Woerle, Elisabeth and Newman, Anthony and D'Silva, Jovita and Burgio, Gaetan and Grohmann, Dina (2022) Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain. NUCLEIC ACIDS RESEARCH, 50 (17). pp. 10153-10168. ISSN 0305-1048, 1362-4962
Full text not available from this repository. (Request a copy)Abstract
Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | SINGLE-MOLECULE FRET; RNA-GUIDED ENDONUCLEASE; R-LOOP COMPLEX; CRYSTAL-STRUCTURE; STRUCTURAL BASIS; EVOLUTIONARY CLASSIFICATION; CRISPR; TARGET; CPF1; DIVERSITY; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Mikrobiologie (Archaeenzentrum) > Prof. Dr. Dina Grohmann |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 31 Oct 2023 07:04 |
| Last Modified: | 31 Oct 2023 07:04 |
| URI: | https://pred.uni-regensburg.de/id/eprint/56546 |
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