Fig. 3.

Target-bound Csy complex recruits Cas1–2/3 and activates the Cas2/3 nuclease for target degradation. (A) EMSA performed with [³²P]-labeled dsDNA oligonucleotides. Lane 1 is dsDNA alone. DNA binding by Csy complex results in a pronounced shift of the target DNA. Addition of MBP-tagged Cas2/3 results in an additional shift (supershift), indicating the formation of a Cas2/3–DNA–Csy supercomplex. The intensity of the supershifted band (i.e., Cas2/3–DNA–Csy) decreases over the time course sampled (2, 5, 10, 30, 60, and 90 min), whereas the intensity of degraded DNA increases. Neither Cas1 alone nor Cas1–2/3 complex show binding or degradation of dsDNA in the absence of the Csy complex. However, addition of Cas1–2/3 to target-bound Csy results in two supershifted species; the bottom band is consistent with a Cas2/3–DNA–Csy supercomplex lacking the 40 kDa MBP fusion used to purify free Cas2/3. The upper band is most consistent with a supercomplex that includes Cas1–2/3. (B) EMSA performed using only enough Csy complex to bind ∼40% of the labeled DNA. Cas2/3 was added in increasing concentrations, resulting in an increase in supercomplex formation. This experiment was repeated at three time points (5, 10, and 20 min), and results were quantified. (C) Formation of the Csy–DNA–Cas2/3 complex increases with Cas2/3 concentration up to 1:1 Cas2/3:Csy. Additional Cas2/3 does not produce more supercomplex. (D) The amount of unbound DNA (∼60%) remains constant over the time course, suggesting that the Csy complex is single-turnover.