PURIFICATION AND CHARACTERIZATION OF A DNA HELICASE FROM PEA CHLOROPLAST THAT TRANSLOCATES IN THE 3'-TO-5' DIRECTION

An ATP-dependent DNA helicase has been purified to near homogeneity fr om 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 ligase 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 poo rly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex w hether it has unpaired single-stranded tails at bath 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 u nidirectionally from 3' to 5' along the bound strand. The unwinding ac tivity is inhibited by the intercalating drugs nogalamycin and daunoru bicine.