臺灣博碩士論文加值系統

人類修復蛋白DDB2為基因損傷結合蛋白複合物其中一個次單元，基因損傷結合蛋白複合物主要是參與在DNA修復機制中的核甘酸切除修復機制，其功能為辨識並結合經紫外線照射或致癌化學藥品造成的DNA傷害。當人類修復蛋白DDB2發生突變時，會導致著色性乾皮症，其主要臨床特徵為對光照極度敏感，且紫外線照射下容易導致皮膚癌化。另外，之前也有研究指出DDB2缺陷的老鼠會發展出自發性腫瘤。同時，DDB2也參與在p53所調控的細胞凋亡機制當中。因此，DDB2在抑制腫瘤機制當中扮演重要角色。然而，對於DDB2在抑制腫瘤其機轉並不是十分清楚。為了進一步了解DDB2在抑制腫瘤機轉當中，其可能包含的一些相關蛋白，我們希望尋找出DDB2的相關蛋白，並進一步研究DDB2與其相關蛋白之間功能上的相關性。本研究中，首先利用酵母菌雜合系統(yeast-two hybrid system)，篩選出可以與DDB2交互作用的結合蛋白，並進一步了解這些DDB2結合蛋白與DDB2在功能上其相關性。利用此系統，我們篩選出---BERG36此蛋白，其為B細胞早期反應基因。而BERG36之前被發現主要與調控含ARE轉錄有關，並且會促進細胞凋亡反應。BERG36與DDB2在細胞內的交互作用也利用免疫共沈澱(co-immunoprecipitation)的方式證明。另外我們也發現BERG36的表現會受到cisplatin及紫外線照射所誘發。由於DDB2為一DNA修復蛋白，因此我們進一步探討是否BERG36也同樣具有DNA修復功能。透過寄主細胞再活化分析(host cell reactivation assay)，結果顯示，BERG36會抑制DNA修復活性及DDB2其DNA修復功能，顯示BERG36對於DDB2在DNA修復上扮演一個拮抗者的角色。此外，之前研究發現BERG36有參與在細胞凋亡路徑，本研究也進一步確認BERG36在細胞凋亡反應中所扮演的角色。藉由流式細胞儀判定凋亡的細胞，我們發現細胞在過量表現BERG36並受到cisplatin處理或紫外線照射下，會加速細胞走向凋亡路徑。另外，由文獻已得知DDB2會促進cFLIP此抗細胞凋亡分子表現，而在我們的結果當中則發現，細胞中過量表現BERG36會抑制cFLIP表現。此外也發現在紫外線照射下，BERG36會大量聚積在粒腺體，顯示其在粒腺體相關的細胞凋亡路徑中扮演重要角色。綜合以上結果，我們推測BERG36藉由與DDB2交互作用，在DNA修復及細胞凋亡機制中，其功能為扮演DDB2拮抗者角色，因此，在未來研究中，我們希望進一步釐清DDB2與BERG36之間其相互拮抗之分子機轉。

DDB2 is an essential subunit of the damaged-DNA binding protein (DDB), which is involved in nucleotide excision repair (NER) in human cells. DDB2, together with DDB1, comprises the UV-DDB complex, which recognizes DNA lesions caused by UV irradiation. Mutations in the human DDB2 gene give rise to xeroderma pigmentosum group E (XPE), a disease characterized by increased skin tumorigenesis in response to UV-irradiation. Similarly to XPE patients, the DDB2-deficient mice developed spontaneous tumors. DDB2 is also shown to involve in p53-mediated apoptosis. Therefore DDB2 participates in tumor suppression through multiple pathways. However, the role of DDB2 in tumor suppression was poorly understood. By screening for the proteins which interact with DDB2 through yeast two-hybrid assays, we found that BERG36, a B cell early response gene encoding a 36kDa protein, is associated with DDB2 protein. BERG36 has been known to mediate apoptosis, which DDB2 is also involved in. BERG36 also regulates an AU-rich element (ARE)-containing reporter transcript. The interaction between DDB2 and BERG36 was detected by co-immunoprecipitation experiments and immunofluorescence assays. BERG36 expression was enhanced by cisplatin and UV treatment, indicating it is a DNA damage- inducible gene. By host cell reactivation assays to detect the DNA repair activities, BERG36 was found to inhibit DNA repair activity, suggesting that it antagonizes DNA repair function of DDB2. We hypothesized that the BERG36 is involved in apoptosis concertedly with DDB2 (or DDB complex). By annexin V staining assays, it was found that BERG36 enhanced apoptosis in human HtTA1 cells treated with UV irradiation or cisplatin, indicating that the BERG36 performs dual effects on DNA repair and apoptosis. We also observed that BERG36 accumulated in mitochondria in response to DNA damage, indicating that BERG36 plays a role in the mitochondrial apoptosis pathway. DDB2 has been found to up-regulate anti-apoptotic gene cFLIP. On the contrary, BERG36 appears to down-regulate cFLIP expression. Moreover, we also found that DDB2 increased cIAP2 expression and BERG36 reduced cIAP2 expression. Thus, BERG36 is suggested to be a DDB2 partner in DNA repair and apoptosis. In the future, we will focus on studies of molecular mechanisms for antagonistic functions between DDB2 and BERG36

