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研究生:蕭珺鈴
研究生(外文):Hsiao, Chun-Ling
論文名稱:Roleofaminoacidresidues(Lys)305and(Arg)306oftumorsuppressorp53innucleotideexcisionrepairofUVC-inducedDNAlesion
論文名稱(外文):抑癌因子p53 蛋白質序列之305 及306 胺基酸殘基對UVC 誘導DNA 損傷的核苷酸切除修復之重要性
指導教授:劉銀樟
指導教授(外文):Liu, Yin-Chang
學位類別:碩士
校院名稱:國立清華大學
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:2010
畢業學年度:98
語文別:英文
論文頁數:42
中文關鍵詞:p53蛋白單點突變
外文關鍵詞:p53site-specific mutation
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Tumor suppressor p53 is an essential component to maintain the genomic
integrity in mammalian cells. Loss p53 compromises the capacity of nucleotide
excision repair (NER). Our previous study has shown that p53 is essential to nuclear
extracts to excise UVC-induced DNA lesions in comet-NE assay, and the positions
299-308 (LPPGSTKRAL) of p53 is indispensable to both the excision activity and the
p53: XPB interaction. In this study, the two positive charged amino acid residues,
K305 and R306 of positions 299-308 were substituted with alanine, individually or
simultaneously, to test the importance of electrostatic contribution in this regard.
Results of the study show that the excision activity of nuclear extract and the
recruitment of XPB and RPA to UV-induced DNA damage sites was reduced in the
order of K305A≧ R306A/K305A> R306A. RPA is a single- DNA binding protein, it
is recruited to the DNA damage sites during NER pathway once the duplex DNA of
the sites are unwound by helicase XPB/XPD of TFIIH complex. This study
demonstrates the importance of electrostatic contribution to p53: XPB interaction in
NER.
Abstract ………………………………………………………………………… 4
中文摘要 …………………………………………………………………………
5
1. Intronduction
1.1. Nucleotide excision repair (NER) …………………………………………
1.2. Tumor suppressor protein, p53 …………………………………………...
1.3. Replication protein A (RPA) ………………………………………….......
1.4. Comet-nuclear extract (Comet-NE) assay ……………………………….
2. Materials and methods
2.1. Cell lines ……………………………………................................................
2.2. Local UV irradiation …………………………………................................
2.3. Expression plasmid ………………………………………………………...
2.4. Transient transfection ……………………………………………………..
2.5. Stable cell line ……………………………………………………………...
2.6. Nuclear extract (NE) preparation ………………………………………...
2.7. Comet-NE assay ……………………………………………………………
2.8. Whole cell extract preparation…………………………………………….
2.9. Western blotting …………………………………………………………...
2.10. Immunofluorescence staining ……………………………………………..
3. Results
3.1. Alanine substitution of K305 or/and R306 (of p53 sequence) attenuates
the excision activity of nuclear extracts toward UVC-induced DNA
damages but not the excision activity toward amoxicillin– induced (or
oxidative) DNA damages. ………………………………………………….
3.2. Alanine substitution of K305A or/and R306A (of p53 sequence)
attenuates the excision activity is resulted from the influence of the
recruitment between XPB and/or RPA proteins at UV-elicited DNA
damage sites …………………………………………………………………
3.3. Alanine substitutions of K305A or/and R306A reduce the recruitment
RPA to CPD sites……………………………………………………………...
4. Discussion ………………………………………………………………………..
5. Figures and figure legends ……………………………………………..........
6. References ……………………………………………………………………….
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