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Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ

Overview of attention for article published in Malaria Journal, February 2016
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Title
Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ
Published in
Malaria Journal, February 2016
DOI 10.1186/s12936-016-1166-0
Pubmed ID
Authors

Jitlada Vasuvat, Atcha Montree, Sangduen Moonsom, Ubolsree Leartsakulpanich, Songsak Petmitr, Federico Focher, George E. Wright, Porntip Chavalitshewinkoon-Petmitr

Abstract

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. 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. 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 Mg(2+) and Mn(2+) 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). 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.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 23 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 23 100%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 7 30%
Student > Bachelor 4 17%
Researcher 3 13%
Student > Master 3 13%
Student > Doctoral Student 1 4%
Other 1 4%
Unknown 4 17%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 8 35%
Agricultural and Biological Sciences 5 22%
Chemical Engineering 1 4%
Nursing and Health Professions 1 4%
Environmental Science 1 4%
Other 4 17%
Unknown 3 13%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 26 February 2016.
All research outputs
#5,501,934
of 7,289,383 outputs
Outputs from Malaria Journal
#2,000
of 2,432 outputs
Outputs of similar age
#197,992
of 282,431 outputs
Outputs of similar age from Malaria Journal
#141
of 173 outputs
Altmetric has tracked 7,289,383 research outputs across all sources so far. This one is in the 13th percentile – i.e., 13% of other outputs scored the same or lower than it.
So far Altmetric has tracked 2,432 research outputs from this source. They receive a mean Attention Score of 4.8. This one is in the 9th percentile – i.e., 9% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 282,431 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 15th percentile – i.e., 15% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 173 others from the same source and published within six weeks on either side of this one. This one is in the 12th percentile – i.e., 12% of its contemporaries scored the same or lower than it.