Yeast DNA helicase A: cloning, expression, purification, and enzymatic characterization

Area of Science:

• Molecular Biology
• Biochemistry
• Enzymology

Background:

• DNA helicases are crucial enzymes for DNA replication and repair.
• Understanding the structure-function relationship of yeast DNA helicase A is essential for elucidating DNA metabolism pathways.

Purpose of the Study:

• To clone, express, and purify yeast DNA helicase A in a soluble form.
• To characterize the enzymatic activities and quaternary structure of recombinant yeast DNA helicase A.
• To compare the properties of recombinant helicase A with known replicative helicases.

Main Methods:

• High-level expression of yeast DNA helicase A in Escherichia coli.
• Two-step purification protocol for homogeneous enzyme preparation.
• Gel-filtration chromatography to determine quaternary structure.
• ATPase and helicase activity assays.
• Kinetic analysis of ATPase activity.

Main Results:

• Homogeneous recombinant yeast DNA helicase A was obtained in high yield.
• The enzyme exists as a hexamer in low salt concentrations, dissociating into smaller units at higher salt.
• ATPase activity is DNA-dependent, with pyrimidine-rich sequences being potent activators.
• Helicase activity is stimulated by yeast replication protein A (RPA) and E. coli single-stranded DNA binding protein (SSB).
• The enzyme exhibits 5' to 3' directionality on DNA templates.

Conclusions:

• The active form of yeast DNA helicase A is likely hexameric.
• Recombinant helicase A shares functional and structural similarities with other replicative helicases.
• This study provides a foundation for further investigations into the role of helicase A in DNA replication.