Contents


Abstract in Chinese…………………………………………………………………..I
Abstract in English…………………………………………………………………...III
Acknowledgements…………………………………………………………………...V
Abbreviations…………………………………………………………………...…….VII
Contents……………………………………………………………………………….VIII
Contents of Tables and Figures……………………………………………………….XI

I. INTRODUCTION……………………………………………………………….….1
1.1 DNA damage and its consequences…………………………………………….......1
1.2 DNA Repair………………………………………………………….………….…..2
1.3 Nucleotide excision repair (NER) ……………………………………………….…4
1.3.1 The mechanism of NER………………………..………………………………4
1.3.2 NER and human genetic diseases………………………………………………5
1.4 The physiological roles of DDB2…………………………………………………...7
1.5 Characteristic of BERG36…………………………………………………………..9


II. SPECIFIC AIMS…………………………………………………… ………..…...11

III. MATERIALS AND METHODS……………………………………………..…..12
3.1 Yeast two-hybrid……………………………………………………………............12
3.2 Cell line and culture………………………………………..……………………….12
3.3 Plasmid constructs…………………………………………………………..……...13
3.4 Transfection……………………………………………………………….………..13
3.5 Immunoprecipitation and Western blotting…………………………………………14
3.6 MTT assays……………………………………………..…………………..….…...14
3.7 Analysis of apoptosis………………………………………………………..……...15
3.8 Host cell reactivation (HCR) assay…………………………..…………………….15
3.9 RNA isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)………………………………………………………………………….........16
3.10 Immunofluorescence and Microscopy………………………………………........17
3.11 Plasmid constructs for yeast screening……………………………….……..........18
3.12 siRNA constructs……………………….…………………………………...........18

IV. RESULTS…………………………………………………………………….….....19
4.1 Identificaiton of DDB2 interactive proteins…………………………………….….19
4.2 In vivo association between DDB2 and putative DDB2 binding protein……….….20
4.3 DDB2 and BERG36 were colocalized in the nucleus……………………….….….20
4.4 BERG36 expression is enhanced by cisplatin and UV treatment in HtTA1 cells………………………………………………………………………………….….21
4.5 DNA repair activities are stimulated by DDB2, but not by BERG36. BERG36 appears to antagonize DDB2 repair function.………………..……………………………...…..22
4.6 BERG36 sensitizes cells to DNA damage……………………………..……….…..23
4.7 BERG36 expression enhanced UV- or cisplatin-induced apoptosis………….….…23
4.8 BERG36 inhibits cFLIP expression and enhances procaspase 8 cleavage………………………………………………………………………….….….23
4.9 BERG36 decreased the level of the inhibitor of apoptosis protein 2 (cIAP2)…......24
4.10 BERG36 shuttles to the mitochondria in response to genotoxic stress……….…..24

V. DISCUSSION…………………………………………………………….………...26
VI. REFERENCES……………………………………………………………………31
VII. TABLES ……………………………………………………………………….....37
VIII. FIGURES…………………………………………………………………..……39
IX. APPENDIX…………………………………………………………………..….…50

