Cas3 nuclease-helicase activity assays

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Type I CRISPR-Cas systems utilize Cas3 protein for foreign DNA degradation.
• Cas3 typically possesses HD phosphohydrolase and Superfamily 2 (SF2) helicase domains.
• The Streptococcus thermophilus DGCC7710 (St-Cas3) CRISPR4 system's Cas3 protein acts as a single-stranded DNA nuclease with ATPase activity.

Purpose of the Study:

• To develop and describe assays for monitoring the nuclease, ATPase, and helicase activities of St-Cas3.
• To investigate these activities both in isolation and in complex with the Cascade ribonucleoprotein complex.
• To provide adaptable methods for studying Cas3 proteins from various microorganisms.

Main Methods:

• Biochemical assays were employed to measure St-Cas3's enzymatic functions.
• Assays were performed on purified St-Cas3 in a stand-alone form.
• Activities were also assessed in the presence of the Cascade ribonucleoprotein complex.

Main Results:

• St-Cas3 exhibits single-stranded DNA nuclease activity.
• The protein possesses single-stranded DNA-stimulated ATPase activity.
• This ATPase activity is coupled to 3' to 5' unwinding of DNA/DNA and RNA/DNA duplexes, facilitating DNA degradation.

Conclusions:

• The combined nuclease and ATPase/helicase activities of St-Cas3 are crucial for DNA degradation.
• The described assays are effective for characterizing St-Cas3's biochemical functions.
• These assays offer a versatile platform for analyzing diverse microbial Cas3 proteins.