人類修復蛋白8-oxoganine DNA glycosylase 1 (hOGG1)可藉由鹼基切除修復(BER) 來移除DNA氧化傷害。hOGG1可經由選擇性剪接機制產生兩種主要的型態：一為alpha-hOGG1，一為beta-hOGG1。alpha-hOGG1會存在在細胞核及粒腺體中，而beta-hOGG1則主要分佈於粒腺體中。在細胞核中，alpha-hOGG1的功能及調控其的機制已較明確。然而，alpha-hOGG1和 beta-hOGG1對於粒腺體DNA的修復仍不明確，需要更進一步的研究。為了要了解alpha-hOGG1和 beta-hOGG1在粒腺體DNA修復所辦演的角色，實驗室之前以酵母菌雙雜合系統來篩選會和hOGG1結合的蛋白質。在這實驗中找到了兩個有興趣的蛋白質：polynucleotide kinase 3'-phosphatase (PNKP) and NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10 (NDUFB10).在本次實驗中，我們以酵母菌雙雜合系統確認了 beta-hOGG1會直接和NDUFB10結合。然而酵母菌雙雜合系統結果指出不論是 alpha-hOGG1或 beta-hOGG1 都不會和PNKP結合。NDUFB10為NADH:ubiquinone oxidoreductase complex I的一部分且位在粒腺體內膜上。另外，我們也以免疫共同沈澱法及免疫螢光染色來確認人類細胞中 beta-hOGG1和NDUFB10的結合關係。此外，免疫共同沈澱法的結果也看到細胞曝露在過氧化氫環境下會增加 beta-hOGG1和NDUFB10的結合。這表示 beta-hOGG1會藉由和NDUFB10結合參與粒腺體DNA的修復。除此之外，alpha-hOGG1可能也會和beta-hOGG1結合且和 beta-hOGG1及NDUFB10形成一粒腺體DNA修復的複合體。雖然，我們也試圖找出 beta-hOGG1和NDUFB10結合的區域，但酵母菌雙雜合系統和免疫共同沈澱法的結果互相矛盾。以酵母菌雙雜合系統找出的beta-hOGG1 之helix-hairpin-helix motif並非是其和NDUFB10結合的位置。雖然如此，其他的結果還是讓我們了解到 beta-hOGG可能在粒腺體DNA修復中辦演的角色。

The human 8-oxoguanine-DNA glycosylase 1 (hOGG1) is an essential base excision repair (BER) for oxidative DNA damage. hOGG1 yields two alternatively spliced isoforms, designated alpha- and beta-hOGG1. The alpha-hOGG1 is localized in nucleus and mitochondria (mt); whereas the beta-hOGG1 is mainly localized in mitochondria. In nucleus, the function and regulation of alpha-hOGG1 is well known. However, the potential functions of alpha- or beta-hOGG1 in mitochondrial DNA repair were not extensively investigated yet. In order to understand the possible roles of alpha- and beta-hOGG1 in mtDNA repair, we previously used yeast two-hybrid screening assays to screen for the proteins that are associated with them. We found that beta- but not alpha-hOGG1 directly interacts with NADH dehydrogenase (ubiquinone) 1 beta subcomplex 10 (NDUFB10). NDUFB10 is a subunit of the NADH:ubiquinone oxidoreductase complex I, which is located on mitochondrial inner membrane. We also confirmed the in vivo interaction between beta-hOGG1 with NDUFB10 by co-immunoprecipitation and immunofluorescence assays. By co-IP studies, it was found that association of ���{hOGG1 with NDUFB10 was enhanced after treatment of hydrogen peroxide, suggesting that beta-hOGG1 repairs mtDNA damages through its interaction with NDUFB10 upon oxidative stress. Furthermore, alpha-hOGG1 also interacts with beta-hOGG1 and might form a repair complex with beta-hOGG1 and NDUFB10 for mtDNA repair. Although, we tried to look for the interaction region of beta-hOGG1 that directly binds with NDUFB10. However, the co-IP result validated either helix-hairpin-helix motif or predictive transmembrane region (C terminus) is not the binding domain. The results of these studies will help us to understand the role of beta-hOGG1 on mitochondrial DNA repair or other relevant functions.

Contents

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

I. Introduction……………………………………………………....…. 1
1.1 DNA damage and its consequence…………………………………………. 1
1.2 DNA repair…………………………………………………………………. 2
1.2.1 Base excision repair (BER)……………………………………………. 3
1.3 Oxidative DNA damage and its repair mechanism…………………………. 4
1.3.1 Oxidative damage and human disease…………………………………. 5
1.4 Human 8-oxoganine DNA glycosylase 1 (hOGG1)………………………... 6
1.4.1 Modification of hOGG1……………………………………………….. 7

II. Research goal……………………………………………………..…. 9
2.1 Characteristic of PNKP……………………………………………………... 9
2.2 Characteristic of NDUFB10………………………………………………… 9

III. Materials and methods……………………………………………. 11
3.1 Yeast two-hybrid assay……………………………………………………… 11
3.1.1 Plasmid constructs……………………………………………………… 11
3.1.2 E. coli transformation…………………………………………………... 12
3.1.3 Isolation DNA plasmid from E. coli………..…………..….…………... 13
3.1.4 Phenol/chloroform extraction………………………………………….. 13
3.1.5 Yeast transformation……………………………………………………. 14
3.1.6 Yeast protein extraction and western blotting………………………….. 14
3.2 Immunoprecipitation assay………………………………………………..... 15
3.2.1 Plasmid constructs……………………………………………………… 15
3.2.2 Cell line and culture……………………………………………………. 17
3.2.3 Transfection……………………………………………………………. 17
3.2.4 Immunoprecipitation and western blotting…………………………….. 17
3.3 Reactive oxygen species treatment…………………………………………. 18
3.4 Immunofluorescence and microscopy……………………………………… 18

IV. Results………………………………………………………………… 20
4.1 Construction of alpha/beta-hOGG1, PNKP and NDUFB10 plasmids…………….. 20
4.2 beta-hOGG1 interacts with NDUFB10 in yeast two-hybrid system…………... 20
4.3 In vivo association between beta-hOGG1 and NDUFB10…………………….. 20
4.4 beta-hOGG1 and NDUFB10 were colocalized in mitochondria…………......... 21
4.5 Association of beta-hOGG1 with NDUFB10 is induced by H2O2 treatment….. 21
4.6 The binding region of beta-hOGG1 that interacts with NDUFB10…………… 22
4.7 In vivo association between alpha-hOGG1 and NDUFB10……………………. 23
4.8 Repair complex of alpha-hOGG1 and beta-hOGG1………………………………. 23

V. Discussion…………………………………………………………….. 25
VI. References…………………………………………………................. 29
VII. Tables………………………………………………………………….. 34
VIII. Figures………………………………………………………………… 36
IX. Appendix……………………………………………………………... 47

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