探討Rec25-Rec27-Mug20複合體與Rec10蛋白的交互作用於減數分裂染色體中的內在功能

Abstract

減數分裂重組是細胞分裂的一個過程，透過同源染色體之間的遺傳物質交換促進遺傳多樣性。減數分裂中，DNA雙股斷裂啟動重組，而對於許多物種來說，聯會複合體（synaptonemal complex）透過液-液相分離（liquid-liquid phase separation）的形成，以連接和排列同源染色體，從而調節交叉(crossover)形成。然而，這種液-液相分離的特性是否在其他生物中也保守存在則仍不清楚。與其他物種中典型的聯會複合體有所不同，裂殖酵母菌(fission yeast)具有線性元素（linear elements）的非典型聯會複合體。線性元素的構造是由四種蛋白質組成：Rec10、Rec25、Rec27和Mug20。Rec25、Rec27和Mug20蛋白統稱為RRM複合體，優先聚集於雙股斷裂的熱點（hotspots）上，並發揮重要作用。過去我們的研究表明，RRM複合體具有DNA結合能力，形成液-液相分離，並與另一線性元素Rec10蛋白相互作用。值得注意的是，Rec10蛋白可以顯著促進RRM介導的液-液相分離形成。然而，RRM生化特性如何促進液-液相分離和減數分裂中的雙股斷裂形成仍不清楚。在本研究中，我確認了幾個在遺傳分析中具有顯著減數分裂表現型的RRM突變基因。我進行了生化分析，並闡明這些突變如何導致所觀察到的減數分裂表現型。結果顯示，這些突變蛋白仍然保留DNA結合和相分離特性；然而，這些突變破壞了Rec10 和RRM彼此蛋白的相互作用。因此，Rec10並無法促進RRM突變蛋白所介導液-液相分離的能力。除此之外，可以與RRM複合體交互作用的Rec10羧基端突變體亦能促進RRM複合體相分離的發生。結合上述，我的研究為RRM-Rec10在減數分裂期間的液-液相分離、雙股斷裂和交叉形成中的相互作用功能提供了重要證據。

Meiotic recombination, a process of cell division, facilitates genetic diversity by exchanging genetic material between homologous chromosomes. DNA double-strand breaks (DSBs) initiate recombination in meiosis, and the synaptonemal complex (SC) forms through liquid-liquid phase separation (LLPS) in many organisms to physically connect and align homologous chromosomes, regulating crossover formation. However, whether this LLPS property is conserved in other organisms remains unclear. In contrast to the typical SCs found in other species, Schizosaccharomyces pombe features atypical SCs known as Linear Elements (LinEs). LinEs comprise four proteins: Rec10, Rec25, Rec27, and Mug20. The Rec25, Rec27, and Mug20 proteins, collectively referred to as the RRM complex, are preferentially enriched in DSB hotspots and play an essential role in DSB formation. Previously, our studies have highlighted that the RRM complex harbors DNA-binding ability, forms LLPS and interacts with the other linear element protein Rec10. Notably, Rec10 could significantly promote the RRM-mediated LLPS formation. However, how those RRM biochemical attributes contribute to LLPS and DSB formation in meiosis remains elusive. In this study, I have identified several RRM missense mutants that exhibit a strong meiotic phenotype in genetic analysis. I conducted biochemical analyses to characterize these mutant variants and to elucidate how these mutations contribute to the observed meiotic phenotypes. The results show that while these mutant variants still retain DNA-binding and phase-separation properties; however, they abolish the physical interaction with Rec10. Consequently, Rec10 lacks the ability to facilitate RRM mutants-mediated LLPS. In addition, the Rec10 C-terminal fragment mutant which can interact with the RRM complex also can promote the RRM condensate formation. In conclusion, my study provides crucial evidence for the functional interaction of the RRM-Rec10 axis in LLPS, DSB, and crossover formation during meiosis.