臺灣博碩士論文加值系統

中文摘要
CDC8為出芽酵母菌 (Saccharomyces cerevisiae) 的essential gene，其基因產物thymidylate kinase為參與去氧核糖核酸生合成途徑上的一個重要的酵素，他的主要作用是以ATP或dATP為phosphate donors，將dTMP轉換為dTDP。 在cdc8的一系列突變株中，cdc8-1為temperature-sensitive mutant，突變株可以在23℃存活，但在高溫36℃下細胞停止生長分裂並且快速死亡，這個現象和一般的cdc mutants截然不同，因為一般的cdc mutants可以在溫度降低以後重新恢復細胞生長分裂的過程；此外，cdc8-1 突變株對於造成DNA damage如UV照射或是干擾DNA生合成的藥物如hydroxyurea的處理相當敏感，顯示CDC8可能參與細胞週期監控點 (cell cycle checkpoint) 功能或是直接影響DNA repair的能力，這些結果也暗示CDC8除了參與核酸合成的酵素活性外，可能還具有其他的功能。 之前已經有研究報告指出，cdc8-1分別與兩個細胞週期監控點突變株，mec1-1與mec2-1，存在synthetic lethal的情形，亦即cdc8-1 mec1-1以及 cdc8-1 mec2-1 double mutants在正常低溫的狀況下仍然無法存活。 這個結果顯示CDC8與checkpoint gene MEC1及 MEC2之間存在有functional interaction的關係。 目前雖然已有許多關於MEC1, MEC2功能的研究，但對於兩者在checkpoint的機制及細胞生長所扮演的角色，仍無清楚的定義。 因此，本研究即利用cdc8-1 mutant在細胞功能上的缺失及其與MEC2基因之間的互動關係來釐清dNTPs生合成步驟和細胞週期監控功能之間的訊息傳遞作用。
本論文的結論發現cdc8-1 mec2-1 之間synthetic lethal的原因並非由於cdc8-1及mec2-1分別造成dNTPs pool不足所引起的加成性損害而致死；CDC8的另一個功用可能在協助細胞有效地利用dNTPs來進行DNA複製，當此機制失去功能時，所造成的replication stress以及過多的dTTP累積，進而引發SWI4與MBP1等基因的表現導致細胞不正常地進入DNA replication的過程，此種不正常的加速進入細胞分裂週期的作用，平常必須藉由活化MEC2的cell cycle checkpoint功能才能協調細胞克服障礙並順利完成細胞分裂，也因此當CDC8與MEC2同時損壞時，導致細胞死亡的結果。

Abstract
Cell-cycle checkpoints are known as surveillance mechanisms designed to ensure proper transmission of genetic information during the progression of cell division. In the budding yeast, the Mec1 and Rad53/Mec2 proteins are involved in the G1, S, and G2 cell cycle checkpoint pathways in response to DNA damage or DNA replicational stress. One of the normal roles of S phase checkpoint functions exerted by Mec1 and Rad53/Mec2 is to properly integrate cell cycle transitions with DNA metabolism. The failure of Mec1 or Rad53/Mec2 in conjunction with the blockage of nucleotide synthesis caused by hydroxyurea treatment could result in lethal events. However, the molecular basis for this lethality is still unclear. The yeast CDC8 gene encodes thymidylate kinase, which phosphorylates dTMP to dTDP in the thymidylate biosynthesis pathway. A previous study has indicated that a cdc8-1 temperature-sensitive mutation is synthetic lethal with mec2-1 mutation even at the permissive temperature. We have used cdc8-1 and mec2-1 as a model to demonstrate that the low dTTP level in cells is not the cause of the synthetic lethality in the double mutant. Quite contrary, the measurement of dTTP pool by HPLC indicates that both cdc8-1 and mec2-1 have higher concentrations of dTTP than those of the wild-type. Base on the results of genetic analysis, we propose that CDC8 might have other roles in regulating the checkpoint activity during S phase in addition to its nucleotide enzyme function.

目錄
頁
中文摘要 1
Abstract 2
縮寫表 3
壹、前言 4
一、細胞週期 4
二、細胞週期的調控 5
三、DNA生合成與細胞控點之間的關聯 5
四、研究目的 7
貳、材料與方法 8
一、實驗材料 8
二、實驗方法 9
參、實驗結果 18
一、cdc8-1 mutant的定性分析 18
二、RNR1的大量表現無法挽救cdc8-1 mec2-1 double mutant 19
三、HPLC定量cdc8-1 mutant中dTTP pool的量 20
四、cdc8-1 mutant提早通過G1/S transition的現象可能導因於SWI4與MBP1的表現量增加 21
五、mec2-1的基因突變分析 21
六、分析MEC2蛋白質立體結構 22
肆、討論 23
一、CDC8可能參與dNTPs channeling的機制 23
二、cdc8-1 mec2-1 synthetic lethal導因於checkpoint功能異常 24
三、dTTP level的上升可能作為細胞通過G1/S transition的訊號 26
伍、參考文獻 27
陸、圖表與圖表說明 30
附錄 45

伍、參考文獻
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