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研究生：	蘇郁虹 Su, Yu-Hung
論文名稱：	探討人類α-與β- 8-oxoguanine DNA glycosylase 1蛋白於粒線體DNA基因修復之功能 Functional studies of the α- and β-human 8-oxoguanine DNA glycosylase 1 in mitochondrial DNA repair
指導教授：	黃溫雅 Huang, Wen-Ya
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	74
中文關鍵詞：	鹼基切除修復系統、8-oxoG 、OGG1
外文關鍵詞：	BER, 8-oxoG, OGG1
相關次數：	點閱：104 下載：0
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7,8-dihydro-8-oxo-guanine (8-oxoG) 是活性氧自由基(ROS)造成的DNA氧化損害中最主要且最具有突變的潛力的損害鹼基，並可同時堆積於細胞核與粒線體DNA中。8-oxoG容易與腺嘌呤進行錯誤配對而造成基因突變，因此被認為與癌症發生相關。細胞內的8-oxoG主要是藉由人類修復蛋白8-oxoguanine DNA glycosylase 1 (hOGG1)經由鹼基切除修復系統(base excision repair, BER)來進行移除。人類細胞中主要有兩種hOGG1(α-hOGG1和β-hOGG1)，其中大部分的α-hOGG1存在於細胞核少部分則存在於粒線體。β-hOGG1 則只存在於粒線體內，是粒線體內主要的hOGG1。我們實驗室之前從酵母菌雙雜合系統與免疫共同沉澱法中發現只有β-hOGG1會直接與粒線體中的NADH dehydrogenase 1 beta sub complex,10 (NDUFB10) 結合，意味著β-hOGG1可能在粒線體中扮演功能。因此，我的研究目的就是要釐清α-與β-hOGG1在粒線體DNA修復當中所扮演的角色。在本次實驗中，我們利用專一的siRNA來分別抑制細胞α-與β-hogg1 mRNA的表現，在給予menadione處理所誘發的氧化壓力下，抑制α-與β-hogg1表現的細胞呈現較低的生存率及較高的細胞凋亡情形。此外，我們利用半定量的聚合脢連鎖反應(semi-quantitative PCR)來評估粒線體DNA中氧化損害的情形，結果發現α-hOGG1與β-hOGG1對於粒線體DNA中氧化傷害的修復同樣重要。另外，我們也利用體外鹼基切除修復活性試驗 (in vitro BER assay)來測試對8-oxoG的修復能力。由結果顯示α-與β-hOGG1同時參與細胞整體的修復功能。但是只有α-hOGG1會參與在細胞核的DNA修復。我們同樣利用粒線體蛋白來進行測試，初步結果發現抑制β-hogg1表現的粒線體蛋白有較低的修復活性，結合半定量的聚合脢連鎖反應結果我們認為β-hOGG1在粒線體當中具有BER的活性且與α-hOGG1同時參與在粒線體DNA的修復。

8-oxo-7,8-dihydroguanine (8-oxoG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA. It is associated with tumorigenesis because its miscoding nature to adenine and it causes G:C to T:A transversion during DNA replication. In mammalian cells, 8-oxodG is primarily removed by human 8-oxoguanine DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are more than 7 alternatively spliced forms of hogg1 gene and α- and β-hogg1 are two major forms in various human tissues. These two hOGG1 proteins have different cell localizations. α-hOGG1 is localized in nucleus and mitochondria; whereas β-hOGG1 is only in mitochondria. Previous studies in our lab have found that β- but not α-hOGG1 protein directly interacts with the mitochondrial protein NADH dehydrogenase 1 beta sub complex 10 (NDUFB10), suggesting that β-hOGG1 is functionally associated with mitochondria. My research goal is to investigate the roles of α- and β-hOGG1 in mitochondrial DNA repair. In the present study, we used specific siRNAs to knock down (KD) the endogenous α- and β-hogg1 expression. My experimental findings showed that the α- and β-hogg1 KD cells were sensitive to oxidative stress induced by menadione treatment, detected by the MTT assays and AnnexinV-FITC apoptosis analyses. By semi-quantitative PCR for mitochondrial DNA (mtDNA), it was found that α- and β-hogg1 KD cells were deficient in removal of oxidative-damaged DNA lesions in mitochondrial genome, suggesting that both α- and β-hOGG1 proteins are involved in mtDNA repair. In addition, by the in vitro BER functional assay it was found that the in vitro BER activity was defective in both α-hOGG1 and β-hOGG1 KD whole-cell extracts (WCE), indicating that α- and β-hOGG1 proteins are both involved in overall oxidative BER in cells. Results of the in vitro BER using the nuclear proteins showed that α- but not β-hogg1 KD cells were deficient in incision activity, indicating that α-hOGG1 mainly contributes to nuclear BER repair. However, in the mitochondrial protein fractions, β-hogg1 KD cells showed a more severe defect in mitochondrial BER activity. In summary, we conclude that both α-hOGG1 and β-hOGG1 were required for mitochondrial DNA repair.

