酵母菌 Ndt80 蛋白抑制 DNA 複製之機制探討

Abstract

在出芽酵母菌(Saccharomyces cerevisiae)中，Ndt80是一個在減數分裂時特定表現的轉錄因子(transcription activator)。剔除Ndt80時會造成細胞停滯在粗絲期 (pachytene)，無法進行減數分裂。有趣的是，在營養細胞中異位表現 (ectopic expression) Ndt80會造成細胞生長週期停滯在G1時期。因此我們推測，Ndt80在第一次減數分裂(meiosis I)和第二次減數分裂(meiosis II)中間可能扮演抑制另一次DNA複製的腳色，使得細胞能夠成功的產生單倍體孢子。 在近期的研究中，我們發現在細胞處於後複製複合體期(post-RC state)表現Ndt80，部分細胞的減數分裂DNA複製(pre-meiotic DNA replication)會受到抑制。而這個現象在持續維持Ndt80表現量時更加明顯。不過我們仍希望能在Ndt80正常表現時機探討Ndt80抑制DNA複製的能力。於是我們構築一系列的Ndt80片段缺失突變株(in-frame deletions)，致力尋找一個能分開活化轉錄功能(transcription activation)及抑制DNA複製能力的突變株，希望可以使酵母菌順利進行減數分裂，但無法抑制第一次與第二次減數分裂之間的DNA複製。 Ndt80抑制DNA複製的機制可能是藉由直接結合到DNA上影響複製，或者誘導其他調控細胞週期的基因表現造成細胞生長停滯。本篇研究中，我們探討了一些Ndt80抑制DNA複製的可能機制。首先，我們在ime2基因剔除的菌株中表現Ndt80，分析Ndt80造成的抑制效果是否透過IME2。結果顯示Ndt80抑制DNA複製並不需透過IME2。再來，我們想要研究Ndt80是否會結合到複製起始點附近的Sum1蛋白結合位，因此我們在sum1基因剔除的菌株中表現Ndt80，觀察缺少Sum1競爭蛋白結合位是否會影響Ndt80抑制DNA複製的效果，但並沒有觀測到明顯的變化。未來將需要進行更多的分析來探討Ndt80抑制DNA複製的機制。

Ndt80 is a meiosis-specific transcription activator in the budding yeast Saccharomyces cerevisiae. ndt80 null mutants arrest at the pachytene stage and fails to complete meiosis. Interestingly, our laboratory found that ectopic expression of NDT80 in vegetative cells causes cell cycle arrest at G1 phase, and we proposed that Ndt80 may be involved in repressing another round of DNA replication between meiosis I and meiosis II. In recent research, our results showed that Ndt80 had the ability to partially repress pre-meiotic DNA replication when expressed at a stage when origin of replication is at a post-RC state. The DNA repression effect grew stronger when continuously expressing Ndt80 throughout pre-meiotic incubation. Despite these discoveries, it is still important to analyze the role of Ndt80 repressing DNA replication when it is normally expressed, meiosis I and meiosis II. Therefore, we screened for a separation-of-function mutant that can promote cells to complete meiosis but cannot cause cell cycle arrest. The repression of DNA replication by Ndt80 could be resulted from a direct binding of Ndt80 to DNA sequences, or indirectly from the effect of cell cycle related genes regulated by Ndt80. In this study, we discussed the possible mechanisms of Ndt80 repressing DNA replication. First, we expressed Ndt80 in a ime2 null strain to test if Ndt80-induced cell cycle arrest rely on IME2. Results showed that IME2 is not responsible for causing Ndt80-induced cell cycle arrest. Second, we tested the possibility of Ndt80 binding to Sum1 binding sites near ARSs to repress DNA replication. We expressed Ndt80 in a sum1 null strain to see if removing Sum1 competition would affect cell cycle arrest effects, but no significant difference was observed. Further analyses are required to explore the mechanisms of Ndt80 repressing DNA replication.