1.Valerie, K. & Povirk, L.F. Regulation and mechanisms of mammalian double-strand break repair. Oncogene 22, 5792-5812 (2003).
2.Shigenaga, M.K., Gimeno, C.J. & Ames, B.N. Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proceedings of the National Academy of Sciences of the United States of America 86, 9697-9701 (1989).
3.Wood, R.D., et al. DNA damage recognition and nucleotide excision repair in mammalian cells. Cold Spring Harbor Symposia on Quantitative Biology 65, 173-182 (2000).
4.Lindahl, T. & Barnes, D.E. Repair of endogenous DNA damage. Cold Spring Harbor Symposia on Quantitative Biology 65, 127-133 (2000).
5.Liu, Y. & Kulesz-Martin, M. P53 regulation and function in normal cells and tumors. Medicina 60 Suppl 2, 9-11 (2000).
6.Wakasugi, M., et al. Damaged DNA-binding protein DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and replication protein A. The Journal of biological chemistry 276, 15434-15440 (2001).
7.Fujiwara, Y., et al. Characterization of DNA recognition by the human UV-damaged DNA-binding protein. The Journal of Biological Chemistry 274, 20027-20033 (1999).
8.Bol, S.A., et al. Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo. Mutagenesis 14, 317-322 (1999).
9.Andressoo, J.O., Hoeijmakers, J.H. & Mitchell, J.R. Nucleotide excision repair disorders and the balance between cancer and aging. Cell Cycle (Georgetown, Tex 5, 2886-2888 (2006).
10.O'Brien, T.J., Brooks, B.R. & Patierno, S.R. Nucleotide excision repair functions in the removal of chromium-induced DNA damage in mammalian cells. Molecular and Cellular Biochemistry 279, 85-95 (2005).
11.Castro, M.A., Mombach, J.C., de Almeida, R.M. & Moreira, J.C. Impaired expression of NER gene network in sporadic solid tumors. Nucleic Acids Research (2007).
12.Ura, K. & Hayes, J.J. Nucleotide excision repair and chromatin remodeling. European journal of Biochemistry / FEBS 269, 2288-2293 (2002).
13.Aboussekhra, A., et al. Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80, 859-868 (1995).
14.Kannouche, P. & Stary, A. Xeroderma pigmentosum variant and error-prone DNA polymerases. Biochimie 85, 1123-1132 (2003).
15.Norgauer, J., Idzko, M., Panther, E., Hellstern, O. & Herouy, Y. Xeroderma pigmentosum. Eur J Dermatol 13, 4-9 (2003).
16.Yu, Y.J. [Genetic modification of porcine organs for human in xenotransplantation]. Yi chuan = Hereditas / Zhongguo yi chuan xue hui bian ji 25, 596-600 (2003).
17.Li, J., et al. DNA Damage Binding Protein Component DDB1 Participates in Nucleotide Excision Repair through DDB2 DNA-binding and Cullin 4A Ubiquitin Ligase Activity. Cancer Research 66, 8590-8597 (2006).
18.Zolezzi, F. & Linn, S. Studies of the murine DDB1 and DDB2 genes. Gene 245, 151-159 (2000).
19.Hirschfeld, S., Levine, A.S., Ozato, K. & Protic, M. A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells. Molecular and Cellular Biology 10, 2041-2048 (1990).
20.Payne, A. & Chu, G. Xeroderma pigmentosum group E binding factor recognizes a broad spectrum of DNA damage. Mutation Research 310, 89-102 (1994).
21.Wittschieben, B.B. & Wood, R.D. DDB complexities. DNA Repair 2, 1065-1069 (2003).
22.Itoh, T. Xeroderma pigmentosum group E and DDB2, a smaller subunit of damage-specific DNA binding protein: proposed classification of xeroderma pigmentosum, Cockayne syndrome, and ultraviolet-sensitive syndrome. Journal of Dermatological Science 41, 87-96 (2006).
23.Sugasawa, K. The xeroderma pigmentosum group C protein complex and ultraviolet-damaged DNA-binding protein: functional assays for damage recognition factors involved in global genome repair. Methods in Enzymology 408, 171-188 (2006).
24.Shiyanov, P., et al. The naturally occurring mutants of DDB are impaired in stimulating nuclear import of the p125 subunit and E2F1-activated transcription. Molecular and Cellular Biology 19, 4935-4943 (1999).
25.Hayes, S., Shiyanov, P., Chen, X. & Raychaudhuri, P. DDB, a putative DNA repair protein, can function as a transcriptional partner of E2F1. Molecular and Cellular Biology 18, 240-249 (1998).
26.Prost, S., Lu, P., Caldwell, H. & Harrison, D. E2F regulates DDB2: consequences for DNA repair in Rb-deficient cells. Oncogene 26, 3572-3581 (2007).
27.Groisman, R., et al. The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell 113, 357-367 (2003).
