Tumor suppressor p53 has been found to facilitate the repair of DNA damages caused by UVC irradiation and enhance the cell survival following the radiation. Nucleotide excision repair (NER) in mammalian cells is the sole mechanism for eliminating the DNA lesions induced by UVC. The involvement of p53, a well-known transactivator, in NER may be via transaction-dependent and transaction-independent manners. Our previous study by comet-assay has shown that p53 is essential for nuclear extracts (NE) to excise UV-induce DNA lesions. In this thesis study, the sequence of p53 protein essential for this activity was further defined to positions 299-308 (LPPGSTKRAL). The peptide derived from the region but not the irrelevant one had inhibitory effect to the excision activity of NE in comet-assay, and interfered the p53:XPB interaction in co-immunoprecipitation experiment. The importance of the only two positive charge bearing residues, Lys (K)305 and Arg (R)306, in this region was examined by substitution with alanine. The inhibitory effect of peptide on NE excision was attenuated. This was supported by the experiments with plasmids expressing the site-specific mutation of p53. The NEs prepared from cells transfected with the respective plasmids showed reduced excision activity, suggesting that positive charges play role in the p53:XPB interaction.

抑癌因子p53蛋白可以促進細胞修復UVC紫外線所造成的DNA損傷，並且增加照射輻射後的細胞存活率。核苷酸切除修復路徑(NER)是哺乳動物細胞去除UVC紫外線造成的DNA損傷的唯一途徑。p53可能藉由轉錄活化(transaction-dependent)或是非轉錄活化(transaction-independent)達成促進NER的功能。我們之前利用彗星分析法的研究指出p53於核萃取物(NE)去除紫外線導致的DNA損傷中扮演必要的角色。在此論文研究中，我們進一步確定p53蛋白是利用第299到308序列(LPPGSTKRAL)來參與此活性。利用彗星分析法發現，由這片段胺基酸所衍生而來的胜肽具有抑制核萃取物的切割核酸活性，並且在共同免疫沉澱分析中可見胜肽會干擾p53:XPB的交互作用。我們利用丙胺酸(Ala)來替代此片段中僅有的兩個帶正電胺基酸：離胺酸Lys (K)305和精胺酸Arg (R)306，以探討其重要性；發現此突變胜肽對於核萃取物的切割抑制作用降低。質體表現的P53點突變株也得到同樣結果。攜帶不同質體的細胞所萃取出的核萃取物皆顯示其切割活性降低。此結果說明帶正電的胺基酸在p53:XPB交互作用中扮演重要的角色。

Adimoolam, S. and J. M. Ford (2002). "p53 and DNA damage-inducible expression of the xeroderma pigmentosum group C gene." Proc Natl Acad Sci U S A 99(20): 12985-90.
Agarwal, M. L., W. R. Taylor, et al. (1998). "The p53 network." J Biol Chem 273(1): 1-4.
Balajee, A. S., A. May, et al. (1998). "Efficient PCNA complex formation is dependent upon both transcription coupled repair and genome overall repair." Mutat Res 409(3): 135-46.
Bates, S. and K. H. Vousden (1999). "Mechanisms of p53-mediated apoptosis." Cell Mol Life Sci 55(1): 28-37.
Beamish, H., K. K. Khanna, et al. (1994). "Ionizing radiation and cell cycle progression in ataxia telangiectasia." Radiat Res 138(1 Suppl): S130-3.
Cadet, J., E. Sage, et al. (2005). "Ultraviolet radiation-mediated damage to cellular DNA." Mutat Res 571(1-2): 3-17.
Chang, Y. C., K. Y. Jan, et al. (2008). "Direct involvement of the tumor suppressor p53 in nucleotide excision repair." DNA Repair (Amst) 7(5): 751-61.
Cho, Y., S. Gorina, et al. (1994). "Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations." Science 265(5170): 346-55.
Conconi, A. (2005). "The yeast rDNA locus: a model system to study DNA repair in chromatin." DNA Repair (Amst) 4(8): 897-908.
Coohill, T. P. (1996). "Stratospheric ozone loss, ultraviolet effects and action spectroscopy." Adv. Space. Res 18(12): 27-33.
Coohill, T. P. and J. L. Sagripanti (2008). "Overview of the inactivation by 254 nm ultraviolet radiation of bacteria with particular relevance to biodefense." Photochem Photobiol 84(5): 1084-90.
Croteau, D. L., Y. Peng, et al. (2008). "DNA repair gets physical: mapping an XPA-binding site on ERCC1." DNA Repair (Amst) 7(5): 819-26.
de Boer, J., J. de Wit, et al. (1998). "A mouse model for the basal transcription/DNA repair syndrome trichothiodystrophy." Mol Cell 1(7): 981-90.
Douki, T. and J. Cadet (2001). "Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions." Biochemistry 40(8): 2495-501.
el-Deiry, W. S., T. Tokino, et al. (1993). "WAF1, a potential mediator of p53 tumor suppression." Cell 75(4): 817-25.
Fields, S. and S. K. Jang (1990). "Presence of a potent transcription activating sequence in the p53 protein." Science 249(4972): 1046-9.
Foord, O. S., P. Bhattacharya, et al. (1991). "A DNA binding domain is contained in the C-terminus of wild type p53 protein." Nucleic Acids Res 19(19): 5191-8.
