探討Rec27、Rec25、Mug20在減數分裂中的同源染色體的配對以及同源重組中的角色與功能

Abstract

在減數分裂中，同源染色體的配對和同源重組對於同源染色體正確的分配到子細胞中是必要的。不正常的同源染色體配對和同源重組會導致非整倍體(aneuploid)的配子(gametes)產生。在分裂酵母(Schizosaccharomyces pombe)中，線性單元(linear elements, LinEs)能幫助同源染色體的配對，並藉由調控Rec12蛋白質產生雙股斷裂(double-strand breaks, DSBs)來起始同源重組反應。喪失參與線性單元中的分子會導致無法正常的產生去氧核醣核酸(DNA)雙股斷裂和配對同源染色體。Fowler和Smith(2013)等人的研究指出線性單元分子：Rec27，Rec25和Mug20對於Rec12產生雙股斷裂的熱點(hot spots)是必須的。作者更進一步推測Rec27，Rec25和Mug20可以形成蛋白質複合體(complex)並促進Rec12的酵素活性而產生DNA雙股斷裂。因此我想要藉由純化Rec27，Rec25和Mug20蛋白並分析他們的生化特性去探討和Rec12的關係。 我們藉由大腸桿菌(E. coli)表現並純化Rec27，Rec25和Mug20重組蛋白。我們初步藉由體外親和性沉降(in vitro affinity pull down)、酵母菌雙雜合分析(yeast two-hybrid analysis)以及膠體電泳位移分析(DNA mobility shift assay)發現：(1) Rec27，Rec25和Mug20之間有直接的交互作用；(2) 只有Rec27擁有和雙股去氧核醣核酸(DNA)結合的能力。除此之外，我們也成功建立了多順反子(polycistronic)表現系統，同時表現並純化Rec27-Rec25-Mug20複合體。我們的研究可以用來進一步分析Rec27-Rec25-Mug20複合體和Rec12產生的雙股斷裂之間的關係。

Homologous pairing and meiotic recombination are essential for proper chromosome segregation during meiosis. Failures of pairing and recombination can lead to the aneuploid of gametes. In Schizosaccharomyces pombe, linear elements (LinEs) are responsible for homologous pairing and programmed DNA double-strand breaks (DSBs) catalyzed by conserved Rec12 protein to initiate meiotic recombination. Loss of LinEs components leads to defects of pairing and DSBs formation. It has been well documented that only Rec27, Rec25, and Mug20 are enriched at DSB hotspots and required for hot spots formation. Those observations suggested that Rec27, Rec25, and Mug20 can form a complex and activate Rec12-mediated DSBs (Fowler et al., 2013). This work aims to investigate the biochemical property of Rec25, Rec27, and Mug20, and their functional relationship with Rec12-mediated DSB. We used E. coli as host cells to express and purify Rec27, Rec25, and Mug20 recombinant proteins. We demonstrated that Rec27, Rec25, and Mug20 have a physical interaction and importantly, only Rec27 harbors a DNA binding ability. We also established the polycistronic expression system to co-express and purify Rec27-Rec25-Mug20 complex to study their biochemical characteristics. Our study provides useful information for further to decipher the functional role of Rec27-Rec25-Mug20 complex in Rec12-mediated DSBs.