CasDinG is a 5'-3' dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity.
In: Nucleic acids research, Jg. 51 (2023-08-25), Heft 15, S. 8115-8132
academicJournal
Zugriff:
CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Here, we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP-dependent 5'-3' DNA translocase that unwinds double-stranded (ds)DNA and RNA/DNA hybrids. The crystal structure of CasDinG reveals a superfamily 2 helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these domains, we identified the preferred PAM sequence for the type IV-A system (5'-GNAWN-3' on the 5'-side of the target) with a plasmid library and performed plasmid clearance assays with domain deletion mutants. Plasmid clearance assays demonstrated that all three domains are essential for type IV-A immunity. Protein expression and biochemical assays suggested the vFeS domain is needed for protein stability and the arch for helicase activity. However, deletion of the N-terminal domain did not impair ATPase, ssDNA binding, or helicase activities, indicating a role distinct from canonical helicase activities that structure prediction tools suggest involves interaction with dsDNA. This work demonstrates CasDinG helicase activity is essential for type IV-A CRISPR immunity as well as the yet undetermined activity of the CasDinG N-terminal domain.
(© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Titel: |
CasDinG is a 5'-3' dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity.
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Autor/in / Beteiligte Person: | Domgaard, H ; Cahoon, C ; Armbrust, MJ ; Redman, O ; Jolley, A ; Thomas, A ; Jackson, RN |
Zeitschrift: | Nucleic acids research, Jg. 51 (2023-08-25), Heft 15, S. 8115-8132 |
Veröffentlichung: | 1992- : Oxford : Oxford University Press ; <i>Original Publication</i>: London, Information Retrieval ltd., 2023 |
Medientyp: | academicJournal |
ISSN: | 1362-4962 (electronic) |
DOI: | 10.1093/nar/gkad546 |
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