進階探討酵母菌 Ndt80 蛋白在減數分裂中對於 DNA 複製之調控

Abstract

有絲分裂會進行一次DNA複製與一次核分裂，而減數分裂被視為由有絲分裂演化而來，但在一次的染色體複製後，卻有連續二次的核分裂。目前對於出芽酵母菌(Saccharomyces cerevisiae)在減數分裂二次核分裂之間如何去防止DNA再複製尚未清楚。Ndt80是一個減數分裂特定轉錄因子，本實驗室發現將其異位表現在營養生長細胞中，會使細胞生長停滯在G1時期，抑制DNA複製。由於Nd80在減數分裂的表現時間以及實驗觀察到的抑制DNA複製效應，因此提出一個假設：Ndt80可能參與防止兩次核分裂間DNA再複製的調控。 在近期的研究中，本實驗室利用半乳糖啟動子將Ndt80在細胞進入減數分裂前提早表現，結果發現細胞的DNA複製會受到抑制。本研究中，我們亦利用偵測減數分裂特定表現蛋白Ime2，確認受到抑制的是減數分裂的DNA複製 (pre-meiotic DNA replication)。然而，相較於異位表現Ndt80在營養生長細胞中完全抑制DNA複製，減數分裂的DNA複製僅受到部分抑制。由於酵母菌會代謝半乳糖，我們推測減數分裂的DNA複製僅受到部分抑制是因為使用減量的半乳糖濃度很快被代謝之故。因此我們剔除GAL1基因試圖讓半乳糖的代謝減緩。結果顯示剔除GAL1基因確實使Ndt80的表現量增加，且使抑制減數分裂DNA複製的效應增強。 此外，本實驗室藉由調控Cdc28活性配合NDT80剔除，發現唯有在完成減數分裂DNA複製的ndt80細胞中短暫抑制Cdc28活性才會造成DNA再複製。然而，NDT80基因的剔除也會使細胞週期停滯在粗絲期，為排除是粗絲期停滯造成DNA再複製的可能性，因此我們利用IME2啟動子將CDC5表現在ndt80基因剔除的細胞，使細胞得以離開粗絲期，結果發現DNA仍會進行再複製，因此推論應當是Ndt80直接參與減數分裂中防止DNA再複製的機制。

Our previous studies showed that ectopic expression of Ndt80 in mitotic cells caused cells to arrest at G1 phase and prohibit DNA replication. Considering the repression of DNA replication by Ndt80 and the expression period of Ndt80 during meiosis, it was proposed that Ndt80 might be involved in repressing another round of DNA replication during the two meiotic divisions. Our recent studies have showed that precocious expression of Ndt80 before induction for meiosis could repress pre-meiotic DNA synthesis. The repressed DNA replication was confirmed again to be pre-meiotic by the induction of Ime2. Compared to the full repression of mitotic DNA replication by Ndt80, the repression of pre-meiotic DNA replication was only partial. Since the concentration of galactose used in meiotic experiment was much less than in the cell. Therefore, we deleted the GAL1 gene to slow down galactose metabolism, and found that both the expression level of Ndt80 and the repression of pre-meiotic DNA replication by Ndt80 were increased. Furthermore, our lab has showed that DNA re-replication occurred after pre-meiotic DNA replication in ndt80 null mutants after transient inhibition of Cdc28 activity. Nevertheless, the ndt80 null cells arrest at the pachytene stage, and we could not rule out the possibility that the DNA re-replication was due to the arrest, but not the absence of Ndt80. To clarify this problem, we constructed the cdc28-as1/cdc28-as1 ndt80Δ/ndt80Δ pIME2-CDC5/CDC5 strain which was assumed to be able to exit from pachytene arrest. The results showed that cells still performed DNA re-replication after transient inhibition of Cdc28 activity, indicating that Ndt80 is involved in regulating DNA replication during meiosis.