單股DNA結合蛋白（single-stranded DNA-binding proteins, SSBs）廣泛地存在於所有的生物體當中，SSBs在細胞DNA複製、修復和重組過程中會保護暫時裸露的單股DNA並召集合適的DNA代謝蛋白以協調基因體的功能與維護，因此對於基因體穩定性的維持至關重要。但目前還未能解釋，在這些基因體維護的過程中，SSB如何選擇性的與各種不同的SSB 相互作用蛋白（SSB interacting proteins, SIPs）進行交互作用。以往的研究認為大腸桿菌的SSB（EcSSB）會通過C端尾部的酸性尖端（TIP）與SIPs結合。然而，另外的假設模型則表示SSB中的內在無序連接器（IDL）亦可能參與調節EcSSB和SIPs的相互作用。因此，我的研究主題將利用DNA解旋酶PriA來研究 SSB/SIPs相互作用的機制。先前有研究表明，SSB會直接與PriA進行相互作用並促進分叉DNA受質上的PriA解旋酶活性。PriA解旋酶是大腸桿菌中用以重新啟動DNA複製的起始子，在細胞複製中，DNA損傷將導致複製複合體過早從DNA上脫離並造成DNA 的複製終止，從而導致基因體複製不完整或甚至是細胞死亡。因此，複製重啟是維持基因完整的必要反應。然而，尚不清楚SSB如何促進PriA所介導的DNA複製重啟的分子機制。因此，本研究旨在闡明SSB/PriA複合物的結構基礎，以期了解IDL在SSB/PriA相互作用中的角色以及SSB刺激PriA解旋酶的機制。在這項研究中，我們成功地在體外環境重建了SSB和PriA的複合物。令人驚訝地是，我們透過體外下拉測定法以及粒徑篩析層析法證實了在SSB/PriA的交互作用中不需要IDL的存在。除此之外，我們還獲得了SSB ΔIDL /PriA複合物的蛋白質晶體，未來的工作可以致力於解析晶體結構，並希望能提供第一個基於結構的證據，探討SSB如何在缺乏IDL的情況仍有功能的與PriA進行交互作用，並進一步了解SSB和SIP之間的交互機制。

Single-stranded DNA-binding protein (SSBs) exists in all organisms and is essential
for genome maintenance by protecting the transiently exposed single-stranded DNA (ssDNA) during DNA replication, repair, and recombination. In addition, SSBs bind to transiently open ssDNA intermediates and recruits appropriate DNA metabolic proteins to coordinate genome maintenance. However, it has not yet been able to explain how SSBs selectively interact with different SSB interacting proteins (SIPs) during genome maintenance. Previously, it was indicated that Escherichia coli SSB (EcSSB) binds to SIPs through the C-terminal acidic tip (TIP). Recently, several hypothetic models suggested that the intrinsically disordered linker (IDL) in SSB is required to regulate the interaction of SSB and SIPs. The profound evidence of how SSB interacts with SIPs is lacking. In this project, we utilized the DNA helicase PriA to investigate the mechanism of SSB/SIPs interaction. Previous studies showed that SSB directly interacts with PriA, one of the SIPs, and stimulates PriA helicase activity on the branched DNA substrate. PriA helicase is an initiator to restart DNA replication in E. coli. In cell replication, DNA damage causes the replication complexes prematurely detach and DNA replication termination, which leads to incomplete genome replication and cell death. Therefore, replication restart is an essential reaction to maintain genome integrity. However, the molecular mechanism of how SSB promotes PriA-mediated DNA replication restart is still unknown. Hence, we aim to solve the structure of the SSB/PriA complex to understand the role of IDL in SSB/PriA interaction, and the mechanism by which SSB stimulates PriA helicase activity. In this study, we successfully reconstituted the in vitro complex formation of SSB and PriA. Interestingly, the biochemical analysis showed that IDL is not required for SSB/PriA interaction. The purified SSB ΔIDL /PriA complex has been successfully crystallized. In the future, structural determination of SSB ΔIDL /PriA complex can provide the structure-based evidence to explore the molecular mechanism by which SSB functionally interacts with PriA in the absence of IDL for understanding the interacting mechanism of SSB-SIP complex.

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