposed as single-stranded DNA (ssDNA) (8). In the type II (9) and type III (10) systems, the ultimate fate of the targeted nucleic acid is cleavage by Cas nucleases. In the type I system, cascade lacks any detectable DNase activity (8). However, ssDNA endonuclease activity has recently been described for Streptococcus thermo...