title: The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum.
abstract: 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.
authors.affiliation: Department of Pathology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Scott R
authors.initials: SR
authors.lastname: Kennedy
authors.forename: Cheng-Yao
authors.initials: CY
authors.lastname: Chen
authors.forename: Michael W
authors.initials: MW
authors.lastname: Schmitt
authors.forename: Cole N
authors.initials: CN
authors.lastname: Bower
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D016054:DNA, Protozoan / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D018503:Genome, Protozoan; D006801:Humans; D016778:Malaria, Falciparum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010963:Plasmodium falciparum / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D016054:DNA, Protozoan; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D001618:beta-Lactamases