NHEJ因子XRCC4、XLF與細胞週期檢查點蛋白SIRT2的蛋白作用力研究

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研究生：	黃建翔 Chien-Hsiang Hwang
論文名稱：	NHEJ因子XRCC4、XLF與細胞週期檢查點蛋白SIRT2的蛋白作用力研究 Protein-protein interaction study of the NHEJ factors, XRCC4 and XLF and the cell-cycle checkpoint protein, SIRT2
指導教授：	賴建勳 Jiann-Shiun Lai 潘榮隆 Rong-Long Pan
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2007
畢業學年度：	96
語文別：	英文
論文頁數：	68
中文關鍵詞：	NHEJ 、XRCC4 、XLF 、SIRT2 、dimerization 、Ligase IV
外文關鍵詞：	NHEJ, XRCC4, XLF, SIRT2, dimerization, Ligase IV
相關次數：	點閱：2 下載：0
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NHEJ是DNA 雙股斷裂修理的主要路徑之ㄧ。在NHEJ中有7個主要的因子。在已知的模式中， Ku70/Ku80 異型二聚體結合到DNA斷點，DNA-PKcs/Artemis 清除DNA 斷點上的多餘核酸。然後XRCC4/Ligase IV 複合體結合至此斷點，而Ligase IV 將二個DNA 斷點接合。 XRCC4 形成二聚體或四聚物對Ligase IV 結合XRCC4 是必須的。最近發現了另一個因子，XLF 。 XLF 顯示了與XRCC4 結構相似。 XLF也能夠形成同型二聚體，也能夠結合至DNA ，這些功能都與XRCC4 類似，並且加強NHEJ 辨認不同型DNA 斷點的能力。現在的NHEJ 模式傾向XRCC4 、XLF 和Ligase IV 形式複合體而不是原本的XRCC4/Ligase IV 複合體。在這份論文，我經由質粒的轉染進293T 細胞與過度表現蛋白，並且經由免疫沉澱和西部墨點來測試蛋白之間是否有作用。結果顯示，XLF 和XRCC4 同型二聚體的形成與XLF/XRCC4 異型二聚體的形成不互相影響。而XRCC4 二聚體形成和XLF 結合到XRCC4 的最小序列都在XRCC4 前134氨基酸的區域。更進一步的片斷突變將失去XRCC4 二聚作用和XLF 結合。這個結果顯示XRCC4 二聚作用和XLF 結合是同時存在的。
SIRT2 是細胞分裂檢查站蛋白質，且能夠防止染色體不穩定。 SIRT2 能夠在有cyclin B/cdc2 的活性下阻止有絲分裂。在G2/M 階段，XRCC4能夠跟SIRT2結合。在這份論文顯示， XLF 也能夠跟SIRT2 結合。且XRCC4 和SIRT2 的結合不影響XRCC4 對XLF 的結合。此外， SIRT2 能夠增強XLF 對XRCC4 的結合能力。所以， SIRT2/XLF/XRCC4 三者可能形成複合體並且提供NHEJ 到細胞週期之間的調節點。

NHEJ is a major pathway of the repair of DNA double strand breaks. There are seven important factors involved in NHEJ. In the original model, Ku70/Ku80 heterodimer binds to DNA break ends and DNA-PKcs/Artemis cleans up the DNA overhanging ends. After XRCC4 is recruited and as a template for Ligase IV binding, Ligase IV joins the two DNA break ends. XRCC4 forms dimer or tetramer in the cell and the dimerization of XRCC4 is required for Ligase IV binding. XLF was identified recently. XLF has been shown structural similarity to XRCC4. As XRCC4, XLF also form homodimer and improves the DNA recognizing ability in the NHEJ. The hypothesis now favors the XRCC4, XLF and Ligase IV form a complex for DNA binding instead of the original proposed XRCC4/Ligase IV complex. In this thesis, I have overexpressed specific proteins by transfection of plasmids into 293T cells and tested the binding activity by co-immunoprecipitation and western blotting. The result shows that the formation of the homodimer of both XLF and XRCC4 does not compete with the formation of the XLF/XRCC4 heterodimer. The minimal sequence of XRCC4 critical for XRCC4 dimerization and XLF binding has been mapped at the region of first 134 amino acids of XRCC4. Further deletion loses both XRCC4 dimerization and XLF binding activity. This result suggests that the XRCC4 dimerization and XLF binding are concomitant.
SIRT2 controls cellular proliferation and is a mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability. SIRT2 blocks the entry of mitosis in the presence of the cyclin B/cdc2 activity. XRCC4 binds to SIRT2 primary at the G2/M phase (Lee et al, submitted). In this thesis, the result shows that XLF also binds to SIRT2. The binding between XRCC4 and SIRT2 does not disrupt the binding of XRCC4 to XLF. Furthermore, SIRT2 enhances the binding of XLF to XRCC4. Therefore, SIRT2 might bind to XLF and XRCC4 as a complex and provides a regulatory point between the cell cycle control and NHEJ repairing machinery.

Abstract    2
Table of Contents    4
Abbreviations    6
Introduction    8
1. NHEJ (nonhomologous end-joining) and V(D)J recombination    8
2. XRCC4 and Ligase IV    10
3. XLF    11
4. NHEJ factors in Saccharomyces cerevisiae    12
5 .SIRT2    13
6. Aims    14
Materials and Methods    15
A General plasmid DNA preparation and construction    15
Mini preparation of DNA    15
Large DNA preparation    15
Preparation of Competent cell (Calcium chloride method)    16
Ligation    17
Bacterial transformation    17
Preservation of bacteria stock at -80℃    17
Plasmid constructs    18
Primers used for plasmid constructions    19
B. Mammalian cell experimental methods and materials    24
The mammalian cells and the ATCC numbers    24
Mammalian cell passage    24
Cell freezing and thawing    24
Transfection (Calcium phosphate transfection method)    25
Transfection (lipofectamine-Plus Invitrogen)    25
Immunoprecipitation    26
SDS- polyacrylamide gel electrophoresis and Western blotting    26
Antibodies    27
Results    28
Summary of the mutants of XRCC4    28
XLF binds to XRCC4 and SIRT2 in the 293T cell    28
The binding between XLF and XRCC4 does not affect the homodimer formation on XRCC4 or XLF.    29
The study of the binding region of XRCC4 to XLF    30
The study of XRCC4 dimerization    32
Binding study of FLAG-XLF and several GST-XRCC4 point mutations    34
Discussion    37
Figures    41
Figure 1: Summary of XRCC4 mutant plasmids used in the thesis    41
Figure 2: XLF binds to XRCC4 and SIRT2 in 293T cell    43
Figure 3: No competitive effect between XRCC4 and XLF while they bind to XLF    44
Figure 4: The interaction between XRCC4 deletion mutants and XLF    46
Figure 5: The dimer formation region study of XRCC4.    50
Figure 6: XRCC4 point mutations do not affect the XLF binding in 293T cell.    53
Figure 7: The model of XRCC4 C130A binds to XLF.    57
Supplemental Figures    58
References    60

                                

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