Characterization of the endonuclease SSO2001 from Sulfolobus solfataricus P2

Abstract 

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) have been suggested to act as an immune system in archaea and bacteria mimicking the eukaryotic RNA interference (RNAi) system. We have investigated the properties of the protein SSO2001 from Sulfolobus solfataricus (Sso) P2, which is part of the cas gene cluster. This study shows that SSO2001 is an endonuclease specifically digesting double-stranded oligonucleotides and preferably cleaving at G:C pairs. Point mutations identify both highly conserved aspartate and glutamate residues as being crucial for the nuclease activity. The catalytic activity shows an optimum at neutral pH and pH 3.

Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats, CAS, CRISPR-associated, COG, cluster of orthologous groups, RNAi, RNA interference, Sso, Sulfolobus solfataricus, ss(ds), single (double)-stranded, SSO2001Est, SSO2001-esterase fusion protein, TFK, trifluoremethyl ketone, PAGE, polyacrylamide gel electrophoresis, nt, nucleotide, bp, base pair, MES, 2-(N-morpholino)ethanesulfonic acid, MSH, mercaptoethanol, NDSB, non-detergent sulfobetaines

Keywords: CRISPR-associated, Clustered regularly interspaced short palindromic repeats, Nuclease, SSO2001