Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease. Hongfan Chen, Jihoon Choi and Scott Bailey.The Journal of Biological Chemistry, 289, 13284-13294 (2014)
4. To evaluate whether other divalent cations besides Mg2+ can activate DNA cleavage by Cas9, the authors performed plasmid cleavage assays in the presence of one of the following divalent cations: Ca2+, Mn2+, Co2+, Ni2+, and Cu2+.
a. Figure A shows cleavage of a target plasmid by Cas9 with either no metal or 1 mm of the indicated metal ions. All reactions were treated with 0.5 mm EDTA prior to metal addition. Interpret the results.
Answer: Reactions containing Ca2+ yielded nicked, instead of linear plasmid, suggesting that Ca2+ activates only one of the Cas9 nuclease domains
b. To identify which domain was activated, we assayed the single active site mutants of Cas9 (D9A or H599A) in a reaction buffer containing Ca2+. In both panels, the positions of negatively supercoiled (nSC), linear (L), and nicked or open circle (OC) plasmid are indicated. The linear control is a digestion of the plasmid target with the restriction enzyme AgeI.
Answer: Little cleavage was with the HNH mutant (H599A) but robust cleavage with the RuvC-like mutant (D9A) , suggesting that the HNH but not the RuvC-like domain was activated by Ca2+ .
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