Cascade-mediated binding and bending of negatively supercoiled DNA.

Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop.