ArticleNucleic acids research2014
The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.
Article in Nucleic acids research, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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22 citing papers in PubMed, 41 citations in OpenAlex.
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- Cyanobacterial Argonautes and Cas4 family nucleases cooperate to interfere with invading DNA.Molecular cell · 2025Article
- CRISPR-Cas Systems: A Functional Perspective and Innovations.International journal of molecular sciences · 2025Review
- Functional insights into Solo-Cas4 inmicroLife · 2025Article
- PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.Molecular cell · 2022Article
- Adaptation by Type III CRISPR-Cas Systems: Breakthrough Findings and Open Questions.Frontiers in microbiology · 2022Review
- The CRISPR-associated Cas4 protein fromCurrent research in microbial sciences · 2021Article
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- Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis.Nucleic acids research · 2021Article
- Cas4 Nucleases Can Effect Specific Integration of CRISPR Spacers.Journal of bacteriology · 2019Article
- The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.eLife · 2019Article
- CRISPR-Cas in Streptococcus pyogenes.RNA biology · 2019Review
- Cas4 Nucleases Define the PAM, Length, and Orientation of DNA Fragments Integrated at CRISPR Loci.Molecular cell · 2018Article
- Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Structural and Mechanistic Analyses Reveal a Unique Cas4-like Protein in the Mimivirus Virophage Resistance Element System.iScience · 2018Article
- Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.Molecular cell · 2018Article
- Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation.Cell reports · 2018Article
- Phylogenomics of Cas4 family nucleases.BMC evolutionary biology · 2017Article
- Structural features of Cas2 from Thermococcus onnurineus in CRISPR-cas system type IV.Protein science : a publication of the Protein Society · 2016Article
- CRISPR-Cas adaptation: insights into the mechanism of action.Nature reviews. Microbiology · 2016Review
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9 authors at 3 institutions in 2 countries.
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Abstract
Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5' to 3' exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5' to 3' and 3' to 5' directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.
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