Structural and Biochemical Characterization of CRISPR-associated Cas4 Nucleases from a Prokaryotic Defense System

Nucleases are an essential component of the prokaryotic CRISPR-Cas immunity as well as repair mechanisms within prokaryotic organisms. To better understand the adaptation step of CRISPR-Cas immunity, I have characterized three Cas4 proteins from hyperthermophilic archaea: SSO0001 and SSO1391 from Sulfolobus solfataricus and Pcal_0546 from Pyrobaculum calidifontis. All three proteins have metal-dependent 5′ to 3′ exonuclease and endonuclease activities, while SSO1391 also demonstrates 3′ to 5′ exonuclease activity. Site-directed mutagenesis confirmed that the conserved RecB motif residues are important for the nuclease activity in all three proteins. SSO0001 and Pcal_0546 also exhibit ATP-independent unwinding and cleavage of splayed arm substrates. Structural analysis of SSO0001 showed it is a toroidal decamer with a [4Fe-4S] cluster and Mn2+ ion bound in the active site located inside the internal tunnel. Our results show that Cas4 proteins have the ability to create 3'-DNA overhangs which may contribute to the addition of novel CRISPR spacers.MAS