The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum

J Mol Biol. 2011 Jul 1;410(1):27-38. doi: 10.1016/j.jmb.2011.04.071. Epub 2011 May 5.


Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an ∼35-kb circular DNA genome of unusually high A/T content (>86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3'→5' exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T→C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism
  • DNA Replication*
  • DNA, Protozoan / genetics*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Genetic Complementation Test
  • Genome, Protozoan*
  • Humans
  • Malaria, Falciparum / enzymology*
  • Malaria, Falciparum / genetics
  • Molecular Sequence Data
  • Mutation / genetics
  • Plasmodium falciparum / genetics*
  • Sequence Homology, Amino Acid
  • beta-Lactamases / metabolism


  • DNA, Protozoan
  • DNA Polymerase I
  • DNA-Directed DNA Polymerase
  • beta-Lactamases