Purified human SUV3p exhibits multiple-substrate unwinding activity upon conformational change

Area of Science:

• Mitochondrial biology
• Molecular enzymology
• Biochemistry

Background:

• Suv3 protein in yeast (Saccharomyces cerevisiae) is classified as a mitochondrial RNA helicase.
• The helicase domain of yeast and human SUV3 differs from typical RNA helicase motifs, necessitating investigation of its enzymatic activities.
• A yeast suv3 null mutant exhibits growth defects on non-fermentable carbon sources and forms petite colonies in glucose medium.

Purpose of the Study:

• To purify and characterize the enzymatic activities of human SUV3.
• To investigate the substrate specificity and functional requirements of human SUV3 helicase activity.
• To explore the role of protein conformation and ATP hydrolysis in SUV3 function.

Main Methods:

• Expression of a truncated human SUV3 (SUV3DeltaN46) in a yeast suv3 null mutant to rescue the null phenotype.
• Purification of SUV3 protein variants (SUV3-83 and SUV3-70) using a five-step chromatographic procedure.
• Assays for ATPase activity, DNA/RNA unwinding, and circular dichroism (CD) spectral analysis.

Main Results:

• Purified SUV3-83 and SUV3-70 proteins exhibit ATPase activity stimulated by single- and double-stranded DNA and RNA.
• SUV3-70 unwinds RNA and DNA duplexes with 5'-3' directionality across a wide pH range, while SUV3-83 requires acidic pH.
• ATP hydrolysis is essential for unwinding, and mutations in the ATP-binding site abolish both ATPase and helicase activities; CD analysis indicates a pH-dependent conformational change in SUV3-83.

Conclusions:

• Human SUV3 possesses broad substrate specificity for DNA and RNA unwinding, dependent on ATP hydrolysis.
• A pH-dependent conformational change is crucial for the helicase activity of the SUV3-83 variant.
• The findings suggest a significant role for human SUV3 in mitochondrial nucleic acid metabolism, potentially involving DNA and RNA processing.