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研究生:譚賢明
研究生(外文):Bertrand Chin-Ming Tan
論文名稱:FACT及其交互作用之蛋白質在細胞週期中角色
論文名稱(外文):A Few FACTs About Interphase: Functional Roles of FACT and Associated Complexes During G1 and S Phases
指導教授:呂勝春
指導教授(外文):Sheng-Chung Lee
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:92
語文別:英文
論文頁數:81
中文關鍵詞:DNA複製基因轉錄磷酸酶染色質結構複合物
外文關鍵詞:FACTNek9transcription elongationkinasesiRNAinterphaseDNA replicationLC/MS
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摘要
FACT為一種調控染色質結構的異雙體,它在DNA複製及基因轉錄中扮演極重要的角色。FACT 被認為在參與基因轉錄延伸步驟中為一重要因子,但此複合物在人類中所具有的功能與機制及其參與細胞過程的關聯性目前並不清楚。在本論文中,我們主要是針對與 FACT 有交互作用的磷酸酶蛋白 Nek9 來作探討。Nek9與FACT在細胞核中形成一個大小約600 kDa的複合物,它在活化的狀態時,其電泳位置稍有偏高,這是由於蛋白被磷酸化所致,特別是位於磷酸酶 activation loop、具保留性的酥胺酸 (Thr210) 上,此位置的磷酸化在 Nek9 的活化過程中扮演不可或缺的角色。當 Nek9 與 FACT 處於同一複合物中時, Nek9 上的磷酸化程度有增加的現象;利用 Nek9dsRNAi 細胞的進行細胞週期之分析時,我們發現 Nek9 對於 G1 和 S 時期的推進是相當重要的,而且同時位在 FACT 複合物中的 Nek9 其磷酸化也有增加的趨勢。我們的實驗提供了一些證據來推論 Nek9 可能是在 FACT 相關的細胞過程中扮演一個酵素活化功能的角色,而此過程的進行對細胞週期有著重要的影響。
本論文的第二部分主要是針對 FACT 與另一交互作用的蛋白 Mcms 之間的關係來做探討。我們發現 FACT 和不同的 Mcms 在交互作用上有不同的組合,可能和參與不同的反應或步驟上有關。接著我們進一步發現與 FACT 結合的 Mcm4 蛋白其磷酸化程度與細胞週期有著時間上的關聯,推測可能是屬於一種功能上的調控機制。在酵母菌中, Spt16/Pob3- 也是 FACT 的相似物,對於細胞週期中 S 時期延伸步驟的進行扮演著重要角色,這些生化及遺傳上的證據更進一步顯示 FACT 在參與 DNA 複製上的可能性。

ABSTRACT
The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulator with important roles in replication and transcription. Although regarded as transcription elongator for chromatin template, little is known about mammalian FACT’s mode of action and involvement in other molecular processes. Here, we report the identification of a novel interacting and functional partner of FACT, Nek9. Nek9 forms a stable, ~600 kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation-dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr210). Phosphorylation at Thr210 is both necessary and sufficient for the activation of Nek9. When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr210. Cell cycle analysis on the Nek9dsRNAi cells directly implicated Nek9 in maintaining proper G1 and S progression, a role temporally correlated to the formation of a phospho-Nek9/FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression.
Minichromosome maintenance proteins (Mcms), which constitute the proposed replicative helicase, represent another group of cellular factors that associate with the FACT heterodimers. The observation that distinct subassemblies of Mcms form complexes with FACT may suggest their participation in discrete reactions or functions during DNA replication. Furthermore, similar to Nek9, the FACT-associated Mcm4 protein undergoes hyperphosphorylation in a cell cycle-dependent manner, implying a temporal mode of regulation for such complexes. Defect in S-phase progression, potentially at the elongation stage, was observed in yeast cells that harbor conditionally functional versions of Spt16 and Pob3. These results are consistent with the known functions of Mcm proteins in DNA replication and further strengthen the link of FACT to this chromatin-based molecular process.

CONTENTS
ABBREVIATIONS AND CHEMICAL SYMBOLS…………………………4
摘要 ……………………..……………………………………………………..5
ABSTRACT…………………………………………………….………………6
I. INTRODUCTION……………………………………………………………8
II. MATERIALS AND METHODS
Molecular cloning of the Nek9 cDNA…………………………………………...15
Generation of Nek9 kinase domain variants by site-directed mutagenesis…………...15
Preparation of recombinant proteins and antibodies……………………………...17
Immunoprecipitation, and western blot analysis………………………………….18
Gel-filtration fractionation…………………………………………………….19
Indirect immunofluorescence and confocal microscopy…………………………...19
Cell culture, transfection, and cell cycle synchronization………………………….20
Colony formation assay……………………………………………………….22
Fluorescence-activated cell sorting (FACS) analysis……………………………..22
Immunocomplex kinase assay………………………………………………….23
Peptide identification by mass spectrometry and bioinformatics analysis…………...23
Yeast strains and media……………………………………………………….25
Yeast cell cycle synchronization and FACS analysis……………………………...25
III. RESULTS
Part I: Nek9
Identification of Nek9 as a novel associated protein of FACT……………………...27
Nek9 interacts with FACT to form a ~600-kDa complex…………………………..28
Nek9 and FACT partially colocalize in the interphase nucleus, are similarly free from condensed chromatin, but dissociate from each other during chromatin decondensation…………………………………………………………….29
The Thr210 residue locates within the activation loop region of Nek9 and is conserved among various kinases……………………………………………………...31
Upon autoactivation, Nek9 undergoes phosphorylation-dependent electrophoretic mobility shift and phosphorylation on Thr210…………………………………..31
Phosphorylation on Thr210 is both necessary and sufficient for the activation of Nek9..33
Phospho-Nek9 associates with FACT in a cell cycle-dependent manner…………….34
RNAi-mediated knock-down of Nek9 proteins triggers interphase delay…………….35
Localization of Nek9 partially overlaps with DNA replication sites and euchromatin regions…………………………………………………………………….37
Part II: The hexameric minichromosome maintenance proteins (MCM)
Subassemblies of Mcms form distinct complexes with FACT heterodimers………….38
FACT-associated MCM4 is phosphorylated and undergoes mitotis-specific hyperphosphorylation……………………………………………………….39
Inactivation of yFACT proteins cause stalled S phase…………………………….40
IV. DISCUSSION
Nek9, a novel FACT-associated protein, modulates interphase progression………...42
Putative role of FACT in DNA replication may be mediated by associating with Mcms
……………………………………………………………………………..48
V. REFERENCES…………………………………………………………….50
VI. TABLES AND FIGURES………………………………………………..54

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