Purification and characterization of a novel 3'-5' DNA helicase from Plasmodium falciparum and its sensitivity to anthracycline antibiotics.
Plasmodium falciparum has developed resistance to most anti-malarials; therefore, an investigation of potential targets should be performed. DNA helicases are enzymes that catalyse the unwinding of double-stranded DNA to provide single-stranded templates for DNA replication, repair and recombination. In this study, a DNA helicase (PfDH A) was purified from a crude extract of Plasmodium falciparum. DNA helicase activity was measured by assaying unwinding activity. The apparent molecular weight of PfDH A as determined by SDS-PAGE was 90 kDa. PfDH A moved unidirectionally in the 3' -to- 5' direction along the bound strand and preferred a fork-like substrate structure and could not unwind blunt-ended duplex DNA. Unwinding activity required Mg2+ and could be inhibited by 200 mM NaCl or KCl and was dependent on hydrolysis of ATP or dATP. Anthracyclines, including daunorubicin, nogalamycin, doxorubicin, and aclarubicin, inhibited PfDH A activity with IC50 values of 2, 5, 8 and 9 microM, respectively. Based on the results, PfDH A differs from all known human DNA helicases. However, its function and roles in parasite DNA replication need to be elucidated in the future.
Suntornthiticharoen
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Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Bangkok 10400, Thailand.
Petmitr
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Chavalitshewinkoon-Petmitr
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D000818:Animals; D018943:Anthracyclines / Q000494:pharmacology*; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D020128:Inhibitory Concentration 50; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D012680:Sensitivity and Specificity; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D018943:Anthracyclines; D012965:Sodium Chloride; D011189:Potassium Chloride; D004265:DNA Helicases; D008274:Magnesium