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標題: | Nucleolin 可促進 DNA Ligase III 之活性 Functional Interaction between Nucleolin and DNA Ligase III |
作者: | Chun-Hua Hsu 徐俊華 |
出版年 : | 1998 |
學位: | 碩士 |
摘要: | Nucleolin 又稱為 Protein C23 ,為快速增生的真核細胞核仁內主要之磷酸化蛋白質( phosphoprotein )。在以往的研究中,nucleolin 的生物功能除了調節 Pre-ribosomal RNA 之轉錄,及初期核糖體的匯聚與生合成,並幫助核糖體蛋白運回核仁內,這可能為其轉錄後調節的一環。就一級結構上而言,nucleolin 具有四個 RNA 結合的蛋白序列,為與 rRNA 作用有關;而報導指 nucleolin 是為轉錄因數且參與 class switch 的LR1,其組成之一 106kDa 的蛋白質為 nucleolin ,皆顯示了其 DNA 結合能力。使我們感興趣的是,這個具多功能的核仁蛋白是否在細胞生長的調控機制: DNA 的複製、修補或重組中、甚至於免疫系統中的 V ( D ) J recombination 與 class switch 內扮演著某種重要的角色。 而 ligase 在上述各種機制中,在最後的連結步驟是不可或缺的,也因此需要一個精密且適切的調控方式。因此我們希望能著手ligase之研究,就先前所知 nuc1eolin 在 class switch 中具備結合 DNA 之能力, nucleolin 與ligase 可能共存在一重組蛋白複合體(recombination protein complex)內。先以 nucleolin 之單株抗體進行免疫沉澱法,之後將沉澱物 adenylation ,且做 DNA ligation 試驗,均指出 nucleolin 與ligase 可共存在同一複合體中。依據分子量大小的推測,可能為 ligase III或 ligase IV 。經過重組之免疫沉澱(reconstitutive immunoprecipitation )後再予 adenylation 或直接偵測其標示之放射性活性,顯示 nucleolin 與 ligase III有直接的交互作用。更進一步的做 ligation 試驗,發現 nucleolin 具有增強 ligase III活性的功能。 這項結果指引出 nucleolin 在 ligase III 參與的機制中有著必要的角色扮演,或許只是幫助 ligase 的傳送,抑或進一步影響修補或重組等蛋白質複體的匯聚,更可能由於訊息傳遞上游之蛋白質影響 nucleolin 的磷酸化程度或裂解的行為而左右其 DNA 結合能力或蛋白質間交互做用的能力,進而調節 ligase 的活性,使得 DNA 之複製、修補或重組得到嚴密的調控。 Nucleolin, also known as protein C23, is a major phosphoprotein present in growing eukaryotic cells. Previous studies have demonstrated that nucleolin is involved in a huge array of molecular processes, including the regulation of pre-ribosomal RNA transcription, the biosynthesis and early assembly of ribosomes, and the nucleolus-bound transportation of ribonucleoprotein. The four RNA-binding motifs of nucleolin deduced from the protein primary structure implicates this protein in the rRNA related cellular events. Nucleolin also acts as a transcription factor. Furthermore, it has been identified as the 106kDa polypeptide in the LR1 complex, which is a protein machinery responsible for carrying out immunoglobulin heavy chain class switch event. All these proposed functions lead to the hypothesis that there might exit a functional relationship between nucleolin and DNA. They further prompted us to focus our investigation on the possible involvement of nucleolin in major cellular processes: DNA replication, repair, recombination, as well as V(D)J recombination and class switch events of the immune system. In order for the aforementioned processes to be executed properly, DNA ligase plays the most critical role as the final step. Hence, the precision of its regulation is highly imperative. We therefore explored the possibility of the potential interaction between nucleolin and DNA ligases. Our preliminary data obtained from the immunoprecipitation experiment, and subsequent adenylylation assays have offered strong evidence of this connection. Further experiment has pinpointed to the participant of this interaction to ligase III. Nucleolin has also been found to be able to stimulate ligase III activity. This report has clearly shown that nucleolin plays an essential role in the activity of ligase III and its related processes. However, the specific mechanism by which this interplay proceeds remains to be solved. The post-translational modification of nucleolin might be responsible for regulating its activity and interaction with other proteins. Such processing and regulator was result in the multifunctional characteristics of nucleolin. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75005 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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