Site specific target binding controls RNA cleavage efficiency by the Kaposi’s sarcoma-associated herpesvirus endonuclease SOX

Aaron S. Mendez; Carolin Vogt; Jens Bohne; Britt A. Glaunsinger

Notably, SOX activity in the presence of Mg 2+ was inhibited in a dose-dependent manner upon competitive addition of Ca 2+ (Fig. 1C and S1B ). This is likely the result of increased coordination partners engaged by Ca 2+ , which decreases the ability of catalytic residues to promote proper base hydrolysis (23-25). Finally, increasing the NaCl concentration above 100 mM led to substantially decreased SOX activity (Fig. 1D ), in accordance with the observation that high salt concentrations frequently inhibit nuclease activity by disrupting protein-protein or protein-substrate interactions (25). Given that recombinant SOX displays robust 5'-3' exonuclease activity (9, 10) , we sought to confirm that LIMD1 54 was subject to endonucleolytic SOX cleavage, as this is the predominant event that directs mRNA turnover in SOX expressing cells (3, 16) . Both the 5' and 3' ends LIMD1 54 were blocked by capping the 5' end with a Cy5 fluorophore and the 3' end with an Iowa Black quencher (LIMD1 54 Flo). We confirmed this RNA was resistant to degradation by the 5'-phosphate dependent exonuclease terminator (Fig. 1E, lane 3) . However, in the presence of SOX, a cleavage product was observed that correlated with an endonucleolytic cut (Fig. 1E, lane 2) .