SWI5-SFR1與RAD51AP1對RAD51重組酵素的作用機轉

The mechanism of SWI5-SFR1 and RAD51AP1 stimulating RAD51-mediated recombination

10.6342/NTU.2013.01899

廖嘉妤

同源重組 ； 相乘作用 ； Rad51重組酵素 ； Swi5-Sfr1 ； Rad51AP1 ； Homologous recombination ； Synergistic effect ； Rad51 recombinase ； Rad51AP1 ； Swi5-Sfr1

臺灣大學生化科學研究所學位論文

2013年

碩士

冀宏源

英文

同源重組(homologous recombination)是修復雙股螺旋去氧核醣核酸斷裂　(DNA double strand breaks)的主要機制。同源重組的功能缺失會引起基因體的不穩定性，甚至造成癌症。當發生雙股去氧核醣核酸斷裂時，雙股去氧核醣核酸會被酵素截切成單股的去氧核醣核酸，接著RAD51重組酵素(recombinase)會以螺旋的方式結合在此單股去氧核醣核酸上，形成一條核蛋白聚合物(nucleoprotein filament)。此核蛋白聚合物會跟同源的雙股去氧核醣核酸配對，接著兩股之間進行交換，完成同源重組反應。在同源重組反應中，RAD51重組酵素會受到一些輔助蛋白的調控，例如：SWI5-SFR1 和 RAD51AP1。抑制哺乳類動物細胞中SWI5-SFR1 或 RAD51AP1 的表現，會提高細胞對去氧核醣核酸損傷藥劑(DNA damaging agents)的敏感度。但目前並不清楚SWI5-SFR1和RAD51AP1如何共同調控同源重組的修復機制。而我們的研究結果發現：SWI5-SFR1與RAD51AP1對於RAD51重組酵素調控的雙股互換，有相乘(synergistic effect)的刺激效果。但過去的研究顯示並非所有的輔助蛋白都可以刺激相乘作用，因此我們想更深入了解SWI5-SFR1與RAD51AP1刺激相乘作用的機制。結果我們發現：此相乘作用需要SWI5-SFR1形成複合體，且此複合體要跟RAD51重組酵素有交互作用；也需要RAD51AP1具有跟去氧核醣核酸以及RAD51重組酵素的交互作用。我們更進一步發現，SWI5-SFR1與RAD51AP1兩者之間並不會有蛋白和蛋白的交互作用，但兩者卻可以跟RAD51重組酵素形成三聚體。代表此相乘作用是SWI5-SFR1與RAD51AP1分別對RAD51重組酵素刺激的結果。我們的研究讓我們更暸解輔助蛋白對於RAD51重組酵素調控的機制。

Homologous recombination (HR) is the major pathway to repair DNA double-strand breaks (DSBs). Dysfunction of HR leads to genome instability and even cancer formation. When a double-stranded DNA (dsDNA) breaks, the break ends undergo a resection process to generate a single-stranded DNA (ssDNA), and then RAD51 recombinase assembles on ssDNA to form a nucleoprotein filament, which is called presynaptic filament. The presynaptic filament captures and invades in dsDNA to search the homologous DNA. Finally, the ssDNA exchanges with their homologous DNA within dsDNA. During this HR reaction, the activity of RAD51 is regulated by accessory factors, including SWI5-SFR1 and RAD51AP1. Previous cell-based studies showed that knockdown SWI5-SFR1 or RAD51AP1 in mammalian cells results in increased sensitivity to DNA damaging agents. It raises an intriguing question how SWI5-SFR1 and RAD51AP1 mechanistically co-regulate Rad51 recombinase activity during HR. To address this question, we use purified proteins and in vitro reconstitution system to study their mechanistic actions in recombination reaction. Our results indicate that mouse SWI5-SFR1 and RAD51AP1 exhibit a synergistic effect on RAD51-mediated strand exchange. Moreover, the synergistic effect not only requires SWI5-SFR1 forming complex interacting with RAD51, but also requires RAD51AP1 binding DNA and RAD51. Furthermore, we found that SWI5-SFR1 can’t associate with RAD51AP1 directly, but SWI5-SFR1, RAD51AP1 and RAD51 form a ternary complex. Taken together, our findings suggest that the synergistic effect stems from the stimulation of SWI5-SFR1 and RAD51AP1 individually, and provide a mechanical insight of accessory factors in homologous recombination.

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