Abstract in Chinese	……………………………………………………………	i
Abstract	……………………….………………………………………………	ii
Acknowledgement	……………………………………………………………	iii
Abbreviation	……………………………………………………………………	iv
Contents	……………………..…………………………………………………	v-vi
List of Tables	…………………………………………………………………	vii
List of Figures	………….………………………………………………………	viii
List of Appendices	……………………………………………………………	ix
		
I.	Introduction………………………………………………………………. 1-10
1.1	DNA damage and cellular response……………………………..   1   
1.1.1   DNA repair mechanisms……………………………………   2
1.1.2   Oxidative DNA damage and associated disease…………….   4
1.1.3	Base excision repair (BER)……………………………………  5
1.2    Mitochondria……………………………………………………...  6
1.2.1	Mitochondrial genome and repair mechanisms ……………...  7
1.2.2	Nuclear repair versus mitochondrial repair…………………….  8
1.3   Human 8-oxoguanine DNA glycosylase 1 (hOGG1)…………….  9
1.3.1   OGG1 in mitochondria…………………………………………  9
II.    Research goal…………………………………………………………… 11-12
2.1	   To identify the functions of α- and β-hOGG1 in overall DNA  
repair.….…………………………………………………………   12
2.2	To study the functions of α- and β-hOGG1 in oxidative stress repair of   
mitochondria…………………………………………………  12
III.	Materials and methods…………………………………………………  13-21
3.1   siRNA construction………………………………………………  13
3.2   RNA extraction and ogg1 RT-PCR……………………………….  13
3.2.1  Cell culture…………………………………………………….  13
3.2.2  Plasmid Transfection……………………………………………  13
3.2.3  Extraction of total RNA from cells……………………………..  14
3.2.4  ogg1 RT-PCR………………………………………………  15
3.3    MTT assay………………………………………………………  15
3.4    Apoptosis assay………………………………………………..   16
3.5    In vitro BER assay……………………………………………….   17
3.5.1  Whole cell extract preparation………………………………..   17
3.5.2  Mitochondrial and nuclear protein extracts preparation..........    17
3.5.3  In vitro BER activity assay…………………………………….  18
3.5.4  In vitro mitochondrial and nuclear BER activity assays..…….  19
3.5.5  Capillary electrophoresis……..………………………………… 19
3.6   Semi-quantitative PCR ………………………………………….  20
3.6.1  Genomic DNA extraction……………………………………   20
3.6.2  Formamidopyrimidine [fapy]-DNA glycosylase digestion….  20
3.7   Western blot……………………………………………………….  21
IV.    Results…………………………………………………………………  22-27
4.1   Selection of specific siRNA constructs…………………………   22
4.2   Cell viability of α- and β-hogg1 knockdown cells under oxidative 
Stress……………………………………………………………   22
4.3   Oxidative DNA damage-induced apoptosis in α- and β-hogg1
 KD cells……………………………………………………..……   22 
4.4   In vitro BER activity of α- and β-hogg1 KD cells….……………  23
4.5   Accumulation of oxidative DNA lesions in α- and β-hogg1 KD cells
     ………………………………………………………………………  26
V.    Discussion………………………………………………………………  28-33
VI.   References………………………………………………………………  34-38

List of Tables
Table 1.  α- and β-hogg1 siRNAs used in this study……………………………  39
Table 2.  Primer sequences of various siRNA clones………………………..  40
Table 3.  α- and β-hogg1 RT-PCR primers…………………………………….  42 
Table 4.  PCR primers used in semi-quantitative PCR………………………….  43
Table 5.  Reagents and Kits ……………………………………………………..  44




















List of Figures
Figure 1.  Screening of specific siRNA for α-hogg1 and β-hogg1………… 46
Figure 2.  α-hogg1 and β-hogg1 knockdown cells are sensitive to oxidative stress 
induced by menadione and H2O2 treatment…………………………….47
Figure 3. Knockdown of α- and β-hogg1 sensitizes HtTA1 cells to oxidative 
stress-induced apoptosis. ……………………………………………….48
Figure 4. In vitro BER products are successfully visualized and measured by 
capillary electrophoresis. ………………………………………………49
Figure 5.  Both α-hOGG1 and β-hOGG1 are involved in overall DNA repair….50
Figure 6.  In vitro BER activity is severely defective in α-, β-, and total hogg1 KD WCE. ………………………………………………………………….51
Figure 7.  β-hOGG1 does not involve in nuclear DNA repair. ………………….52
Figure 8.  β-hOGG1 is the major hOGG1 for mitochondrial BER. …………… …53
Figure 9.  Semi-quantitative PCR of menadione-induced oxidative DNA lesions in 
mitochondrial DNA………………………………………………….54
Figure 10.  The efficiency of removal of oxidative damage lesions from mtDNA is 
 deficient in α-, β-, and total hogg1 KD cells………………………55










List of Appendices
Appendix 1.  Cellular response to DNA damage, and repair mechanisms for DNA 
damage……………………………………………………………….56
Appendix 2.  Mechanism of base excision repair (BER)…………………………..57
Appendix 3.  Mitochondrial dynamics, mitochondrial genome, and mitochondrial 
electron transport chain………………………………………………58
Appendix 4.  Summary of repair Proteins identified in eukaryotic mitochondria…59
Appendix 5.  DNA repair pathways in nucleus and mitochondria of mammalian 
cells……………………………………………………………….…60
Appendix 6.  Structural comparison of hOGG1 proteins…………………………61
Appendix 7.  The localization of β-hOGG1 and NDUFB10 in mitochondria……62
Appendix 8.  Oxidative stress induced by H2O2 enhanced the association between 
β-hOGG1 and NDUFB10…………………………………………..63

                                    

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