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Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system
CRISPR-Cas adaptive immune systems uptake short “spacer” sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integra...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10504553/ https://www.ncbi.nlm.nih.gov/pubmed/37607619 http://dx.doi.org/10.1016/j.jbc.2023.105178 |
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author | Dhingra, Yukti Sashital, Dipali G. |
author_facet | Dhingra, Yukti Sashital, Dipali G. |
author_sort | Dhingra, Yukti |
collection | PubMed |
description | CRISPR-Cas adaptive immune systems uptake short “spacer” sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn(2+) dependent, Cas1 preferentially uses Mg(2+) over Mn(2+). The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration. |
format | Online Article Text |
id | pubmed-10504553 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-105045532023-09-17 Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system Dhingra, Yukti Sashital, Dipali G. J Biol Chem Research Article CRISPR-Cas adaptive immune systems uptake short “spacer” sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn(2+) dependent, Cas1 preferentially uses Mg(2+) over Mn(2+). The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration. American Society for Biochemistry and Molecular Biology 2023-08-20 /pmc/articles/PMC10504553/ /pubmed/37607619 http://dx.doi.org/10.1016/j.jbc.2023.105178 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Dhingra, Yukti Sashital, Dipali G. Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system |
title | Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system |
title_full | Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system |
title_fullStr | Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system |
title_full_unstemmed | Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system |
title_short | Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system |
title_sort | cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type i-g crispr-cas system |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10504553/ https://www.ncbi.nlm.nih.gov/pubmed/37607619 http://dx.doi.org/10.1016/j.jbc.2023.105178 |
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