Fritsche, M., C. Haessler, et al. (1993). "Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents." Oncogene 8(2): 307-18.
Funk, W. D., D. T. Pak, et al. (1992). "A transcriptionally active DNA-binding site for human p53 protein complexes." Mol Cell Biol 12(6): 2866-71.
Geyer, R. K., H. Nagasawa, et al. (2000). "Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest." Cell Growth Differ 11(3): 149-56.
Harris, C. C. (1996). "Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies." J Natl Cancer Inst 88(20): 1442-55.
Jayaraman, L., E. Freulich, et al. (1997). "Functional dissection of p53 tumor suppressor protein." Methods Enzymol 283: 245-56.
Kan, L. S., L. Voituriez, et al. (1992). "The Dewar valence isomer of the (6-4) photoadduct of thymidylyl-(3'-5')-thymidine monophosphate: formation, alkaline lability and conformational properties." J Photochem Photobiol B 12(4): 339-57.
Kern, S. E., K. W. Kinzler, et al. (1991). "Identification of p53 as a sequence-specific DNA-binding protein." Science 252(5013): 1708-11.
Kraemer, K. H., M. M. Lee, et al. (1984). "DNA repair protects against cutaneous and internal neoplasia: evidence from xeroderma pigmentosum." Carcinogenesis 5(4): 511-4.
Lakin, N. D. and S. P. Jackson (1999). "Regulation of p53 in response to DNA damage." Oncogene 18(53): 7644-55.
Leveillard, T., L. Andera, et al. (1996). "Functional interactions between p53 and the TFIIH complex are affected by tumour-associated mutations." Embo J 15(7): 1615-24.
Levine, A. J. (1990). "Tumor suppressor genes." Bioessays 12(2): 60-6.
Levine, A. J. (1997). "p53, the cellular gatekeeper for growth and division." Cell 88(3): 323-31.
Lin, J., J. Chen, et al. (1994). "Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein." Genes Dev 8(10): 1235-46.
Mazur, S. J., K. Sakaguchi, et al. (1999). "Preferential binding of tumor suppressor p53 to positively or negatively supercoiled DNA involves the C-terminal domain." J Mol Biol 292(2): 241-9.
Mitchell, D. L. and R. S. Nairn (1989). "The biology of the (6-4) photoproduct." Photochem Photobiol 49(6): 805-19.
Miura, M. (1999). "Detection of chromatin-bound PCNA in mammalian cells and its use to study DNA excision repair." J Radiat Res (Tokyo) 40(1): 1-12.
Mocquet, V., J. P. Laine, et al. (2008). "Sequential recruitment of the repair factors during NER: the role of XPG in initiating the resynthesis step." Embo J 27(1): 155-67.
Nance, M. A. and S. A. Berry (1992). "Cockayne syndrome: review of 140 cases." Am J Med Genet 42(1): 68-84.
Niedernhofer, L. J. (2008). "Tissue-specific accelerated aging in nucleotide excision repair deficiency." Mech Ageing Dev 129(7-8): 408-15.
Oren, M. and V. Rotter (1999). "Introduction: p53--the first twenty years." Cell Mol Life Sci 55(1): 9-11.
Patrick, S. M. and J. J. Turchi (2002). "Xeroderma pigmentosum complementation group A protein (XPA) modulates RPA-DNA interactions via enhanced complex stability and inhibition of strand separation activity." J Biol Chem 277(18): 16096-101.
Pfeifer, G. P. (1997). "Formation and processing of UV photoproducts: effects of DNA sequence and chromatin environment." Photochem Photobiol 65(2): 270-83.
Pfeifer, G. P., Y. H. You, et al. (2005). "Mutations induced by ultraviolet light." Mutat Res 571(1-2): 19-31.
Pietenpol, J. A., T. Tokino, et al. (1994). "Sequence-specific transcriptional activation is essential for growth suppression by p53." Proc Natl Acad Sci U S A 91(6): 1998-2002.
Sancar, A. (1994). "Mechanisms of DNA excision repair." Science 266(5193): 1954-6.
Schaeffer, L., R. Roy, et al. (1993). "DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor." Science 260(5104): 58-63.
Seo, Y. R. and H. J. Jung (2004). "The potential roles of p53 tumor suppressor in nucleotide excision repair (NER) and base excision repair (BER)." Exp Mol Med 36(6): 505-9.
Steele, R. J., A. M. Thompson, et al. (1998). "The p53 tumour suppressor gene." Br J Surg 85(11): 1460-7.
Sturzbecher, H. W., R. Brain, et al. (1992). "A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization." Oncogene 7(8): 1513-23.
Sugasawa, K., J. M. Ng, et al. (1997). "Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity." Mol Cell Biol 17(12): 6924-31.
Tsodikov, O. V., D. Ivanov, et al. (2007). "Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA." Embo J 26(22): 4768-76.
Wang, X. W., K. Forrester, et al. (1994). "Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3." Proc Natl Acad Sci U S A 91(6): 2230-4.
Wang, X. W., H. Yeh, et al. (1995). "p53 modulation of TFIIH-associated nucleotide excision repair activity." Nat Genet 10(2): 188-95.