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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/3126
Title: Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ
Authors: Jitlada Vasuvat
Atcha Montree
Sangduen Moonsom
Ubolsree Leartsakulpanich
Songsak Petmitr
Federico Focher
Wright, George E.
Porntip Chavalitshewinkoon‑Petmitr
Mahidol University. Faculty of Tropical Medicine. Department of Protozoology
Keywords: Open Access article;Plasmodium falciparum;DNA polymerase δ;Drug target;Malaria;Biochemical characterization;Functional characterization
Issue Date: 2016
Citation: Malaria Journal. Vol.15, (2016), 116
Abstract: Background: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase δ 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 δ catalytic subunit (PfPolδ-cat), DNA polymerase δ small subunit (PfPolδS) 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. PfPolδ-cat was biochemically characterized. The roles of PfPolδS and PfPCNA in PfPolδ-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPolδ-cat activity and on in vitro parasite growth using SYBR Green I assay. Results: The purified recombinant protein PfPolδ-cat, PfPolδS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPolδ-cat and PfPolδS had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPolδ-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). PfPolδS stimulated PfPolδ-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPolδ-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 μM) and 7-acetoxypentyl-( 3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 μM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 μM). Conclusions: Recombinant PfPolδ-cat, PfPolδS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPolδ-cat. The high sensitivity of PfPolδ 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.
URI: http://repository.li.mahidol.ac.th/dspace/handle/123456789/3126
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