DIRECT REPEATS AND DELETIONS IN MITOCHONDRIAL DNA: CAUSAL AND EVOLUTIONARY ASPECTS LAKSHMI NARAYANAN LAKSHMANAN (M.Tech. IIT Madras, India) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 ii DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _______________________________________ Lakshmi Narayanan Lakshmanan 23 December 2013 iii iv I wish to express my love, respect, gratitude and devotion to MAHAVISHNU MOTHER SARASWATHI MAHADEV I wish to pay my respects to BRAHMARISHI VISHWAMITHRA LEONARDO DI SER PIERO DA VINCI v ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my thesis supervisor Dr. Rudiyanto Gunawan for his motivation and guidance throughout my thesis work. He has been very kind and understanding to me and taught me a number of valuable lessons in research. I specifically like to acknowledge his patience towards me during the uncertain situations that occurred during the project. I consider myself lucky for having pursued my Ph.D. under his guidance. I like to convey my sincere thanks to Dr. Jan Gruber (Assistant Professor, Yale- NUS) with whom I have been collaborating for my thesis work. Dr. Gruber always adds a new perspective to my research work and had played critical role in my manuscript preparations. I learnt a lot during our discussions about new research ideas and manuscripts. I sincerely wish to continue my association with Dr. Gunawan and Dr. Gruber and bring out some impactful scientific publications in future. I would also like to thank Dr. Saif A. Khan, who kindly accepted me as his student during the last 2 years of my Ph.D. Dr. Khan has been very considerate and kind to me whenever I approached him for support. I am also thankful to the 2 internal examiners and 1 external examiner who have kindly agreed to evaluate my thesis report. I am grateful to NUS and ETH Zurich for providing me education and for allowing me to use their established infrastructure. Finally I wish to convey my sincere thanks to all my family members, lab students and friends for their support and concern for me. vi Table of Contents Table of Contents ........................................................................................................ vii Summary ....................................................................................................................... x List of tables ................................................................................................................ xii List of figures .............................................................................................................. xiii List of symbols ............................................................................................................ xiv Abbreviations .............................................................................................................. xv Chapter 1: Introduction ................................................................................................ 1 1.1. Mitochondria ................................................................................................ 1 1.2. Mitochondrial DNA ....................................................................................... 2 1.3. Mitochondrial DNA Replication .................................................................... 3 1.3.1. Strand Displacement Model ................................................................. 4 1.3.2. Strand Coupled Model .......................................................................... 5 1.3.3. RITOLS Model ........................................................................................ 6 1.4. Mitochondrial DNA Repair ............................................................................ 6 1.5. Mitochondrial DNA Mutations ...................................................................... 8 1.6. Deletion Mutations ....................................................................................... 9 1.7. Mechanisms of Deletion Formation ........................................................... 11 1.7.1. Models based on errors during mtDNA replication ............................ 11 1.7.2. Models based on errors during mtDNA repair ................................... 13 1.8. Sequence Motifs ......................................................................................... 14 1.8.1. Direct Repeat ...................................................................................... 15 1.8.2. Stem-loop ............................................................................................ 17 1.8.3. Homopolymeric Run ........................................................................... 17 1.9. Mutations in Nuclear Genes ....................................................................... 18 1.10. Thesis Outline .......................................................................................... 19 1.10.1. Role of Sequence Motifs in Deletion Formation ................................. 19 vii 1.10.2. Origin and Evolution of mtDNA DR Motifs.......................................... 20 1.10.3. Theoretical Analysis of DNA Misalignments in mtDNA....................... 21 Chapter 2: Role of Sequence Motifs in mtDNA Deletions ......................................... 23 2.1. Introduction ................................................................................................ 23 2.2. Materials and Methods ............................................................................... 24 2.2.1. Mitochondrial DNA and Random DNA Sequences ............................. 24 2.2.2. Analysis of Direct Repeats in Mitochondrial Genomes....................... 25 2.2.3. Analysis of Stem-loop Motifs in Mitochondrial Genomes .................. 26 2.2.4. Analysis of Deletion Breakpoints ........................................................ 27 2.3. Results ......................................................................................................... 28 2.3.1. Abundance, Distribution and Stability of DR Motifs ........................... 28 2.3.2. Stem-loop Motifs in Mitochondrial Genomes .................................... 31 2.3.3. Age Associated mtDNA Deletion Breakpoint Spectra ......................... 33 2.4. Discussion.................................................................................................... 38 Chapter 3: Origin and Evolution of DRs in mtDNA ...................................................... 42 3.1. Introduction ................................................................................................ 42 3.2. Materials and Methods ............................................................................... 44 3.2.1. Mitochondrial DNA sequences, Lifespan and Body mass Dataset ..... 44 3.2.2. Analysis of DR Count ........................................................................... 44 3.2.3. Generation of Random DNA Sequences ............................................. 45 3.2.4. Analysis of Synonymous Codon Usage Bias ........................................ 46 3.2.5. Phylogenetically Independent Contrast Analysis ................................ 46 3.2.6. Statistical Analysis ............................................................................... 46 3.3. Results ......................................................................................................... 47 3.3.1. DR Counts in Mammalian mtDNA ....................................................... 47 3.3.2. Long DRs and Longevity of Mammals ................................................. 48 3.3.3. Determinants of DR counts in mtDNA ................................................ 51 viii 3.4. Discussion.................................................................................................... 57 3.4.1. Do long DRs Constrain the Longevity of Mammals? ........................... 57 3.4.2. Determinants of DR Counts in mtDNA ................................................ 58 3.4.3. Origin and Evolution of DRs in Mammalian mtDNA Sequences ......... 60 Chapter 4: Theoretical Analysis of mtDNA Misalignments ......................................... 64 4.1. Introduction ................................................................................................ 64 4.2. Materials and Methods ............................................................................... 66 4.2.1. Mitochondrial DNA Sequences ........................................................... 66 4.2.2. Deletion Breakpoints Data .................................................................. 66 4.2.3. Calculation of Partition Functions for DNA Hybridization .................. 68 4.3. Model Description....................................................................................... 69 4.4. Results ......................................................................................................... 71 4.5. Discussion.................................................................................................... 74 Chapter 5: Conclusions and Future Works ................................................................. 78 Bibliography ................................................................................................................ 83 Appendix A