title: Toroidal structure and DNA cleavage by the CRISPR-associated [4Fe-4S] cluster containing Cas4 nuclease SSO0001 from Sulfolobus solfataricus.
abstract: Cas4 proteins, a core protein family associated with the microbial system of adaptive immunity CRISPR, are predicted to function in the adaptation step of the CRISPR mechanism. Here we show that the Cas4 protein SSO0001 from the archaeon Sulfolobus solfataricus has metal-dependent endonuclease and 5'→3' exonuclease activities against single-stranded DNA, as well as ATP-independent DNA unwinding activity toward double-stranded DNA. The crystal structure of SSO0001 revealed a decameric toroid formed by five dimers with each protomer containing one [4Fe-4S] cluster and one Mn(2+) ion bound in the active site located inside the internal tunnel. The conserved RecB motif and four Cys residues are important for DNA binding and cleavage activities, whereas DNA unwinding depends on several residues located near the [4Fe-4S] cluster. Our results suggest that Cas4 proteins might contribute to the addition of novel CRISPR spacers through the formation of 3'-DNA overhangs and to the degradation of foreign DNA.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto , Toronto, Ontario M5S 3E5, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Lemak
authors.forename: Natalia
authors.initials: N
authors.lastname: Beloglazova
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Nocek
authors.forename: Tatiana
authors.initials: T
authors.lastname: Skarina
authors.forename: Robert
authors.initials: R
authors.lastname: Flick
authors.forename: Greg
authors.initials: G
authors.lastname: Brown
authors.forename: Ana
authors.initials: A
authors.lastname: Popovic
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053837:DNA Cleavage; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D048229:Sulfolobus solfataricus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D007506:Iron-Sulfur Proteins; D004247:DNA