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研究生：	嚴涵誼 Yen, Han-Yi
論文名稱：	人類粒線體基因組維持外切酶之結構解析 Structural Study of Human Mitochondrial Genome Maintenance Exonuclease 1
指導教授：	吳權娟 Wu, Chyuan-Chuan
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	76
中文關鍵詞：	MGME1 、粒線體 DNA 、粒線體疾病、核酸外切酶、X 射線晶體學
外文關鍵詞：	MGME1, mitochondrial DNA, mitochondrial disease, exonuclease, X-ray crystallography
相關次數：	點閱：244 下載：0
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Abstract I
中文摘要 III
致謝 IV
Table of Contents VI
List of Tables VIII
List of Figures IX
Abbreviation X
Introduction 1
1 The function of mitochondria 1
2 mtDNA and its maintenance 2
2.1 Key mtDNA maintaining proteins 3
2.2 The mechanism of mtDNA replication 5
2.3 mtDNA repair 7
2.4 Damaged-induced mtDNA degradation 8
3 Physiological function of MGME1 9
3.1 Loss-of-function of MGME1: observations from patient and knockout mice 9
3.2 Loss-of-function of MGME1: observations from cultured cells 10
3.3 The role of MGME1 in mtDNA replication 11
3.4 The role of MGME1 in mtDNA degradation 13
4 Molecular structure of MGME1 13
Specific Aims 16
1 Expression and purification of recombinant human MGME1 16
2 Crystallization of MGME1 in complex with a 5′-overhang DNA duplex 17
Materials and Methods 18
1 Materials 18
1.1 Plasmids and Constructs 18
1.2 Host cell lines 18
1.3 Synthetic oligonucleotides 18
1.4 Buffers and Solutions 19
2 Methods 19
2.1 protein expression 19
2.2 Protein purification 20
2.3 Crystallization 22
2.4 Electrophoretic mobility shift assay (EMSA) 22
2.5 Gel electrophoresis for examining crystal composition 23
2.6 Thermal shift assay 24
Results 26
1 Protein expression and purification of catalytically inactive MGME1 26
2 The design of DNA substrate for co-crystallization with MGME1-KH 29
3 X-ray crystallography 30
3.1 To assemble the protein-DNA complex for co-crystallization 30
3.2 Crystallization screen and crystal optimization 31
3.3 Identification of DNA crystals in some specific conditions 34
4 Thermal shift assay 35
Discussion 37
1 Low protein purification yield of MGME1-KH 37
2 Poor quality of the grown crystals 38
Conclusion 40
Tables 42
Figures 47
References 68
Appendix Figures 74
                                    

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校內：2027-09-23公開
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