肺炎鏈球菌是伺機性的病原菌，通常潛伏在上呼吸道，當幼兒或免疫力低下的病人發病時，其病菌會入侵下呼吸道及其他部位，造成腦膜炎、敗血症及群聚的肺炎。隨著抗藥性的肺炎鏈球菌的急遽上升，已經引發了全球百萬人民的死亡。肺炎鏈球菌獲得抗藥性基因的方式是透過DNA的重組，當感受到外在環境給予的壓力時(例如：抗生素的投藥)，會啟動重組的機制將對之有利的外在基因嵌進細菌的基因體中以此存活。諷刺的是，本該殺菌的抗生素在歷經世代投藥的篩選，導致抗藥基因快速傳播，使肺炎鏈球菌儼然成為多重抗藥性的「超級細菌」。然而基因重組及修復機制尚未明瞭。先前研究指出肺炎鏈球菌的DNA 聚合酶I，本研究簡稱SpPol I，對於DNA 複製、修復、重組的過程極為重要，但其真正的角色是如何參與其中還未被完整討論。透過蛋白質序列的比對，我們發現SpPol I 與大腸桿菌DNA 聚合酶I (EcPol I) 互為同源關係。SpPol I 擁有5端核酸外切酶及DNA 聚合酶的特性，唯獨缺少3端往5端的核酸外切酶功能，表示無校正DNA 合成時產生錯誤的能力。而在先前基因剔除的研究內容證實，SpPol I 上的5端核酸外切酶區域對於肺炎鏈球菌的生長及存活是不可或缺的，但此區域的真正特性及是否參與DNA複製過程都還未被明確定義。在本篇研究中，我們首先表達並純化SpPol I 及其5端外切酶上帶有突變的蛋白。在DNA 引子延伸的實驗中，SpPol I 表現出DNA 切口平移、DNA鏈置換、5端外切酶、5端flap核酸內切酶以及相似於核糖核酸酶H的活性。而將5端外切酶區域上可能與金屬離子契合的天門冬胺酸殘基進行突變 (D116A與D139A)，會大幅去除其5端外切酶、5端flap核酸內切酶以及相似於核糖核酸酶H的酵素活性。特別的是，在輔因子二價鎂離子催化下，SpPol I傾向DNA合成反應，但換成二價錳離子時，SpPol I則傾向降解反應，說明5端外切酶區域及DNA 聚合區域可能需要不同輔因子來催化能力。除此之外，實驗結果也展現了SpPol I上的5端外切酶區域及DNA 聚合區域在DNA複製過程能一起合作填補間隙，從雙股DNA或RNA-DNA結構置換下游的DNA或RNA鏈，留下可黏的切點供肺炎鏈球菌連接酶 (SpLigA) 進行連接，恢復了完整的雙股DNA結構。總歸而言，我們推測SpPol I 可能對於肺炎鏈球菌內基因體複製中岡崎片段的成熟及基因修復重組過程，扮演移除DNA或RNA引子的重要角色。

Streptococcus pneumoniae (S. pneumoniae) is the opportunistic pathogen that normally colonizes the upper respiratory tract and causes lethal community-acquired pneumonia, meningitis, and sepsis in children or immunocompromised adults. The acquisition of multidrug-resistant genes among drug-resistant S. pneumoniae strains have transformed the bacteria to become “superbugs”. The surge of antibiotic-resistant S. pneumoniae annually causes millions of deaths worldwide. The acquisition of drug-resistance by S. pneumoniae is commonly via the direct exchange of its own genomic DNA with exogenous drug-resistant genes by DNA recombination. However, the mechanism of DNA replication and recombination in S. pneumoniae remains unknown. Previous studies have shown that the DNA polymerase I of S. pneumoniae (SpPol I) is required for DNA replication, repair, and recombination pathways. How SpPol I involves in these processes is still unclear. SpPol I is homologous to Escherichia coli DNA polymerase I (EcPol I) and contains a functional 5’→ 3’ exonuclease (5’-Exo) and a DNA polymerase domain (Pol). SpPol I naturally lack a 3’→5’ exonuclease, or error-proofreading function. The gene-deletion study has confirmed the requirement of 5’-Exo domain of SpPol I for the growth and viability of S. pneumoniae. However, the intrinsic property and function of this domain is not well characterized. In this study, we cloned, expressed, and purified SpPol I and its 5’-Exo mutants. In the in vitro biochemical assays, SpPol I shows multiple enzymatic activities, including DNA nick-translation, strand-displacement synthesis, 5’-exonuclease (5’-Exo), 5’-flap endonuclease (5’-Fen), and RNase H-like activities. The mutations at the conserved aspartate residues (D116A and D139A) of 5’-Exo domain greatly abolish the 5’-Exo, 5’-Fen, and RNase H-like activities. Moreover, the 5’-Exo and Pol functions of SpPol I prefer different divalent metal cations as cofactors. The divalent Mg2+ ions greatly stimulate the DNA polymerase activity of SpPol I. On the contrary, the Mn2+ ions enhance the 5’-Exo functions of SpPol I. The 5’-Exo and Pol functions of SpPol I can work together to fill in the DNA gap and remove the downstream DNA or RNA primer from the duplex DNA or hybrid RNA/DNA structure. The cooperative reactions of 5’-Exo and Pol functions of SpPol I leave a ligatable DNA nick, which can be sealed by SpLigA to restore the integrity of double-stranded DNA. Taken together, SpPol I may play a critical role in DNA replication, DNA repair and recombination in S. pneumoniae.

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