臺灣博碩士論文加值系統

在真核細胞中，ＤＮＡ複製起始的詳細機制仍不明確，特別是複製起始是否具有特定的位置與如何決定了解不多，目前被認為染色體構造與轉錄活性有關。果蠅的卵泡細胞的蛋殼蛋白基因增生，是個具有多重細胞複製起始的過程，而可以用於研究複製起始的生物模式，目前已知道染色體構造與轉錄因子皆可以調控此複製起始的活性與專一性，但詳細機制仍不明確。泛素黏合酵素Ｃul4與ＤＤＢ1可以調控複製所需的蛋白Cdt1的降解，另外在染色體上也有許多重要的功能。在本論文我們以卵泡細胞的蛋殼蛋白基因增生作為題材，探討Ｃul4是否參與此系統中Cdt1蛋白降解的調控，以及是否參與複製起始的其他步驟。此系統中，果蠅的Cdt1蛋白會聚集在這些正在複製的基因上，而我們發現在大部分的Ｃul4突變細胞中，Cdt1蛋白卻會有累積在細胞核內的現象，然而，ＤＤＢ1突變細胞卻只有部分會如此。進一步分析Ｃul4與ＤＤＢ1突變細胞中，都觀察到蛋殼蛋白基因增生發生缺失及不正常基因複製的現象，以及複製起始的Ｏrc2蛋白也無法坐落於蛋殼蛋白基因起始的位置。為了找出Ｃul4的角色，我們找到Ｃul4可以在細胞內外與另外一個起始蛋白ＭＣＭ2-7結合，我們利用基因的交互作用進行實驗發現Ｃul4與ＭＣＭ2-7是協同作用的;然而，Ｃul4卻可以藉由負調節Ｅ2F1的功能來使在蛋殼蛋白基因增生的複製起始能正常進行。

In high eukaryotes, the mechanism of DNA replication initiation remains unclear, and evidences indicate that the chromatin structure and transcription activity are important. Chorion gene amplification of Drosophila follicle cells is a good model system to address how the initiation starts. It has been shown that transcriptional factors (E2F/RB and the Myb complex) and chromatin regulate origin activity in chorion loci, but the detail mechanisms are still unclear. The Cul4-DDB1- based ubiquitin ligase is known to regulate the replication licensing factor Cdt1 level and also execute multiple functions on chromatin. In this study, I investigated if Cul4 participates in Cdt1 degradation and the process of replication initiation during gene amplification. During the initiation of chorion gene amplification, Double-parked (Dup), the Drosophila ortholog of Cdt1, is restricted to chorion gene foci. I found that Dup accumulated in nuclei in Cul4 mutant follicle cells, and the accumulation was less prominent in DDB1 mutant cells. Loss of Cul4 or DDB1 activity in follicle cells also compromised chorion gene amplification and induced ectopic genomic DNA replication. The focal localization of Orc2, a subunit of the origin recognition complex, is frequently absent in Cul4 mutant follicle cells. Furthermore, double-labeling BrdU and Orc2 showed that two replication phenotypes (inappropriate genomic DNA replication and origin firing ceased, respectively) in Cul4 mutant cells could exist at the same time. To reveal how Cul4 functions, I identified Cul4 complexes with another licensing factor MCM2-7 in vivo and in vitro, and the genetic interaction suggests that Cul4 and MCM2-7 function together. Interestingly, reducing E2F1 genomic dose suppresses the Cul4 mutant phenotype suggesting that Cul4 negatively regulated E2F1 activity for initiation process. Taken together, our results indicate that the Cul4-DDB1 based ubiquitin ligase functions in DNA replication initiation during gene amplification.

中文摘要 III
ABSTRACT IV
INTRODUCTION 1
DNA REPLICATION INITIATION 1
DROSOPHILA GENE AMPLIFICATION IN FOLLICLE CELLS 1
CUL4-DDB1 UBIQUITIN LIGASE FUNCTIONS ON CHROMATIN 3
CUL4 MEDIATES CDT1 DEGRADATION 6
MATERIALS AND METHODS 9
FLY STOCKS AND GENETICS 9
IMMUNOLABELING AND MICROSCOPY 10
BRDU INCORPORATION ASSAYS 10
Q-PCR OF CHORION GENES AND BRDU INCORPORATION IN HEAT SHOCK INDUCED CUL4 KR FLIES 11
RESULTS 13
GENERATION OF CUL4 MUTANTS 13
DUP ACCUMULATED DIFFERENTIALLY IN CUL4-DDB1 MUTANTS 14
CUL4-DDB1 MUTANT CELLS EXHIBIT DISTINCT REPLICATION PHENOTYPES 16
LOCALIZATION OF ORC2 IS DIFFERENTIALLY ALTERED IN CUL4-DDB1 MUTANT 18
CUL4 COMPLEXES WITH MCM IN VIVO AND IN VITRO 19
THE GENETIC INTERACTIONS OF CUL4 DURING GENE AMPLIFICATION 20
CUL4 IS REQUIRED FOR PROLIFERATION DURING DEVELOPMENT 21
CYCE IS ACCUMULATED IN CUL4 MUTANT IMAGINAL DISC CELLS 22
DISCUSSION 24
ABNORMAL DUP ACCUMULATION IN CUL4 MUTANT CELLS IN DNA REPLICATION 24
INVOLVEMENT OF CUL4 AND DDB1 IN CHORION GENE AMPLIFICATION 25
ROLE CUL4 IN CONTROLLING ORC2 PROTEIN LOCALIZATION 27
REFERENCE 29

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