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Impact of DNA structure and Aeropyrum pernix single - strand DNA binding protein on oxidative damage to DNA
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Tables
List of Figures
List of Schemes
List of Abbreviations
1. Introduction
1.1 DNA Replication
1.2 Single-Stranded DNA Binding Proteins
1.3 DNA Damage and the Impact of Protein Binding
1.4 DNA Structure
1.5 Goal and Specific Aims
2. Experimental
2.1 Synthesis of 1-[5-O-(4,4!-Dimethoxytrityl)-3-C-(2,2-dimethylpropanoyl)-3-O-(triethylammoniumphosphonyl)-2!-Deoxy-"-D-threo-pentofuranosyl)thymidine H-phosphonate and 1-(2!-Deoxy-"-
D-threo-pentofuranosyl)thymidine Phosphoramidite
2.2 Synthesis of Modified Oligonucleotides
2.2.1 Semi-Automated Synthesis of Oligonucleotides Containing the C3!-Pivaloylthymidine Radical Precursor
2.2.2 Fully Automated DNA Synthesis of Oligonucleotides Containing 1-(2!-Deoxy-"-D-threopentofuranosyl) thymidine
2.2.3 Fully Automated DNA Synthesis of 3!-(6-FAM) Labeled Oligonucleotides Containing 1-(2!-Deoxy-"-D-threopentofuranosyl) thymidine
2.2.4 Semi-Automated Synthesis of 3!-(6-FAM) Labeled Oligonucleotides Containing C3!-Pivaloylthymidine Radical Precursor
2.2.5 Preparation of H-Phosphonate Oxidant I (4% Iodine in THF/Pyridine/Water (8:1:1))
2.2.6 Preparation of H-Phosphonate Oxidant II (THF/Triethylamine/Water (8:1:1))
2.3 Nucleoside Deprotection and Oligonucleotide Cleavage from Solid Supports
2.4 Purification of Modified Oligonucleotides
2.4.1 Purification of Oligonucleotides Containing the C3!-Pivaloylthymidine Radical Precursor
2.4.2 Purification of Oligonucleotides Containing 1-(2!-Deoxy-"-Dthreo-pentofuranosyl) thymidine
2.4.3 Purification of 3!-(6-FAM) Labeled Oligonucleotides Containing 1-(2!-Deoxy-"-D-threopentofuranosyl) thymidine
2.4.4 Purification of of 3!-(6-FAM) Labeled Oligonucleotides Containing C3!-Pivaloylthymidine Radical Precursor
2.5 Desalting of IEX Purified Oligonucleotides
2.6 Quantification of Oligonucleotides
2.7 MALDI-TOF MS Preparation and Data Collection of Oligonucleotides
2.7.1 Preparation of Matrix
2.7.2 Desalting of Samples
2.7.3 Spotting of Samples
2.7.4 Calibration
2.7.5 Data Collection
2.8 Annealing of DNA Duplexes
2.9 Circular Dichroism of DNA Substrates
2.10 Anaerobic Photolysis of Oligonucleotides Containing the C3!-Pivaloylthymidine Radical Precursor
2.11 HPLC Optimization and Analysis of Photolysis Samples
2.12 Enzymatic Digestion and HPLC Analysis of Isolated Photolysis Products
2.13 MALDI-TOF MS Preparation and Data Collection of Isolated Nucleosides Resulting from Enzymatic Digestion
2.13.1 Preparation and Spotting of Samples
2.13.2 Preparation of Matrix
2.13.3 Calibration
2.13.4 Data Collection
2.14 Melting Temperature Analysis of DNA Duplexes
2.15 Differential Scanning Calorimetry of DNA Damage Substrates
2.16 Cloning of the Ape"C Plasmid
2.17 Expression and Purification of Ape"C
2.18 Quantification of Ape"C
2.19 MALDI-TOF MS Preparation and Data Collection of Intact Ape"C
2.19.1 Preparation of Matrix
2.19.2 Desalting of Samples
2.19.3 Spotting of Samples
2.19.4 Calibration
2.19.5 Data Collection
2.20 In-Gel Trypsin Digest of Ape"C
2.21 MALDI-TOF MS Preparation and Data Collection and Analysis of In-Gel Trypsin Digest Samples
2.21.1 Preparation of Matrix
2.21.2 Desalting of Samples
2.21.3 Spotting of Samples
2.21.4 Calibration
2.21.5 Data Collection
2.21.6 MASCOT Online Utility Database Search
2.22 Fluorescence Anisotropy
2.22.1 Buffer Exchange of Ape"C
2.22.2 DNA Preparation
2.22.3 DNA-Protein Binding Titrations
2.23 Quaternary Structure Determination of Ape"C
2.24 Crystal Screening of a Dickerson-Drew Dodecamer (DDD) Duplex Containing 1-(2!-Deoxy-"-D-threo-pentofuranosyl) thymidine
2.25 Photolysis of SSB-DNA Complex
2.25.1 Determination of Ape"C SSB Concentration
2.25.2 Buffer Exchange of Ape"C SSB
2.25.3 DNA Preparation
2.25.4 Ape"C SSB-DNA Complex Formation
2.25.5 Anaerobic and Aerobic Photolysis of Ape"C SSB-DNA Complexes
2.25.6 TBE-Urea Denaturing Gel Electrophoresis of Photolysate
3. Results and Discussion
3.1 Utility of C3!-Pivaloylthymidine for Selective Radical Generation in Architectures Relevant to DNA Replication
3.2 Impact of DNA Structure on the Formation of Oxidative Damage Products
3.3 Thermodynamics and Structural Analysis of DNA Damage Architectures
3.4 Effect of Oxidative Damage on SSB-DNA Binding
3.5 Oxidative Damage of DNA in the Presence of the Ape"C SSB
3.6 Stability and Crystallization of Modified Dickerson-Drew Dodecamer
4. Conclusions
5. Future Work
5.1 Radiolabeling C3!-Pivaloylthymidine Modified ODNs for Selective Radical Generation
5.2 Nucleic Acid Capture Assays for Specific and Non-Specific Radical
Generation
References
Appendices
A. Utility of C3!-Pivaloylthymidine for Selective Radical Generation in Architectures Relevant to DNA Replication
B. Impact of DNA Structure on the Formation of Oxidative Damage Products
C. Thermodynamics and Structural Analysis of DNA Damage Architectures
D. Effect of Oxidative Damage on SSB-DNA Binding
E. Oxidative Damage of DNA in the Presence of the Ape#C SSB
F. Stability and Crystallization of Modified Dickerson-Drew Dodecamer
G. Protein Preparation MaterialsBài viết liên quan
