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標題: | DNA拓樸異構酶II對於染色體高度結構的組織及功能之探討 DNA Topoisomerase II in Higher-Order Chromosome Organization and Function |
作者: | Ya-Han Hsu 許雅涵 |
指導教授: | 李財坤(Tsai-Kun Li) |
關鍵字: | 拓樸異構酶,染色體結構,細胞核基座, DNA topoisomerase,chromosomal structure,nuclear matrix, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 我們實驗室致力於研究DNA拓撲異構酶的細胞生理功能。此論文的研究重點便是想探討第二類DNA拓樸酶的兩種同功酶 (isozymes) 於染色體DNA放射環的結構與功能上扮演之角色。染色體DNA線性長度遠大於細胞核的長度,所以 DNA 需大量濃縮以便置入核中。細胞核基座 (nuclear matrix) 在組成染色體的高度結構上具有舉足輕重的地位。前人發現第二類拓撲酶位於染色體DNA放射環 (chromosomal loop domains) 的連結基座 (nuclear matrix attachment regions) 上並參與染色質 (chromatin) 高度結構的調控。已知細胞中第二類拓樸酶有兩種isozyme - TOP2α及TOP2β(真核);gyrase及TopoIV(原核)。本論文便是想利用RNA interference (RNAi)及其它基因遺傳技術來探討其於染色體 DNA 放射環的結構與功能上扮演之角色。結果顯示,原核細胞中亦可能存在有類似DNA放射環的結構;而gyrase在此結構扮演較主要的角色。另外,利用RNAi技術製造出的第二類拓撲酶isozyme-specific knockdown的細胞株,指出 hTOP2β 於染色體DNA放射環的結構扮演較決定性的角色。另外,第二類拓撲酶isozyme-specific knockdown的細胞株亦提供了獨一的機會來研究TOP2α 及TOP2β分別及共有的功能與細胞對TOP2α 或TOP2β造成的DNA斷裂之訊息傳導和修復機制。 We studied the roles of topoisomerase II (TOP2) isoforms in chromosomal DNA loop domain organization/function. The eukaryotic nuclear DNA is packed with histones to form nucleosome, and further partitioned into topological/ chromatin loops emanating from the nuclear matrix/scaffold. Interestingly, DNA TOP2 has been shown to be a structural component of mitotic chromosome scaffolds. Recently, two isoforms of TOP2 have been identified (TOP2α and TOP2β) in mammalian cells and in bacterial cells [gyrase and topoisomerase IV (Topo IV)]. Here, we used the RNA interference technology and other genetic approaches to create TOP2 isozyme-deficient conditions for studying the structural/enzymatic roles of TOP2 isoforms in organizing the chromosomal DNA loop domains. Both of the isozyme-specific, RNAi-knockdown clones had been isolated and verified by Western blot analysis. In addition, all of the RNAi knockdown clones confer resistance to TOP2-targeting drugs. Preliminary results using PFGE analysis have also suggested that TOP2β might play a more dominant role in loop organization. To further support the notion that TOP2 is involved in the loop organization of the chromosome, we also explored the potential existence of chromosomal loops in prokaryotic cells. Our results showed that TOP2-targeting antibiotics can effectively cleave bacterial chromosome into 50-100 kb DNA fragments, indicating the existence of loop domains. Strains with genetic manipulations of drug-resistant gyrase and/or Topo IV provide a unique opportunity for dissecting their roles in bacterial chromosomal DNA loop organization. Using the loop-excision protocol and TOP2 drug-resistant bacteria, we have been able to identify gyrase, rather than TopoIV, as the main player in the organization of loop domains in bacterial nucleoid. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35724 |
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顯示於系所單位: | 微生物學科所 |
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