title: Cas3 nuclease-helicase activity assays.
abstract: Cas3 is a signature protein of the type I CRISPR-Cas systems and typically contains HD phosphohydrolase and Superfamily 2 (SF2) helicase domains. In the type I CRISPR-Cas systems Cas3 functions as a slicer that provides foreign DNA degradation. Biochemical analysis indicate that Cas3 of the Streptococcus thermophilus DGCC7710 (St-Cas3) CRISPR4 system is a single-stranded DNA nuclease which also possesses a single-stranded DNA-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes in 3' to 5' direction. The interplay between the nuclease and ATPase/helicase activities of St-Cas3 results in DNA degradation. Here, we describe assays for monitoring of St-Cas3 nuclease, ATPase and helicase activities in a stand-alone form and in the presence of the Cascade ribonucleoprotein complex. These assays can be easily adapted for biochemical analysis of Cas3 proteins from different microorganisms.
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, 02241, Vilnius, Lithuania.
authors.forename: Tomas
authors.initials: T
authors.lastname: Sinkunas
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D006868:Hydrolysis; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D013194:Staining and Labeling; D048249:Streptococcus thermophilus / Q000201:enzymology
chemical_list: D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012261:Ribonucleoproteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases