惡性瘧原蟲 (Plasmodium falciparum)，屬於Apicomplexa門的單細胞寄生性原生動物，是造成惡性瘧疾及死亡的主要瘧原蟲。Apicomplexa動物門的寄生蟲有個特點，它們大多含有一個特殊的質體樣細胞器 (apicoplast)，參與原蟲細胞許多重要的代謝途徑，對原蟲的存活來說不可或缺。P. falciparum質體樣細胞器有屬於自己的一個環狀DNA，AT序列的組成含量特別地高 (86.9%)，而DNA複製由唯一一個DNA聚合酶Pom1負責。由於apicoplast是獨特唯一的胞器，與原蟲細胞生存息息相關，負責複製DNA的唯一DNA聚合酶與哺乳類動物不同源，因此我們認為Pom1會是具有潛力的藥物標的，本研究的目的即在探討DNA聚合酶Pom1的生化特性與功能。Pom1是個很大的蛋白質複合體，可區分成三個結構域 (domain) ：DNA引子酶、DNA解旋酶和DNA聚合酶。其中DNA聚合酶結構域簡稱為KPom1，可區分為兩個次結構域 (subdomain) ：5'→3' DNA 聚合酶，以及3'→5' DNA核酸外切酶。KPoml與大腸桿菌DNA 聚合酶 I的Klenow片段同源，在我們的研究中發現，野生型KPom1WT可在缺失了Pol I的大腸桿菌體內替補Pol I的功能，使之存活，但無法有效複製ColE1型質體，與之相反的是缺失外切酶活性的KPom1exo-則能有效率複製。純化出蛋白之後，藉由活性測試，我們發現KPom1WT缺乏nick-translation活性，以及在AT-DNA模板上沒有strand-displacement活性，這是複製ColE1型質體所必需的活性，然而KPom1exo-則是兩個活性都具備。此外，無論下游引子是DNA或RNA都不影響結果。此結果也表示KPom1在複製DNA的過程中有能力分辨AT或GC-DNA模板，而這個特性是Klenow和同樣是質體樣細胞器 DNA聚合酶的ToxoPol所沒有的，因此是KPom1獨有的特性。另外，我們在KPom1exo- DNA結構域的motif B上做1848位置組胺酸轉精胺酸(H1848R)的點突變，發現這個點突變會提升KPom1exo-對核苷酸的選擇性，同時也發現KPom1exo-本身就能有效率地在DNA複製的過程中使用雙去氧核甘酸和抗病毒藥物更昔洛韋。最後，我們確定KPom1和Klenow 片段一樣具有能夠在雙股DNA平滑末端加上一至多個腺嘌呤的能力。

Plasmodium falciparum, a unicellular parasite belongs to the phylum of Apicomplexa, is a major causative agent of human severe malaria, which threatens millions of life globally. The apicomplexan parasites commonly harbor a unique non-photosynthetic plastid, apicoplast, which involves in various, metabolic pathways and is vital for the parasite survival. The apicoplast has its own genome, which is highly AT-rich (~86.9%) in P. falciparum. The replication of the apicoplast genome is solely achieved by the polymerase of malaria 1 (Pom1), which is a single polypeptide that contains three distinct, functional domains: DNA primase, DNA helicase, and DNA polymerase, respectively. Since Pom1 is essential to parasite and has no orthologs in mammals, we consider it is a potential antimalarial drug target. Our objective is to elucidate the biochemical functions and properties of Pom1 from P. falciparum. The DNA polymerase domain of Pom1, designed as KPom1 in this study, is homologues to the Klenow fragment of E. coli DNA polymerase I (Pol I), which contains a 3'→5' exonuclease (exo) and 5'→3' DNA polymerase (pol) domain. In our study, we demonstrated that KPom1WT can functionally substitute for E. coli Pol I in vivo. However, KPom1WT only partially, while KPom1exo- efficiently, maintains the ColE1 plasmid. In the biochemical studies, both KPom1WT and KPom1exo- proteins were purified and characterized. The results showed that KPom1WT lacks both DNA nick-translation and strand-displacement synthesis functions, which is essential for the efficient initiation of DNA replication of ColE1-type plasmid. In the primer extension assays, KPom1 can differentiate AT- from GC-rich template regardless of the presence of downstream DNA or RNA primer. This enzymatic property is unique to KPom1, since homologous Klenow and ToxoPol, apicoplast DNA polymerase from T. gondii, do not have this feature. In separate experiments, I discovered that KPom1 can naturally incorporate sugar-modified nucleotide analogs, such as dideoxynucleosides and anti-viral ganciclovir, and a H1848R mutation in the motif B of KPom1 increases the base-analogous selectivity of KPom1. Finally, like Klenow enzyme, KPom1 also has an A-tailing function, which adds an extra dATP to the recessed DNA ends.

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