2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38576Initiation of chromosomal DNA replication in bacteria for genome duplication is a complex process that relies on divergent multi-protein assembly for entry of the replicative DNA helicase at the replication origin. In General, loading the replicative DNA helicase DnaB for oriC-dependent DNA replication is carried out with the aid of two other proteins, DNA initiator protein DnaA and the helicase loader protein DnaC. The replication forks could accidentally be arrested anywhere along the DNA, so reloading the DnaB helicase for oriC-independent DNA replication is required. Nevertheless, essential components responsible for the helicase-recruiting process previously found in Gram-negative bacteria, such as PriB, PriC, DnaT, and DnaC proteins are not found in Gram-positive bacteria. Instead, three other proteins, DnaD, DnaB, and DnaI, have been genetically and biochemically proven to be required for DNA replication initiation of Gram-positive bacteria. Here we will focus on the study of DnaD. The DnaD protein is composed of two major structural domains: an N-terminal oligomerization domain and a C-terminal ssDNA binding domain. It has been reported that the DNA-remodeling function of DnaD is the sum of a scaffold-forming activity on the N-terminal domain and a DNA-dependent oligomerization activity on the C-terminal domain. In an effort to a better understanding of how remodeling and untwisting the supercoiled DNA is associated with the initiation of DNA replication in Gram-positive bacteria, we propose to study the structure and function of the DnaD protein by use of the crystallographic analysis and the DNA binding assay. The resultant information is expected to provide the structure-function knowledge of the DnaD protein.在細菌的基因體複製為二 (genome duplication) 的過程中, 如何開始染色體DNA 的複製是一個很複雜的程序, 主要是靠著多個相異蛋白質的聚集使得主要用於複製 DNA 的解旋. (helicase) 進入一個特定的序列, 稱之為複製起點 (replication origin, OriC)。一般來說, 對於需要複製起點的DNA 複製, 載入此解旋.主要要有以下兩個蛋 白質, 分別是起始蛋白質 (initiator) DnaA 以及解旋.載入蛋白質 (helicase loader) DnaC。然而, DNA 複製過程中的 DNA 複製叉 (replication forks) 常會因意外而停止移 動使得DNA 複製中止, 因此能夠不在複製起點來重新載入DNA 解旋.是必須的。儘 管如此, 之前在格蘭氏陰性菌中所發現能不在複製起點來重新載入DNA 解旋.的一些 必要的蛋白質, 如PriB, PriC, DnaT, 以及DnaC, 在格蘭氏陽性菌中並沒有發現。取而 代之的是其他三個不同的蛋白質如 DnaD, DnaB 與 DnaI, 且在基因與生化研究的層 面上都證明了這三個獨特的蛋白質在格蘭氏陽性菌的DNA 複製是必須的。在DnaD 的 研究中目前已知 DnaD 有兩個主要的功能區域, 分別是N 端的多套體聚合區域以及C 端的單股DNA 結合區域。已有文獻報導 DnaD 具有可以重新調整DNA 構造 (remodeling) 的能力，且這個解開超螺旋DNA 的能力是來自於N 端具有的骨架形成的 能力與C 端與DNA 相關的聚合能力兩者綜合而成的。為了能更多的瞭解此活性是如 何與格蘭氏陽性菌的DNA 複製有關連, 因此我們想要利用DnaD-DNA 結合的測量方 法與蛋白質晶體結構的分析來研究DnaD 的結構與功能的相關性，我們預期此計畫執 行的結果將可提供DnaD 在功能與結構方面的相關知識。DnaD起始蛋白質去氧核醣核酸DnaDinitiatorDNAProject