Biochemical and functional characterization of Plasmodium falciparum DNA polymerase delta

Background: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase delta is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. Methods: The coding sequences of DNA polymerase delta catalytic subunit (PfPol delta-cat), DNA polymerase delta small subunit (PfPol delta S) and proliferating cell nuclear antigen (PfPCNA) from chloroquine-and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPol delta-cat was biochemically characterized. The roles of PfPoldS and PfPCNA in PfPol delta-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPol delta-cat activity and on in vitro parasite growth using SYBR Green I assay. Results: The purified recombinant protein PfPol delta-cat, PfPoldS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPol delta-cat and PfPol delta S had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPol delta-cat possessed both DNA polymerase and 3'-5' exonuclease activities. It used both Mg2+ and Mn2+ as cofactors and was inhibited by high KCl salt (> 200 mM). PfPol delta S stimulated PfPol delta-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPol delta-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 mu M) and 7-acetoxypentyl-( 3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 mu M). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 mu M). Conclusions: Recombinant PfPol delta-cat, PfPol delta S and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPol delta-cat. The high sensitivity of PfPol delta to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.