title: Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ.
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 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).
  
  
  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.
authors.affiliation: Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand. jitlada.vasuvat@gmail.com.
authors.forename: Jitlada
authors.initials: J
authors.lastname: Vasuvat
authors.affiliation: Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand. tomorn_at@hotmail.com.
authors.forename: Atcha
authors.initials: A
authors.lastname: Montree
authors.affiliation: Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand. saengduen.moo@mahidol.ac.th.
authors.forename: Sangduen
authors.initials: S
authors.lastname: Moonsom
authors.affiliation: National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Pahonyothin Rd, Pathumthani, 12120, Thailand. ubolsree@biotec.or.th.
authors.forename: Ubolsree
authors.initials: U
authors.lastname: Leartsakulpanich
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand. songsak.pet@mahidol.ac.th.
authors.forename: Songsak
authors.initials: S
authors.lastname: Petmitr
authors.affiliation: Institute of Molecular Genetics, CNR, 27100, Pavia, Italy. focher@igm.cnr.it.
authors.forename: Federico
authors.initials: F
authors.lastname: Focher
authors.affiliation: GLSynthesis Inc., One Innovation Drive, Worcester, MA, 01605, USA. george.wright@glsynthesis.com.
authors.forename: George E
authors.initials: GE
authors.lastname: Wright
authors.affiliation: Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand. porntip.pet@mahidol.ac.th.
authors.forename: Porntip
authors.initials: P
authors.lastname: Chavalitshewinkoon-Petmitr
mesh_terms: D000962:Antimalarials / Q000494:pharmacology; D002478:Cells, Cultured; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004351:Drug Resistance; D004912:Erythrocytes / Q000469:parasitology; D006801:Humans; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000962:Antimalarials; D018809:Proliferating Cell Nuclear Antigen; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D004258:DNA Polymerase III