PMID:23056615

Citation	Zhang, J, Kasciukovic, T and White, MF (2012) The CRISPR associated protein Cas4 Is a 5' to 3' DNA exonuclease with an iron-sulfur cluster. PLoS ONE 7:e47232
Abstract	The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the prokaryotic CRISPR system for antiviral defence. Cas4 is thought to play a role in the capture of new viral DNA sequences for incorporation into the host genome. No biochemical activity has been reported for Cas4, but it is predicted to include a RecB nuclease domain. We show here that Cas4 family proteins from the archaeon Sulfolobus solfataricus utilise four conserved cysteine residues to bind an iron-sulfur cluster in an arrangement reminiscent of the AddB nuclease of Bacillus subtilis. The Cas4 family protein Sso0001 is a 5' to 3' single stranded DNA exonuclease in vitro that is stalled by extrahelical DNA adducts. A role for Cas4 in DNA duplex strand resectioning to generate recombinogenic 3' single stranded DNA overhangs is proposed. Comparison of the AddB structure with that of a related bacterial nuclease from Eubacterium rectales reveals that the iron-sulfur cluster can be replaced by a zinc ion without disrupting the protein structure, with implications for the evolution of iron-sulfur binding proteins.
Links	PubMed PMC3466216 Online version:10.1371/journal.pone.0047232
Keywords	Archaeal Proteins/chemistry; Archaeal Proteins/classification; Archaeal Proteins/genetics; Archaeal Proteins/metabolism; Chromatography, Gel; Exodeoxyribonucleases/chemistry; Exodeoxyribonucleases/classification; Exodeoxyribonucleases/genetics; Exodeoxyribonucleases/metabolism; Iron-Sulfur Proteins/chemistry; Iron-Sulfur Proteins/classification; Iron-Sulfur Proteins/genetics; Iron-Sulfur Proteins/metabolism; Phylogeny; Sulfolobus solfataricus/enzymology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SULSO:CAS4	enables	GO:0045145: single-stranded DNA 5'-3' exodeoxyribonuclease activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
SULSO:CAS4		GO:0045145: single-stranded DNA 5'-3' exodeoxyribonuclease activity	ECO:0000314:		F		Figures 4,5- the exonuclease activity of the gene was studied. A DNA oligonucleotide substrate was labeled at the 5' and 3' terminus with fluorescence. When labeled at the 5' end, the product had a size of around 4 nucleotides, while the 3' end had products that got progressively smaller until they reached 1-2 nucleotides in length. This shows that the gene is involved in the 5' to 3' exodeoxyribonuclease activity and initiates cleavage at the 5' end.	complete CACAO 9333

References

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