Biochemical and functional characterization of Plasmodium falciparum DNA polymerase delta

Background The emergence of drug-resistant Plasmodium falciparum has increased the urgent need for new drug targets. DNA polymerase delta is an essential enzyme required for chromosomal DNA replication and repair, and may therefore be a potential target for antimalarial drug development. However, a little is known about its characteristics and function in this deadly parasite. Methods The coding sequences of DNA polymerase delta catalytic subunit (PfPoldelta-cat), a small subunit (PfPoldeltaS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine and pyrimethamine resistant P. falciparum strain K1 were amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant proteins were analyzed by SDS-PAGE and identified by LC-MS/MS. PfPoldelta-cat was biochemically characterized. Roles of PfPoldeltaS and P. falciparum proliferating cell nuclear antigen (PfPCNA) on PfPoldelta-cat were investigated. Inhibitory effects of 11 compounds were determined on PfPoldelta-cat activity and in vitro parasite growth using SYBR Green I. Results The purified recombinant proteins PfPoldelta-cat, PfPoldeltaS and PfPCNA showed the expected sizes of 143 kDa, 57 kDa and 34 kDa on SDS-PAGE, respectively. Predicted amino acid sequences of the PfPoldelta-cat and PfPoldeltaS were 59.2% and 24.7 % similarity to that of the human counterparts. The PfPoldelta-cat possessed both DNA polymerase and associated 3' to 5' exonuclease activities. It could use both Mg2+ and Mn2+ as cofactors and was inhibited by high KCl salt (>200 mM). PfPoldeltaS stimulated PfPoldelta-cat activity 3-folds and up to 4-folds when PfPCNA was also included. PfPoldelta-cat was inhibited by 4 compounds but only 2 compounds were potent namely Butylphenyl-dGTP (BuPdGTP, IC50 38 µM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (IC50 55 µM). However, the latter compound showed higher inhibition on parasite growth (IC50 of 4.1 µM). Conclusions The recombinant PfPoldelta-cat, PfPoldelta and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA can increase DNA polymerase activity of PfPoldelta-cat. The high sensitivity of PfPoldelta to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-(3, 4 dichlorobenzyl)guanine showed inhibitory effects on both enzyme activity and parasite growth. Therefore, 7-acetoxylpentyl-DCBG can be a potential candidate for development of new antimalarial agents targeting parasite replicative DNA polymerase in the future.