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.