28.Matsuda, N., et al. DDB2, the xeroderma pigmentosum group E gene product, is directly ubiquitylated by Cullin 4A-based ubiquitin ligase complex. DNA Repair 4, 537-545 (2005).
29.Nag, A., Bondar, T., Shiv, S. & Raychaudhuri, P. The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells. Molecular and Cellular Biology 21, 6738-6747 (2001).
30.El-Mahdy, M.A., et al. Cullin 4A-mediated proteolysis of DDB2 protein at DNA damage sites regulates in vivo lesion recognition by XPC. The Journal of Biological Chemistry 281, 13404-13411 (2006).
31.Datta, A., et al. The p48 subunit of the damaged-DNA binding protein DDB associates with the CBP/p300 family of histone acetyltransferase. Mutation Research 486, 89-97 (2001).
32.Martinez, E., et al. Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo. Molecular and Cellular Biology 21, 6782-6795 (2001).
33.Fitch, M.E., Cross, I.V. & Ford, J.M. p53 responsive nucleotide excision repair gene products p48 and XPC, but not p53, localize to sites of UV-irradiation-induced DNA damage, in vivo. Carcinogenesis 24, 843-850 (2003).
34.Hwang, B.J., Ford, J.M., Hanawalt, P.C. & Chu, G. Expression of the p48 xeroderma pigmentosum gene is p53-dependent and is involved in global genomic repair. Proceedings of the National Academy of Sciences of the United States of America 96, 424-428 (1999).
35.Tan, T. & Chu, G. p53 Binds and activates the xeroderma pigmentosum DDB2 gene in humans but not mice. Molecular and Cellular Biology 22, 3247-3254 (2002).
36.Adimoolam, S. & Ford, J.M. p53 and regulation of DNA damage recognition during nucleotide excision repair. DNA Repair 2, 947-954 (2003).
37.El-Deiry, W.S. Transactivation of repair genes by BRCA1. Cancer Biology & Therapy 1, 490-491 (2002).
38.Hartman, A.R. & Ford, J.M. BRCA1 induces DNA damage recognition factors and enhances nucleotide excision repair. Nature Genetics 32, 180-184 (2002).
39.Takimoto, R., et al. BRCA1 transcriptionally regulates damaged DNA binding protein (DDB2) in the DNA repair response following UV-irradiation. Cancer Biology & Therapy 1, 177-186 (2002).
40.Sun, N.K., Kamarajan, P., Huang, H. & Chao, C.C. Restoration of UV sensitivity in UV-resistant HeLa cells by antisense-mediated depletion of damaged DNA-binding protein 2 (DDB2). FEBS letters 512, 168-172 (2002).
41.Gomperts, M., Pascall, J.C. & Brown, K.D. The nucleotide sequence of a cDNA encoding an EGF-inducible gene indicates the existence of a new family of mitogen-induced genes. Oncogene 5, 1081-1083 (1990).
42.Barnard, R.C., et al. Coding sequence of ERF-1, the human homologue of Tis11b/cMG1, members of the Tis11 family of early response genes. Nucleic Acids Research 21, 3580 (1993).
43.Maclean, K.N., See, C.G., McKay, I.A. & Bustin, S.A. The human immediate early gene BRF1 maps to chromosome 14q22-q24. Genomics 30, 89-90 (1995).
44.Liao, Y., Moir, R.D. & Willis, I.M. Interactions of Brf1 peptides with the tetratricopeptide repeat-containing subunit of TFIIIC inhibit and promote preinitiation complex assembly. Molecular and Cellular Biology 26, 5946-5956 (2006).
45.Stoecklin, G., et al. Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover. The EMBO journal 21, 4709-4718 (2002).
46.Martinez, M.J. & Sprague, K.U. Cloning of a putative Bombyx mori TFIIB-related factor (BRF). Archives of Insect Biochemistry and Physiology 54, 55-67 (2003).
47.Schmidlin, M., et al. The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B. The EMBO journal 23, 4760-4769 (2004).
48.Carballo, E., Lai, W.S. & Blackshear, P.J. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science (New York, N.Y 281, 1001-1005 (1998).
49.Kontoyiannis, D., Pasparakis, M., Pizarro, T.T., Cominelli, F. & Kollias, G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 10, 387-398 (1999).
50.Schuler, G.D. & Cole, M.D. GM-CSF and oncogene mRNA stabilities are independently regulated in trans in a mouse monocytic tumor. Cell 55, 1115-1122 (1988).
51.Ning, Z.Q., Norton, J.D., Li, J. & Murphy, J.J. Distinct mechanisms for rescue from apoptosis in Ramos human B cells by signaling through CD40 and interleukin-4 receptor: role for inhibition of an early response gene, Berg36. European journal of Immunology 26, 2356-2363 (1996).
52.Lee, S.K., et al. Butyrate response factor 1 enhances cisplatin sensitivity in human head and neck squamous cell carcinoma cell lines. International journal of Cancer 117, 32-40 (2005).
