A CHLOROPLAST DNA HELICASE-II FROM PEA THAT PREFERS FORK-LIKE REPLICATION STRUCTURES

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purifi ed to apparent homogeneity from pea (Pisom sativum). The enzyme contai ned intrinsic, single-stranded, DNA-dependent ATPase activity and an a pparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylami de gel electrophoresis. The DNA helicase was markedly stimulated by DN A substrates with fork-like replication structures. A 5'-tailed fork w as more active than the 3'-tailed fork, which itself was more active t han substrates without a fork. The direction of unwinding was 3' to 5' along the bound strand, and it failed to unwind blunt-ended duplex DN A. DNA helicase activity required only ATP or dATP hydrolysis. The enz yme also required a divalent cation (Mg2+ > Mn2+ > Ca2+) for its unwin ding activity and was inhibited at 200 mM KCl or NaCl. This enzyme cou ld be involved in the replication of ctDNA. The DNA major groove-inter calating ligands nogalamycin and daunorubicin were inhibitory to unwin ding (K-i approximately 0.85 mu M and 2.2 mu M, respectively) and ATPa se (K-i approximately 1.3 mu M and 3.0 mu M, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may b e useful in further studies of the mechanisms of chloroplast helicase activities.