探討酵母菌Ndt80蛋白在減數分裂中對於DNA複製之調控

Abstract

減數分裂在一次的DNA複製之後，連續進行兩次的核分裂。目前對於出芽酵母菌 (Saccharomyces cerevisiae)在減數分裂時期如何在兩次分裂期之間去防止DNA再複製的機制並不清楚，但是一些研究指出出芽酵母菌中的週期蛋白依賴性激酶 (cyclin-dependent Kinase) 蛋白Cdc28可能參與其中。在本實驗室過去的研究發現Ndt80與抑制DNA再複製可能也有關。Ndt80為一個減數分裂時特定表現的轉錄因子 (transcription factor) ，可以調控下游至少150個與細胞核分裂以及孢子形成相關的基因。有趣的是，在營養生長的細胞中異位表現 (ectopic expression) Ndt80時會抑制DNA複製，因此我們推測Ndt80除了扮演轉錄因子的角色之外，可能也參與了在兩次核分裂之間抑制DNA再次複製。為了證明此一假設，我們藉由調控Cdc28的活性配合剔除Ndt80，觀察在減數分裂複製其後是否會發生DNA再次複製的現象。我們發現正常表現Ndt80的細胞在減數分裂的DNA複製期之後短暫的抑制Cdc28的活性，並不會造成DNA再複製，然而在缺乏Ndt80的細胞中短暫的抑制Cdc28活性卻能夠造成DNA再複製。這說明在減數分裂中Cdc28和Ndt80都具有防止DNA再複製的功能，確保能夠產生單倍體的配子。 我們也利用相同的策略針對另一個被懷疑可能也參與在控制DNA複製的蛋白激酶Ime2進行研究。我們發現，在細胞完成減數分裂的DNA複製後，將Ime2的活性短暫的移除並不會造成DNA的再複製。即使在缺乏Ndt80的菌株中短暫移除Ime2活性也不會發生；而同時抑制Cdc28和Ime2的活性只能讓極小部分的細胞進行再複製。另外，我們也發現在缺乏Ndt80的菌株中單獨抑制Cdc28活性和同時抑制Cdc28和Ime2的活性時，DNA再複製的能力無明顯差異。因此Ime2可能在此階段不參與抑制DNA 複製。

Meiosis consists of one round of DNA replication and two consecutive nuclear divisions without an intervening DNA synthesis. The mechanism to prevent another round of DNA replication in yeast is not clear, but Cdc28, the CDK in budding yeast, was supposed to be responsible for the control. Here we show the evidence for that Ndt80 is also involved in preventing another round of DNA replication. Ndt80 is a meiosis-specific transcription factor, which transcribes more than 150 genes required for meiotic division and spore formation. Interestingly, our lab found that ectopic expression of Ndt80 in mitotic cells inhibits DNA replication. We proposed that Ndt80 might be involved in preventing DNA replication between the two meiotic nuclear divisions. To verify this hypothesis, we constructed cdc28-as1 strains in which the Cdc28 activity could be attenuated by the 1-NM-PP1 inhibitor. Transient inhibition of Cdc28 activity alone after the pre-meiotic S phase did not cause DNA re-replication. On the other hand, DNA re-replication did occur when Cdc28 activity was transiently inhibited in the absence of Ndt80. These results support our hypothesis that Ndt80 could be involved in regulation of meiotic DNA replication. Since it was speculated that Ime2 may also be involved in preventing DNA re-replication, we constructed ime2-as1 strains in which the Ime2 activity is sensitive to the 1-NA-PP1 inhibitor. Transient inhibition of Ime2 activity alone after the pre-meiotic S phase did not lead to DNA re-replication, whereas inhibition of both Ime2 and Cdc28 only caused a very minor proportion of the cells to undergo another round of DNA duplication. Moreover, in Ndt80-depleted strains, transient inhibition of both Cdc28 and Ime2 activity did not enhance the re-replication efficiency compared with that of only Cdc28 inhibited.