Inhibition of pea chloroplast DNA helicase unwinding and ATPase activities by DNA-interacting ligands

Abstract

DNA helicases unwind the duplex DNA in an ATP dependent manner and thus play an essential role in DNA replication, repair, recombination and transcription. Any DNA-interacting ligand which will modulate DNA helicase activity may interrupt practically all kinds of DNA transactions. There are no studies on the effect of various cytotoxic DNA-interacting ligands on organelle helicases. We have determined the effect of camptothecin, VP-16 (etoposide), ellipticine, genistein, novobiocin, m-AMSA, actinomycin C, ethidium bromide, daunorubicin and nogalamycin on unwinding and ATPase activities of purified chloroplast DNA helicase from pea (Pisum sativum). Our study has shown that DNA-intercalating ligands actinomycin C, ethidium bromide, daunorubicin and nogalamycin were inhibiting the DNA unwinding activity with an apparent Ki of 2.9 μM, 3.0 μM, 1.4 μM and 1.0 μM, respectively. These four inhibitors also inhibited the ATPase activity of pea chloroplast DNA helicase. These results indicate that the intercalation of the inhibitors into DNA generates a complex that impedes the translocation of chloroplast DNA helicase, resulting in both inhibition of unwinding activity and ATP hydrolysis. This study would be useful for understanding the mechanism of organelle DNA helicase unwinding and the mechanism by which these DNA-interacting ligands inhibit cellular function.