探討 ID complex和RAD51之間的交互作用與對RAD51蛋白絲的影響

Abstract

DNA 股間交聯（DNA interstrand crosslinks, ICL）會阻礙 DNA 鏈分離，從而抑制 DNA 複製，導致細胞損傷。修復 ICL 的主要機制是范可尼貧血（Fanconi anemia, FA）途徑，是維持基因組穩定性至關重要的途徑。在 FA 途徑中FANCI 和 FANCD2 會形成蛋白複合物（ID complex）並扮演活化途徑的重要角色，當ID complex ICL存在時會誘發泛素化同時會鎖定在受損的 DNA 上，並招募核酸內切酶，將ICL 移除，再透過同源重組 (homologous recombination, HR) 進行DNA修復。先前的研究顯示，FANCI、FANCD2和RAD51 會在停滯的複製叉處大量聚集，以防止 DNA 被外切酶降解。最近的發現進一步指出，ID complex可以穩定 RAD51-DNA核蛋白絲，並協同保護停滯的複製叉。然而，ID complex與 RAD51 之間相互作用的結構基礎以及這種相互作用如何穩定停滯的複製叉仍然未知。我們的目標是了解 ID-RAD51 複合物的結構和功能以及ID complex如何穩定 RAD51蛋白絲。利用ID complex與RAD51在脊椎動物間的高度同源性，我們首先通過下拉實驗(pull down assay)確認了Gallus gallus ID complex（GgID complex）和 Homo sapiens RAD51（HsRAD51）在生理溫度下能較好的相互作用。並藉由電泳遷移率實驗（Electrophoretic Mobility Shift Assays, EMSA），確認了 HsRAD51 和 GgID 複合物可同時存在於 5'-flap DNA 上。我們還透過單分子 TPM 實驗 (single-molecule TPM experiment)，證明了 GgID complex可藉由促進 HsRAD51核蛋白絲形成和降低核蛋白絲解離的速率，來穩定 HsRAD51核蛋白絲。最後為了進一步了解 ID complex與 RAD51 之間的相互作用，我們重組了 ID-RAD51 complex，並以負染電鏡(negative staining EM) 和冷凍電鏡（cryo-electron microscopy, cryo-EM）進行分析。結果顯示GgID complex存在時會延長HsRAD51 形成的蛋白絲比，未來也將繼續優化組成複合物的條件，以提高 ID-RAD51 complex在cryo-EM分析中的顆粒的品質，並透果結構解析，更深入ID complex和RAD51交互作用機制。

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that block DNA strand separation, thereby inhibiting DNA replication and transcription. The primary mechanism for repairing ICLs is the Fanconi anemia (FA) pathway, which is crucial for maintaining genome stability. The protein complex formed by FANCI and FANCD2 (ID complex) plays an essential role in the FA pathway; when monoubiquitinated, the ID complex locks onto damaged DNA and recruits endonucleases, to introduce DNA double-strand breaks (DSBs). After ICL removal, the DNA lesion is repaired by homologous recombination. Recent findings have revealed that the ID complex can stabilize the RAD51-DNA filament and cooperatively protect the stalled replication fork. However, the structural basis underlying the interaction between the ID complex and RAD51 and how this interaction stabilizes stalled replication forks remains unknown. Our aim is to understand the structure and function of the ID-RAD51 complex and how the ID complex stabilizes the RAD51 filament. Since the ID complex and RAD51 are well conserved in vertebrates, we first confirmed that the Gallus gallus ID complex (GgID complex) and Homo sapiens RAD51 (HsRAD51) interact more effectively at physiological temperatures using a pull-down assay. Electrophoretic mobility shift assay (EMSA) was conducted to confirm the simultaneous presence of HsRAD51 and the GgID complex on a 5’-flap DNA. We also employed single-molecule TPM experiments to demonstrate that the GgID complex stimulates HsRAD51 filament formation and reduces filament disassembly, thereby stabilizing the HsRAD51 filament. To gain further insight into the interaction between the ID complex and RAD51, we reconstituted the ID-RAD51 complex for structural characterization using negative staining EM and cryo-electron microscopy (cryo-EM). Preliminary analysis with negative staining EM revealed that HsRAD51 forms longer filaments in the presence of the GgID complex compared to when it is alone. We will continue to optimize incubation conditions to improve the homogeneity of the ID-RAD51 complex to facilitate structural analysis using in cryo-EM analysis.