Purification and characterization of a DNA helicase from pea chloroplast that translocates in the 3'-to-5' direction

Abstract

An ATP-dependent DNA hecase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA gase or nuclease activities. The enzyme requires Mg²⁺ or Mn²⁺ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utized. Pea chloroplast DNA hecase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.