Purification and characterization of a novel 3′-5′ DNA helicase fromPlasmodium falciparumand its sensitivity to anthracycline antibiotics

Abstract

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.

Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3

“…All the effective inhibitors bind strongly to DNA [39], and so they most likely interfere with PfRuvB3 unwinding function, whose domain ought to be distant from that of ATPase. PfRuvB3 is more sensitive to daunorubicin (IC 50 of 0.76 μM) than other helicases, such as P. falciparum DNA helicase A (PfDH A) [22], P. falciparum UvrD helicase (PfUDN) [40], P. falciparum 45 kDa helicase (PfH45) [41], P. falciparum 60 kDa DNA helicase (PfDH60) [42] and human DNA helicase (HDH II) [43] (IC 50s of 2.0, 4.4, 5.0 3.0, 6.23 μM, respectively). However, it is less sensitive to netropsin (IC 50 of 7.09 μM) than PfUDN, PfH45, PfDH60 and P. falciparum Dbp5/DDX19 homolog (PfD66) [44] (IC 50s of 3.3, 0.8, 0.5, 3.2 μM, respectively).…”

“…However, it is less sensitive to netropsin (IC 50 of 7.09 μM) than PfUDN, PfH45, PfDH60 and P. falciparum Dbp5/DDX19 homolog (PfD66) [44] (IC 50s of 3.3, 0.8, 0.5, 3.2 μM, respectively). Inhibition of PfRuvB3 activity by daunorubicin (IC 50 of 0.76 μM) and doxorubicin (IC 50 of 2.6 μM) did not correlate with parasite growth inhibition (IC 50s of 2.5 and 1.5 μM respectively) [22] suggesting that they may have different cell permeability and metabolic properties.…”

“…Oligodeoxynucleotides were 5′-end labelled using T4 polynucleotide kinase and [γ- 32 P] ATP (800 CimMol −1 ; PerkinElmer, MA, USA). Partial duplex of either short or long oligonucleotide was produced by annealing 34 μM [ 32 P]-labelled oligodeoxynucleotide type 1 or 4 (Table 1) to single-stranded circular M13mp18 (+) DNA (New England Biolabs, MA, USA) in 20 mM Tris–HCl pH 7.5 containing 10 mM MgCl 2 , 100 mM NaCl and 1 mM DTT, incubating at 95 °C for 5 min and then cooling down slowly to room temperature over a period of 1 h [22]. Blunt ended DNA duplex was constructed by annealing 5 ng of 5′ end [ 32 P]-labelled oligonucleotide type 5–50 ng of oligodeoxynucleotide type 6 in 40 mM Tris–HCl pH 7.5 containing 20 mM MgCl 2 , and 50 mM NaCl at 95 °C for 5 min and then cooling to room temperature as described above.…”

Helicases − feasible antimalarial drug target for Plasmodium falciparum

“…To the best of our knowledge only a few helicases have been characterized from P. falciparum . These include two members of the DEAD‐box family namely PfDH60 and P. falciparum DNA helicase 45 (PfDH45) [20,21] (A. Pradhan and R. Tuteja, unpublished) and PfDHA, a 90 kDa DNA helicase which has been purified from P. falciparum [18]. Our studies indicated that PfDH60 contains helicase and ssDNA‐dependent ATPase activities and is expressed in schizont stages of the development of parasite [20,21].…”

“…PfDH45 is a homolog of eukaryotic initiation factor 4A contains helicase and ssDNA‐dependent ATPase activities and is expressed in all the developmental stages of the parasite (A. Pradhan and R. Tuteja, unpublished). PfDHA moves in the 3′ to 5′ direction and prefers a fork‐like substrate for its unwinding activity [18].…”