title: The primase domain of PfPrex is a proteolytically matured, essential enzyme of the apicoplast.
abstract: The apicoplast of Plasmodium is an essential organelle with its own circular genome that must be faithfully replicated and segregated to its progeny during parasite sporogony and schizogony. DNA replication proteins are not encoded by its genome. Instead, the replication machinery must be imported from nuclear-encoded genes. A likely apicoplast DNA replication factor, PfPrex, bears a bipartite leader sequence for apicoplast trafficking and contains several DNA replication-related enzymatic domains. Here we analyze the domain structure of PfPrex and examine its trafficking and maturation within the parasite. A minimal primase domain of PfPrex is shown to contain functional zinc-binding and TOPRIM-fold domains, which in a recombinant form are sufficient to produce RNA primers from a single-stranded DNA template. PfPrex is shown to be extensively proteolytically matured within the parasite, which effectively separates its functional domains. Gene targeting attempts to knockout the Plasmodium yoelii ortholog of Prex were unsuccessful, indicating the apparent essentiality of this protein to the parasite. Finally, overexpression in Plasmodium falciparum of PfPrex's trafficking and primase sequences yielded specific and dynamic localization to foci within the apicoplast. Taken together, these observations strongly suggest an essential role of PfPrex primase in the production of RNA primers for lagging strand DNA synthesis of the apicoplast genome.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 550 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706, USA. Scott.Lindner@SeattleBioMed.org
authors.forename: Scott E
authors.initials: SE
authors.lastname: Lindner
authors.forename: Manuel
authors.initials: M
authors.lastname: Llinás
authors.forename: James L
authors.initials: JL
authors.lastname: Keck
authors.forename: Stefan H I
authors.initials: SH
authors.lastname: Kappe
mesh_terms: D000818:Animals; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology*; D051379:Mice; D015388:Organelles / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D019915:DNA Primase; D015032:Zinc