미생물 글라이코실레이즈의 특성분석과 이를 이용한 SNP 확인 : Characterization of the bacterial glycosylase and its application to SNP detection
- 발행기관 고려대학교 대학원
- 발행년도 2004
- 학위명 박사
- 학과 대학원:생명공학과
- 식별자(기타) DL:000014914347
- 서지제어번호 000045212972
초록/요약
Damaged DNA strands are repaired by base excision in organisms, a process initiated by repair enzymes, which include DNA glycosylases and endonucleases. We expressed and characterized two putative endonuclease genes from Methanobacterium thermoautotrophicum, Mt0764 and Mt1010, encoding homologues of the endonuclease III (endo III) and endonuclease IV (endo IV) of Escherichia coli. The Mt0764 and Mt1010 proteins showed endo III activity by removing thymine glycol from DNA strand and AP endonuclease activity, respectively. The Mt0764 protein not only cleaved the oligonucleotide duplex containing a thymine glycol/adenine pair efficiently but also showed activity on the 8-oxoguanine-containing oligonucleotide duplex. In this study we report upon the stimulation of endo III activity by endo IV using two recombinant proteins (Mt1010 and Mt0764) from M. thermoautotrophicum. Mt1010 stimulated the DNA glycosylase activity of Mt0764 for DNA substrates containing 8-oxoguanine residues and increasing the formation of the Mt0764 protein-DNA complex. The interaction between Mt1010 and Mt0764 was observed by using an in vitro binding assay. These results suggest that association between endo III and endo IV may occur in vivo, and this contributes to efficient base excision repair for the oxidative damage of DNA. A new approach is presented for the sensitive and selective scanning for DNA mutations, based on the use of the A/G mismatch glycosylase. We expressed and characterized putative glycosylase gene from Shewanella oneidensis MR-1, So3368, encoding homologue of the A/G mismatch glycosylase (MutY) of Escherichia coli. The So3368 proteins showed A/G mismatch glycosylase activity removing adenine from A/G containing oligonucleotide duplexes strand. The resulting apurinic/apyrimidinc sites at the position of mismatches are treatment heat converted to 3′- OH containg strand breaks. Recent developments have applied a DNA array-type approach to single strand oligonucleotides on a s
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CONTENTS
Abstract ⅰ
Contents ⅳ
List of Figures ⅶ
List of Tables xi
Abbreviations xii
Part I. Endonuclease Ⅳ enhances base excision repair of endonuclease Ⅲ from Methanobacterium thermoautotrophicum
1. Introduction
1.1. Sequence-Based Approach to Functional Identification 2
1.1.1. Protein sequence motif 2
1.1.2. DNA glycosylases and HhH motif 5
1.2 Oxidative Damage to DNA 10
1.3. Base excision repair 13
1.4. Endonuclease III 20
1.5. Endonuclease IV 25
1.6. Characteristics of Methanobacterium thermoautotrophicum 31
1.7. -Lactamase protein fragment complementation assays as sensors of protein-protein interactions 32
1.8. Purpose of This Study 34
2. Materials and Methods
2.1. Enzymes and Other Materials 36
2.2. Preparation of Oligonucleotide Duplex Substrates 36
2.3. Protein Expression and Purification 38
2.4. Assay of Mt0764 and Mt1010 activity 41
2.5.DNA Trapping Assay with NaBH4 42
2.6. In vitro Binding Assay 43
2.7.β-lactamase Complementation Assay
2.7.1. Schematic representation of vectors used for expression of -lactamase fragment fusion proteins. 44
2.7.2. β-lactamase activity assay in E.coli 45
3. Results
3.1. Purification of Mt0764 and Mt1010 47
3.2. Substrate Specificity of Mt0764 50
3.3. Enzyme Activity of Mt1010 55
3.4. Stimulation of Mt0764 activity by Mt1010 57
3.5. Direct Association between the Mt1010 and Mt0764 proteins in vitro
62
3.6.β-lactamase Complementation Assay 64
4. Discussion 65
5. References 71
Part II. Gene detection using the A/G mismatch Glycosylase from Shewanella oneidensis MR-1
1. Introduction
1.1. MutY-Dependent Mismatch Repair in E.coli 85
1.2. Techniques to Identify Polymorphisms and Mutations 88
1.2.1. Technologies to detect known polymorphisms 89
1.2.2. Techniques to identify unknown polymorphisms 91
1.3. Characteristics of Shewanella oneidensis MR-1 93
1.4. Purpose of This Study 95
2. Materials and Methods
2.1. Enzymes and Other Materials 101
2.2. Preparation of Genomic DNA of S. oneidensis MR-1 1
