A Saccharomyces cerevisiae DNA helicase associated with replication factor C

Area of Science:

• Molecular Biology
• Biochemistry
• Yeast Genetics

Background:

• DNA helicases are crucial enzymes involved in DNA replication, repair, and recombination.
• Understanding the properties and functions of novel helicases can elucidate fundamental cellular processes.

Purpose of the Study:

• To isolate and characterize a novel DNA helicase from Saccharomyces cerevisiae.
• To determine the enzymatic properties, cofactor requirements, and subunit composition of the purified helicase.

Main Methods:

• Isolation and purification of DNA helicase using various chromatography techniques (S-Sepharose, HPLC, Affi-Gel Blue, heparin-agarose, ssDNA-cellulose, FPLC MonoS, hydroxyapatite HPLC).
• Separation from Replication Factor C (RF-C) via glycerol gradient sedimentation.
• Biochemical assays to determine ATPase activity, NTP/dNTP cofactor preference, and DNA helicase activity.
• UV cross-linking to assess ATP binding and subunit composition.

Main Results:

• A novel DNA helicase was successfully isolated from Saccharomyces cerevisiae.
• The helicase co-purified with RF-C but was separable by glycerol gradient sedimentation.
• The enzyme is likely a homodimer of a 60-kDa polypeptide, binds ATP, and exhibits single-stranded DNA-dependent ATPase activity.
• Optimal activity was observed with ATP and dATP, with 5' to 3' directionality.

Conclusions:

• A novel ATP-dependent DNA helicase with 5' to 3' directionality has been identified in yeast.
• This helicase possesses distinct biochemical properties and cofactor preferences.
• Further characterization may reveal its specific role in DNA metabolism within Saccharomyces cerevisiae.