CRISPR/Cas9
RuvC Nuclease
Figure 1: RuvC Nuclease active site with catalytic residues and magnesium shown (original figure) (crystal image rendered from PDB: 4UN3, Anders et al. 2014)
The RuvC Nuclease initiates cleavage of the DNA strand not complementary to the guide RNA. The crystal structure of the active site is shown above, and catalyzes single-stranded DNA cleavage through a mechanism similar to the two-metal mechanisms of RuvC holiday junction resolvases (Nishimasu et al. 2014; Gorecka et al. 2013).
Figure 2: RNase H fold motif of RuvC nuclease domain (original figure) (crystal image rendered from PDB: 4UN3, Anders et al. 2014)
The RuvC nuclease shares an RNase H fold structure, shown to right, with other nucleases in the retroviral integrase superfamily (Nishimasu et al. 2014).
Mutation of any of the catalytic amino acids – histidine 983, aspartate 986, aspartate 10, or glutamate 762 – resulted in loss-of-function of the RuvC nuclease domain, and the creation of a functional Cas9 nickase. This data further supported the two-metal mechanism of RuvC (Nishimasu et al. 2014).
Below is a putative mechanism based on the active site information from a RuvC holiday junction resolvase and the RuvC domain of the Cas9 nuclease (Nishimasu et al. 2014; Gorecka et al. 2013).
(original figure)
(original figure)
I appreciate the work you have put into this site. It has been very useful for an upcoming seminar I am presenting related to the chemistry of Cas9 enzyme. Do you know of any details or do you know a proposed mechanism for release of the phosphate from the Mg ion and release of the cut DNA from the enzyme?
Thank you!
You may want to alter this mechanism to show a two-metal catalyzed reaction. As you pointed out in your text, the active site consists of a two-metal system, where cleavage is facilitated by the lone pair electrons of a OH- attacking the phosphorus center. The Jienk et al. 2014 Science paper provides an excellent structure of the Cas9 RuvC active site in this context. PDB: 4OGC
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