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Abstract
The phylum Apicomplexa comprises a group of intracellular parasites of significant global health and economic concern. Most apicomplexan parasites possess a relict plastid organelle, which no longer performs photosynthesis but still retains important metabolic functions. This organelle, known as the apicoplast, also contains its own genome which is required for parasite viability. The apicoplast and its genome have been shown to be useful as therapeutic targets. However, limited information is available about the replication and maintenance of plastid DNA, not just in apicomplexan parasites but also in plants and algae. Here we use the genetic tools available in the model apicomplexan Toxoplasma gondii to examine putative apicoplast DNA replication and condensation factors, including homologs of DNA polymerase I, single-stranded DNA binding protein, DNA gyrase, and the histone-like protein HU. We confirm targeting to the apicoplast of these candidates, which are all encoded in the nucleus and must be imported. We created a genetic knockout of the Toxoplasma HU gene, which encodes a homolog of a bacterial protein that helps condense the bacterial nucleoid. We show that loss of HU in Toxoplasma results in a strong decrease in apicoplast DNA content, accompanied by biogenesis and segregation defects of the organelle. We were also interested in examining the roles of enzymes that might be more directly involved in DNA replication. To this end we constructed conditional mutants of the Toxoplasma gyrase B homolog and the DNA polymerase I homolog, which appears to be the result of a gene fusion and contains multiple different catalytic domains. We find that these proteins are essential to the parasite and required for apicoplast DNA replication. Together these data highlight the importance of the apicoplast DNA in apicomplexan cell biology and increase our understanding of plastid genome biology.