Developing New Afm Imaging Technique and Software for Dna Mismatch Repair

DEVELOPING NEW AFM IMAGING TECHNIQUE AND SOFTWARE FOR DNA MISMATCH REPAIR Zimeng Li A dissertation submitted to the faulty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Physics and Astronomy. Chapel Hill 2019 Approved by: Dorothy Erie Tom Clegg Richard Superfine Wu Yue Tom Kunkel Paul Modrich ©2019 Zimeng Li ALL RIGHTS RESERVED ii ABSTRACT Zimeng Li: Developing New AFM Imaging Technique and Software for DNA Mismatch Repair (Under the direction of Dorothy Erie) Atomic Force Microscopy (AFM) is a powerful technique to study the assembly and function of multiprotein-DNA complexes, such as the MutS-MutL-DNA complex in DNA mismatch repair. As a high-resolution, single-molecule imaging technique, AFM has the advantage of directly visualizing individual protein-DNA complexes in their native conformations, but it has two severe limitations. First, it is unable to resolve the location of the DNA inside the protein. Second, it lacks a comprehensive software that is tailored for single- molecule studies and high-throughput analyses. To tackle these issues, we developed DREEM (Dual-Resonance-frequency-Enhanced Electrostatic Force Microscopy). DREEM is a new AFM imaging method that is capable of resolving DNA path inside the protein-DNA complex. With DREEM, we can reveal the path of the DNA wrapping around histones in nucleosomes and the path of DNA through multiprotein mismatch repair complexes. We also developed Image Metrics, a full-featured AFM software package that excels in high-throughput shape analysis and single-molecule analysis. Built using MATLAB, Image Metrics is uniquely positioned in single- molecule studies with its specially designed single-molecule analysis and shape analysis modules. It also blends unique flexibility into a user-friendly interface that allows for easy customization and automation of user workflows. Finally, as a case study, we used AFM and Image Metrics to study DNA mismatch repair (MMR) in the context of trinucleotide repeat (TNR) expansion diseases. Our data show that when interacting with heteroduplex DNAs with iii the TNR context, MMR proteins adopt conformations that are different from a repair signaling capable species, suggesting the altered conformations may not be competent for repair. iv TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x LIST OF ABBREVIATIONS ...................................................................................................... xiv CHAPTER 1. OVERVIEW ............................................................................................................ 1 CHAPTER 2. VISUALIZING THE PATH OF DNA THROUGH PROTEINS USING DREEM IMAGING ............................................................... 4 2.1. Introduction ...................................................................................................................... 5 2.2. Design............................................................................................................................... 8 2.3. Results and Discussion ................................................................................................... 13 CHAPTER 3. IMAGE METRICS, A NEXT-GEN AFM IMAGE ANALYSIS SOFTWARE ..................................................................................... 24 3.1. Introduction .................................................................................................................... 25 3.2. Interface .......................................................................................................................... 29 3.3. Basic Operations ............................................................................................................ 32 3.4. Image Processing............................................................................................................ 36 3.4.1. Surface Correction .................................................................................................. 36 3.4.2. Cross-correlation ..................................................................................................... 46 3.4.3. Fast Fourier Transform ........................................................................................... 48 v 3.5. Image Analysis ............................................................................................................... 52 3.5.1. Particle Detection .................................................................................................... 52 3.5.2. Shape Analysis ........................................................................................................ 57 3.5.3. Single-Molecule Analysis ....................................................................................... 68 3.5.4. Compare Measurement to Other AFM Software .................................................... 81 3.6. Macros and User Extensions .......................................................................................... 92 3.7. Author’s Remarks and Future Directions....................................................................... 94 3.7.1. Motivations on Creating Image Metrics ................................................................. 94 3.7.2. Particle Detection .................................................................................................... 96 3.7.3. Shape Analysis ........................................................................................................ 99 3.7.4. Single-Molecule Analysis ..................................................................................... 103 3.7.5. Open Development Model .................................................................................... 106 3.8. License and Distribution .............................................................................................. 108 CHAPTER 4. STRUCTURAL AND FUNCTIONAL STUDY OF DNA MISMATCH REPAIR IN THE CONTEXT OF TRINUCLEOTIDES REPEATS EXPANSION.................................................. 109 4.1. Introduction .................................................................................................................. 110 4.1.1. DNA Mismatch Repair (MMR) ............................................................................ 113 4.1.2. Trinucleotides Repeat Expansion (TNR) .............................................................. 118 4.1.3. Molecular Mechanisms of TNR ............................................................................ 120 4.1.4. Relations between TNR and MMR....................................................................... 126 vi 4.2. Materials and Methods ................................................................................................. 128 4.2.1. Proteins and DNAs ............................................................................................... 128 4.2.2. AFM Sample Preparation ..................................................................................... 129 4.2.3. Deposition, Imaging, and Analysis ....................................................................... 130 4.3. Results .......................................................................................................................... 134 4.3.1. (CTG)1................................................................................................................... 135 4.3.2. (CTG)5................................................................................................................... 139 4.3.3. (CTG)56/(CAG)54 .................................................................................................. 142 4.4. Discussion .................................................................................................................... 155 APPENDIX A. SUPPLEMENTAL INFORMATION FOR DREEM ....................................... 163 A.I. Supplemental Figures ....................................................................................................... 163 A.II. Theoretical Basis of DREEM Measurements ................................................................. 166 A.III. Supplemental Experimental Procedures ........................................................................ 169 APPENDIX B. AUTHOR’S CONTRIBUTION TO DREEM................................................... 174 APPENDIX C. DREEM OPTIMIZATION ............................................................................... 176 APPENDIX D. SUPPLEMENT FIGURES FOR IMAGE METRICS ...................................... 184 APPENDIX E. SUPPLEMENT FIGURES FOR MMR AND TNR .......................................... 195 APPENDIX F. IMAGE ARTIFACTS ........................................................................................ 197 APPENDIX G. PARTICLE CLASSIFICATION ALGORITHMS ........................................... 201 G.I. Classification through Eigenanalysis ............................................................................... 202 vii G.II. Classification through Clustering Analysis .................................................................... 212 G.III. Verifications and Best Practices .................................................................................... 214 APPENDIX H. IMAGE SIMULATION ...................................................................................

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