以結構生物學探討FANCI-FANCD2與RAD51交互作用

Structural and functional characterization of the interactions between Rad51 and FANCI-FANCD2

汪宏遠(Hong-Yuan Wang)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202202027

摘要

去氧核醣核酸 (DNA) 受損對於所有生物的生存而言是持續存在的威脅，無論是外源性之化學藥物或輻射線、以及內源性形成的代謝產物皆可能導致DNA鹼基發生錯誤配對、缺失、異常的插入、DNA附加物、DNA股間交聯作用 (DNA interstrand crosslinks, ICL) 、單股或雙股 DNA斷裂等損傷。因此，啟動 DNA 損傷反應和 DNA 修復路徑以維持遺傳穩定性和完整性極為重要。在眾多DNA 修復機制中，一個被稱為范可尼貧血 (Fanconi anemia, FA) 的獨特路徑，主要負責在細胞週期中的染色質複製期 (S phase) 執行ICL的修復。當 ICL 在染色質複製週期形成時，DNA 複製叉會在ICL的位置停止。由於 ICL 的修復相當複雜，其中包含因核苷酸切除而伴隨產生的 DNA 雙股斷裂 (DNA double-stranded breaks, DSBs)，因此 FA 路徑具備能夠招募並協調下游不同的DNA 修復路徑，包含核苷酸切除修復(nucleotide excision repair, NER)、跨病灶 DNA 合成 (translesion DNA synthesis, TLS)和同源重組 (Homologous recombination, HR)等。 其中HR路徑主要可藉由修復 DSBs、ICLs 和停滯的複制叉引發的DNA 損傷來維持基因完整性。 RAD51 重組酶是真核生物中高度保守的核蛋白，HR 的核心步驟之一便是由 RAD51 於DNA受損處形成核蛋白絲來介導同源序列的搜尋以達成DNA修復。先前的研究指出RAD51 核蛋白絲會形成在單股DNA (ssDNA)上，也稱為突觸前複合物，藉由構型改變來激活其 ATP 水解活性，促進ssDNA入侵(invasion)並與姊妹染色體上的互補股配對來進行修復。
在 FA 路徑的初始步驟中，FANCM 會結合至 ICL位點並調控下游 FA 蛋白（A、B、C、E、F、G、L）組裝構成E3泛素連接酶複合物(E3 ubiquitin ligase complex)又被稱為 FA 核心複合物，進而將FA 路徑的中心樞紐FANCI 和 FANCD2泛素化。 FANCI-FANCD2複合物(ID2 complex)通過促進核酸酶的募集以切除 DNA 損傷，此為 FA 路徑修復DNA的關鍵步驟。 FA 蛋白 FANCD1/J/N/O 可被 ID2 complex募集至DNA受損位點，然後促進 RAD51 與ssDNA結合，讓同源DNA完成配對並修復。目前已知 ID2 complex和 RAD51 的交互作用在 FA 路徑修復 ICL 中的重要性。先前的研究亦揭示RAD51 會累積在 ICL 引發的停滯複製叉，且 ID2 complex能與 RAD51 共同保護受損處的 DNA ，使停滯複製叉不受外切核酸酶過度切割而產生降解。然而，ID2 complex如何招募 RAD51 並協同保護 DNA 的結構機制仍待探討。
為了進一步了解ID2 complex和 RAD51的交互作用及其介導 RAD51 核蛋白絲形成的機制，我們在大腸桿菌 BL21 (DE3) Condon(+)RIL 菌株中表達了Gallus gallus RAD51、FANCD2 和 FANCI。 ID2 complex和 RAD51核蛋白複合物將通過冷凍電鏡或 X-射線晶體學進行分析。 另外，我們還構建並表現在形成自體複合體有缺陷的RAD51 F86E突變以便在純化後獲得更多的RAD51單體。在凝膠過濾層析和 His pull-down 結果中，我們證實了 RAD51 與 ID2 complex的直接結合。 近期，我們成功地通過電子顯微鏡負染觀察到了ID2 complex與 RAD51 F86E 的蛋白顆粒。總之，這些結果將有助於揭示 FA 下游路徑和 HR 修復之間的未知機制或可能的結構。

關鍵字

DNA 損傷修復 ； 范可尼貧血修復路徑 ； FANCI-FAND2 複合物 ； 同源重組修復 ； 同源重組蛋白 RAD51

並列摘要

DNA damage represents a constant threat to all living organisms. The exogenous agents, such as hazardous chemicals and ionization radiation, or endogenously formed reactive metabolites may cause base mismatches, deletions, insertions, bulky adducts, DNA interstrand crosslinks (ICL), or single or double-stranded DNA breaks. As a result, DNA damage responses and DNA repair pathways are vital to the maintenance of genetic stability and genome integrity. Among cellular DNA repair pathways, the Fanconi anemia (FA) pathway is specialized for repairing ICLs formed predominantly during the S-phase, whose presence is known to cause stalled DNA replication forks. Since the repair of ICL involves nucleotide excision and the resultant DNA double-stranded breaks (DSBs), the FA pathway recruits and orchestrates several downstream DNA repair pathways, including nucleotide excision repair, translesion DNA synthesis, and homologous recombination (HR). HR is an essential pathway for maintaining genome integrity by participating in the repair of DSB, ICL, and collapsed replication forks. One of the central steps of HR is the ATP-dependent DNA strand exchange mediated by the RAD51, a highly conserved nuclear protein in eukaryotes. Previous studies have shown that the formation of RAD51 nucleoprotein filament on single-stranded DNA (ssDNA), also termed the presynaptic complex, would allosterically activate its ATP hydrolysis activity to facilitate homology search and DNA strand exchange.
Upon the activation of FA pathway, FANCM would bind to ICL site and recruit additional FA proteins (A, B, C, E, F, G, L) to form a multisubunit ubiquitin E3 ligase complex, referred to as the FA core complex. This supramolecular complex will perform monoubiquitination of the FANCI-FANCD2 (ID2) complex, which is to function as the central hub for FA pathway. The assembly of ID2 complex at ICL site represents a key step in the FA pathway by facilitating the recruitment of nucleases to remove the DNA lesion. The recruitment of FA downstream proteins FANCD1/J/N/O by ID2 complex then promotes RAD51 loading to initiate subsequent search for homologous sequences, strand paring and strand exchange. Given that the interaction between ID2 complex and RAD51 plays important roles in FA pathway, and that the ID2 complex is known to work with RAD51 to protect the damaged DNA from undergoing undesired degradation by exonucleases, it is important to understand how the ID2 complex-mediated recruitment of RAD51 is achieved and how this interaction promotes the assembly of RAD51 nucleoprotein filament at the ICL-induced stalled replication forks.
The goal of this work is to characterize the structures of ID2-RAD51 complexes. To this end, we had expressed Gallus gallus RAD51, FANCD2 and FANCI in the Escherichia coli BL21 (DE3) Condon(+)RIL strain. The reconstituted nucleoprotein complexes will be characterized by cryo-EM or X-ray crystallography. We also constructed and expressed the RAD51 F86E mutant, which is defective in self-association, in order to harvest more RAD51 monomer. Using gel-filtration chromatography and His pull-down assay, we confirmed that RAD51 can interact directly with ID2 complex. We have also visualized the particles formed by ID2 complex and RAD51 F86E by negative-staining EM. Together, these results lay a solid ground for the proposed structural studies.

並列關鍵字

DNA repair pathway ； DNA interstrand crosslinks ； FA pathway ； RAD51 ； FANCI-FANCD2 complex

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