發展單分子螢光顯微平台研究RAD51重組酶核蛋白絲的解離動態

Developing Single-Molecule Fluorescence Imaging Platform to Study Dissociation Dynamics of RAD51 Recombinase Filament

廖庭尉(Ting-Wei Liao)

指導教授 ： 李弘文

國立臺灣大學/理學院/化學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802384

摘要

真核細胞中，重組酶RAD51在DNA雙股斷裂的同源修復中扮演關鍵的角色，其調控步驟為RAD51核蛋白絲的穩定性。核蛋白絲的穩定性主要受到兩個機制調控，一則重組酶的核蛋白絲形成，另一則為重組酶自核蛋白絲上的解離。目前已知RAD51核蛋白絲的形成牽涉兩個步驟，即較緩慢的成核反應與蛋白絲延長反應，然而對重組酶在核蛋白絲的解離步驟卻瞭解甚少。本研究透過建立單分子螢光顯像平台，量測標記螢光分子的重組酶老鼠RAD51在ATP與AMPPNP兩種不同條件下核蛋白絲解離情形。實驗觀察到在ATP條件下，其核蛋白絲在觀測時間內完全解離比例極高，受限於其解離速率目前尚無法精準量測完整的解離過程；然而在水解被抑制的AMPPNP條件下，其核蛋白絲穩定性雖有提高，但不如預期的穩定，重組酶仍有一階或多階螢光變化。根據實驗的統計結果，螢光變化比例大致與螢光強度正相關，此一量測平台提供更清晰的重組酶動態變化，未來亦極具潛力研究輔助蛋白調控機制。

關鍵字

同源重組修復 ； RAD51 ； 單分子螢光顯微

並列摘要

RAD51 plays a crucial role in homologous recombination to repair double strand break damage in eukaryotes. The stability of RAD51 protein filament is one of the most key regulatory processes. The formation of RAD51 protein filament includes two processes, slow nucleation and fast extension process. However, there is limited understanding of protein filament stability and protein dissociation kinetics.Here, we developed single-molecule fluorescence imaging platform to investigate the stability of labeled mouse RAD51 protein filament. We characterized the stability of RAD51 filament and RAD51 dissociation from the protein filament under two different conditions, ATP or AMPPNP. Most RAD51 filament under ATP fully dissociates within our observation time, which made us difficult to track the complete dissociation process, and thus we move to AMPPNP condition. Surprisingly, RAD51 filaments under AMPPNP condition, although expected stably bound, show one or multiple intensity drops, and the drop ratio is approximately positive correlated with the fluorescence intensity. This single-molecule fluorescence platform provides more detailed RAD51 filament dynamics, and has potential to be used to measure the regulation of accessory proteins.

並列關鍵字

homologous recombination repair ； RAD51 ； single-molecule fluorescence imaging

參考文獻

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