53.Moser, J., et al. The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions. DNA Repair 4, 571-582 (2005).
54.Alekseev, S., et al. Enhanced DDB2 expression protects mice from carcinogenic effects of chronic UV-B irradiation. Cancer Research 65, 10298-10306 (2005).
55.Lai, W.S., Carballo, E., Thorn, J.M., Kennington, E.A. & Blackshear, P.J. Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA. The Journal of Biological Chemistry 275, 17827-17837 (2000).
56.Sun, N.K., Lu, H.P. & Chao, C.C. Overexpression of damaged-DNA-binding protein 2 (DDB2) potentiates UV resistance in hamster V79 cells. Chang Gung medical journal 25, 723-733 (2002).
57.Sun, C.L. & Chao, C.C. Potential attenuation of p38 signaling by DDB2 as a factor in acquired TNF resistance. International journal of Cancer 115, 383-387 (2005).
58.Cottet, S., et al. cFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells. Diabetes 51, 1805-1814 (2002).
59.Chau, H., et al. Cellular FLICE-inhibitory protein is required for T cell survival and cycling. The Journal of Experimental Medicine 202, 405-413 (2005).
60.Hainsworth, A.H., et al. Expression of cellular FLICE inhibitory proteins (cFLIP) in normal and traumatic murine and human cerebral cortex. J Cereb Blood Flow Metab 25, 1030-1040 (2005).
61.Jonsson, G., Paulie, S. & Grandien, A. High level of cFLIP correlates with resistance to death receptor-induced apoptosis in bladder carcinoma cells. Anticancer Research 23, 1213-1218 (2003).
62.Fujiwara, Y., Inase, A., Kawasaki, Y., Yoshikawa, S. & Iwai, S. Characterization of the binding of distamycin A to damaged DNA: a comparison with the DNA recognition of the human DDB protein. Nucleic Acids Symposium Series (2004), 235-236 (2006).
63.Reardon, J.T., et al. Comparative analysis of binding of human damaged DNA-binding protein (XPE) and Escherichia coli damage recognition protein (UvrA) to the major ultraviolet photoproducts: T[c,s]T, T[t,s]T, T[6-4]T, and T[Dewar]T. The Journal of Biological Chemistry 268, 21301-21308 (1993).
64.Shiyanov, P., Nag, A. & Raychaudhuri, P. Cullin 4A associates with the UV-damaged DNA-binding protein DDB. The Journal of Biological Chemistry 274, 35309-35312 (1999).
65.Herouy, Y., Krutmann, J., Norgauer, J. & Schopf, E. [Xeroderma pigmentosum: children of the moon]. J Dtsch Dermatol Ges 1, 191-198 (2003).
66.Lehmann, A.R. DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie 85, 1101-1111 (2003).
67.Park, S. & Dock, M. Xeroderma pigmentosum: a case report. Pediatric Dentistry 25, 397-400 (2003).
68.Bondar, T., et al. Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome. Molecular and Cellular Biology 26, 2531-2539 (2006).
69.Sun, C.L. & Chao, C.C. Cross-resistance to death ligand-induced apoptosis in cisplatin-selected HeLa cells associated with overexpression of DDB2 and subsequent induction of cFLIP. Molecular Pharmacology 67, 1307-1314 (2005).
70.Stanelle, J., Stiewe, T., Theseling, C.C., Peter, M. & Putzer, B.M. Gene expression changes in response to E2F1 activation. Nucleic Acids Research 30, 1859-1867 (2002).
71.Jiang, Z. & Clemens, P.R. Cellular caspase-8-like inhibitory protein (cFLIP) prevents inhibition of muscle cell differentiation induced by cancer cells. Faseb J 20, 2570-2572 (2006).
72.Guicciardi, M.E., Bronk, S.F., Werneburg, N.W. & Gores, G.J. c-FLIPL Prevents TRAIL-Induced Apoptosis of Hepatocellular Carcinoma Cells by Inhibiting the Lysosomal Pathway of Apoptosis. Am J Physiol Gastrointest Liver Physiol (2007).
73.Bartke, T., et al. p53 upregulates cFLIP, inhibits transcription of NF-kappaB-regulated genes and induces caspase-8-independent cell death in DLD-1 cells. Oncogene 20, 571-580 (2001).
74.Lee, T.J., Lee, J.T., Park, J.W. & Kwon, T.K. Acquired TRAIL resistance in human breast cancer cells are caused by the sustained cFLIP(L) and XIAP protein levels and ERK activation. Biochemical and Biophysical Research Communications 351, 1024-1030 (2006).
75.Liu, X., et al. Cellular cIAP2 gene expression associated with anti-HBV activity of TNF-alpha in hepatoblastoma cells. J Interferon Cytokine Res 25, 617-626 (2005).
76.Esposito, I., et al. cIAP2 Overexpression is an early event in pancreatic cancer progression. J Clin Pathol (2006).