Ribonucleotide Misincorporation and Reverse Transcriptase Activities of Plasmodium falciparum Apicoplast DNA Polymerase.

Abstract

Plasmodium falciparumis the most common and harmful causative agent of malaria worldwide. As a member of the phylum Apicomplexa, P. falciparum is characterized by the presence of a unique and essential organelle called the apicoplast. Reminiscent of an algal chloroplast, the apicoplast possesses its own genome, which is maintained by a single apicoplast DNA polymerase (apPol). Ribonucleotides misincorporated into the genome are among the most common lesions encountered by DNA polymerases, and the ability to replicate past these lesions varies widely among characterized enzymes. Here, we have investigated the ribonucleotide (rNTP) misincorporation frequency of apPol and determined its reverse transcriptase (RT) activity across templating ribonucleotides. Pre-steady-state kinetic experiments indicate that apPol does not have an unusually high discrimination between deoxy and ribonucleotides, with frequencies ranging between 104 and 106 depending on the identity of the ribonucleotide. Once incorporated into its template, apPol can replicate across ribonucleotides using its RT activity, but extension of a deoxynucleotide basepaired with the ribonucleotide is slow relative to a canonical basepair. Exonuclease assays indicate that apPol proofreads ribonucleotides an order of magnitude faster than extension, suggesting that most, but not all, misincorporated ribonucleotides will be excised. Although the components have not been identified, ribonucleotide excision repair or other tolerance mechanisms may exist in the P. falciparum apicoplast, and more targeted proteomic efforts will be needed to elucidate them.