Fidelity, Mismatch Extension, and Proofreading Activity of the Plasmodium falciparum Apicoplast DNA Polymerase

Abstract

Plasmodium falciparum, a parasitic organism and one of the causative agents of malaria, contains an unusual organelle called the apicoplast. The apicoplast is a nonphotosynthetic plastid responsible for supplying the parasite with isoprenoid units and is therefore indispensable. Like mitochondria and the chloroplast, the apicoplast contains its own genome and harbors the enzymes responsible for its replication. In this report, we determine the relative probabilities of nucleotide misincorporation by the apicoplast polymerase (apPOL), examine the kinetics and sequence dependence of mismatch extension, and determine the rates of mismatch removal by the 3' to 5' proofreading activity of the DNA polymerase. While the intrinsic polymerase fidelity varies by >50-fold for the 12 possible nucleotide misincorporations, the most dominant selection step for overall polymerase fidelity is conducted at the level of mismatch extension, which varies by >350-fold. The efficiency of mismatch extension depends on both the nature of the DNA mismatch and the templating base. The proofreading activity of the 12 possible mismatches varies <3-fold. The data for these three determinants of polymerase-induced mutations indicate that the overall mutation frequency of apPOL is highly dependent on both the intrinsic fidelity of the polymerase and the identity of the template surrounding the potential mismatch.

Replication and maintenance of the Plasmodium falciparum apicoplast genome

“…1), which was referred to as KPom1 based on sequence similarity to the Klenow fragment of E. coli Pol I [24]. Most recently, the Nelson laboratory designed a construct based on sequence alignments (referred to as apPOL) and identified a possible polymerase boundary spanning residues 1389 to 2016 that is conserved across the Plasmodium genus [41,42] (Fig. 1).…”

“…The apPOL construct behaves more like the E. coli Klenow fragment, and its fidelity is reminiscent of other replicative DNA polymerases [41]. Differences in the reported fidelity of KPom1 and apPOL may not be solely due to the structural variances between the constructs, as the methods of analysis differed between the two proteins [41].…”

“…Differences in the reported fidelity of KPom1 and apPOL may not be solely due to the structural variances between the constructs, as the methods of analysis differed between the two proteins [41]. Mutation rates are not exclusively determined by polymerase fidelity, as the ability of a polymerase to extend a mismatch and removal of misincorporated nucleotides also plays a role.…”

“…By combining misincorporation, exonuclease, and mismatch extension rate data, the most likely mutations caused by apPOL were ranked. apPOL is most likely to mutate a G to an A if the downstream base is a T, followed by C replacing a T if a T, C, or G is downstream [41]. Of course, these observations do not take into account potential DNA repair enzymes which could impact mutation probabilities in the apicoplast.…”

Crystallization and preliminary X-ray analysis of thePlasmodium falciparumapicoplast DNA polymerase

“…Following import, Prex is cleaved by an unknown protease into three separate proteins (Seow et al, 2005). The P. falciparum apicoplast DNA polymerase (apPOL) is the only known DNA polymerase targeted to the apicoplast, and we have previously demonstrated that the activity and fidelity of apPOL is consistent with that of a replicative DNA polymerase (Wingert et al, 2013).…”