Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation

Killelea, Tom; Dimude, Juachi U; He, Liu; Stewart, Alison L; Kemm, Fiona E; Radovčí, Marin; Ivančić-Baće, Ivana; Rudolph, Christian J; Bolt, Edward L

Abstract

Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on naive adaptation-Cas1-Cas2 targeting of MGE DNA without the aid of pre-existing immunity 'interference' complexes-by mechanisms that are not clear. Using E. coli we show that the chaperone DnaK inhibits DNA binding and integration by Cas1-Cas2, and inhibits naive adaptation in cells that results from chromosomal self-Targeting. Inhibition of naive adaptation was reversed by deleting DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage λ) protein. We also imaged fluorescently labelled Cas1 in living cells, observing that Cas1 foci depend on active DNA replication, and are much increased in frequency in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naive adaptation from DNA self-Targeting, until DnaK is triggered to release Cas1-Cas2 to target MGE DNA.

Citation

Killelea, T., Dimude, J. U., He, L., Stewart, A. L., Kemm, F. E., Radovčí, M., Ivančić-Baće, I., Rudolph, C. J., & Bolt, E. L. (2023). Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation. Nucleic Acids Research, 51(13), 6914-6926. https://doi.org/10.1093/nar/gkad473

• Journal Article Type: Article
• Acceptance Date: May 16, 2023
• Online Publication Date: Jun 2, 2023
• Publication Date: Jul 21, 2023
• Deposit Date: May 25, 2023
• Publicly Available Date: May 31, 2023
• Journal: Nucleic Acids Research
• Print ISSN: 0305-1048
• Electronic ISSN: 1362-4962
• Publisher: Oxford University Press
• Peer Reviewed: Peer Reviewed
• Volume: 51
• Issue: 13
• Pages: 6914-6926
• DOI: https://doi.org/10.1093/nar/gkad473
• Keywords: Genetics
• Public URL: https://nottingham-repository.worktribe.com/output/21107847
• Publisher URL: https://academic.oup.com/nar/article/51/13/6914/7188753
• Additional Information: © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Res.earch.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.