Inhibition of unwinding and ATPase activities of Plasmodium falciparum Dbp5/DDX19 homologue

Abstract

We have recently reported the isolation and characterization of Plasmodium falciparum Dbp5/DDX19 homologue PfD66 and the results indicate that it contains ATP-dependent bipolar DNA and RNA unwinding activity, intrinsic nucleic acid-dependent ATPase and RNA-binding activities (Molecular and Biochemical Parasitology, (In press) 10.1016/j.molbiopara.2010.12.003). In the present study we report the effect of a number of compounds such as actinomycin D, aphidicolin, camptothecin, cyclophosphamide, 4’,6’-di-amidino-2-phenylindole (DAPI), daunorubicin, distamycin, ethidium bromide, ellipticine, genistein, mitoxantrone, nalidixic acid, netropsin, nogalamycin, novobiocin and VP-16 on the DNA unwinding and ATPase activities of PfD66. The results indicate that DAPI, ethidium bromide, netropsin and nogalamycin efficiently inhibited the helicase and ATPase activities of PfD66. These studies will make an important contribution in understanding the mechanism of DNA unwinding by Plasmodium falciparum helicase PfD66. 

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Acknowledgements

J.M. is supported by fellowship from the Department of Biotechnology, Govt. of India. This work in R.T.'s laboratory is partially supported by the Department of Biotechnology and Defence Research and Development Organization grants. Infrastructural support from the Department of Biotechnology, Government of India is gratefully acknowledged.

Figures and Tables

Figure 1 The effect of different DNA intercalating compounds on the DNA unwinding activity of PfD66. The reaction was performed using purified enzyme PfD66 and 50 µM of the compound. The DNA-interacting compounds used in the present study were purchased from Topogene Inc., (Columbus, OH), Sigma Chemical Co., (St. Louis, MO) and BDH (E. Merck, Mumbai, India). All of these compounds were dissolved in dimethylsulfoxide (DMSO). The compound added is mentioned at the top of the autoradiogram and (C) is control, no inhibitor is control having enzyme without inhibitor and (B) is the heat denatured substrate. (B–I) Concentration curves of netropsin (B), ethidium bromide (D), DAPI (F) and nogalamycin (H). The DNA helicase reactions were performed by using the same substrate and enzyme as in (A) in the presence of increasing concentration of various compounds. In each (C) is control without enzyme and (B) is the boiled substrate and lanes 1–6 are reactions in the presence of increasing amounts of various compounds. (C, E, G and I) show the quantitative data.

Figure 2 The effect of different DNA intercalating compounds on the ATPase activity of PfD66. The reaction was performed using purified enzyme and 50 µM of the compound. The compound added is mentioned at the top of the autoradiogram and (C) is no enzyme control and lane 1 is control having enzyme without inhibitor. (B–I) Concentration curves of netropsin (B), ethidium bromide (D), DAPI (F) and nogalamycin (H). The assays were performed by using the same substrate and enzyme as in (A) in the presence of increasing concentration of various compounds. In each (C) is control without enzyme and lanes 1–6 are reactions in the presence of increasing amounts of various compounds. (C, E, G and I) show the quantitative data.

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