CRISPR pp 277-291 | Cite as

Cas3 Nuclease–Helicase Activity Assays

  • Tomas Sinkunas
  • Giedrius Gasiunas
  • Virginijus Siksnys
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)

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.

Key words

Cas3 CRISPR Cascade Streptococcus thermophilus 

Notes

Acknowledgement

We thank Rodolphe Barrangou and Philippe Horvath for discussions. The work on CRISPR systems in Siksnys’s laboratory was funded by the European Social Fund under Global Grant measures.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Tomas Sinkunas
    • 1
  • Giedrius Gasiunas
    • 1
  • Virginijus Siksnys
    • 1
  1. 1.Institute of BiotechnologyVilnius UniversityVilniusLithuania

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