Publication: Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ
Issued Date
2016
Resource Type
Language
eng
Rights
Mahidol University
Rights Holder(s)
BioMed Central
Bibliographic Citation
Malaria Journal. Vol.15, (2016), 116
Suggested Citation
Jitlada Vasuvat, Atcha Montree, Sangduen Moonsom, Ubolsree Leartsakulpanich, Songsak Petmitr, Federico Focher, Wright, George E., Porntip Chavalitshewinkoon‑Petmitr Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ. Malaria Journal. Vol.15, (2016), 116. doi:10.1186/s12936-016-1166-0 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/3126
Research Projects
Organizational Units
Authors
Journal Issue
Thesis
Title
Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ
Other Contributor(s)
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.