An investigation into the effect of DNA structural polymorphism and single-stranded DNA binding proteins on repair of disease-associated slipped-DNA repeats by Jennifer Jing Luo A thesis submitted in conformity with the requirements for the degree of Master of Science Molecular Genetics University of Toronto © Copyright by Jennifer Jing Luo 2015 An investigation into the effect of DNA structural polymorphism and single-stranded DNA binding proteins on repair of disease- associated slipped-DNA repeats Jennifer Jing Luo Master of Science Molecular Genetics University of Toronto 2015 Abstract Gene-specific repeat expansions are the cause of a growing list of neurological diseases, including myotonic dystrophy type 1 and Huntington's disease. The formation of slipped-DNA structures in the expanded repeat sequences is thought to drive repeat instability and pathogenesis by impairing normal DNA metabolic processes. Here I show that slipped-DNAs with nicks located within the repeat tract displayed increased structural heterogeneity relative to slipped-DNAs with nicks located in the flanking sequence. Nick-in-repeat slipped-DNAs were repaired better than nick-in-flank slipped-DNAs, likely due to increased amounts of single- stranded DNA at the nicked repeat ends allowing for better repair factor binding. Single-stranded DNA binding proteins RPA and aRPA seem to play an important part in tissue-specific instability as both complexes are overexpressed in the brains of HD patients. Neither RPA nor aRPA was required for slipped-DNA repair, although they both enhanced slipped-DNA repair efficiency. ii Acknowledgments I would like to thank my supervisor Dr. Christopher Pearson for his guidance, enthusiasm and encouragement over the years. I have learned much about science and life in your lab and I will always be grateful for the opportunities that you have given me. I would like to thank my committee members, Dr. David Bazett-Jones and Dr. Irene Andrulis, for the much-appreciated advice and feedback that you have always given me. I would like to thank our collaborators, especially Dr. Marc Wold, who has provided valuable resources and expertise for my project. A big thank you for all the members of the Pearson lab, both past and present, for the help and support you have given me. I am so grateful that I had such wonderful colleagues to depend on, to turn to for help, and most importantly I am grateful for the laughs. I would like to thank all of my GGB and Mol Gen friends who all contributed to make my graduate school experience a fun and productive one. Thank you to my friends and family who gave me love, support and encouragement. Thank you to my parents, Hui Luo and Aishe Sun. I am so grateful for everything that you have given me and all the sacrifices you have made for me. I hope to make you proud in everything I do. Thank you to Petro, the best partner-in-life I could ask for. Thank you for your love and support. Thank you for feeding me when I forget to eat. Thank you for making me sleep when work becomes all-consuming. Thank you for the walks and the heated debates. Most importantly, thank you for making me want to be better in everything I do. iii Table of Contents Acknowledgments .......................................................................................................................... iii List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Abbreviations ..................................................................................................................... ix 1 Introduction ................................................................................................................................ 1 1.1 Disease-causing trinucleotide repeats ................................................................................. 1 1.2 Pathogenic mechanisms in trinucleotide repeat diseases .................................................... 3 1.2.1 Gain of toxic protein function: Huntington’s disease ............................................. 3 1.2.2 Gain of toxic RNA function: myotonic dystrophy type 1 ....................................... 5 1.3 DNA structures and repeat instability ................................................................................. 6 1.3.1 CTG/CAG slipped-DNA ........................................................................................ 7 1.4 Mechanisms of repeat instability ........................................................................................ 9 1.4.1 DNA repair and repeat instability ......................................................................... 11 1.4.2 DNA replication and repeat instability ................................................................. 14 1.5 Replication protein A: eukaryotic singled-stranded DNA binding protein ...................... 16 1.5.1 RPA in DNA replication ....................................................................................... 16 1.5.2 RPA in nucleotide excision repair ........................................................................ 17 1.5.3 RPA in base excision repair .................................................................................. 17 1.5.4 RPA in mismatch repair ........................................................................................ 18 1.5.5 An alternative form of RPA: aRPA ...................................................................... 18 1.6 Thesis Goals ...................................................................................................................... 19 2 Effect of DNA structural hyper-polymorphism and single-stranded DNA binding proteins on repair of disease-associated slipped-DNA repeats .............................................................. 22 2.1 Introduction ....................................................................................................................... 22 2.2 Materials and Methods ...................................................................................................... 25 iv 2.2.1 Slipped-intermediate DNA (SI-DNA) substrate preparation ................................ 25 2.2.2 Human cell extract preparation ............................................................................. 26 2.2.3 SV40 DNA replication .......................................................................................... 27 2.2.4 In vitro DNA repair ............................................................................................... 27 2.2.5 Western blotting .................................................................................................... 28 2.2.6 DNA binding assays ............................................................................................. 29 2.3 Results ............................................................................................................................... 29 2.3.1 Slipped-DNAs with increased single-stranded potential ...................................... 29 2.3.2 RPA4 expression is elevated above RPA2 in HD patient brains .......................... 38 2.3.3 RPA and aRPA can enhance slipped-DNA repair ................................................ 40 2.3.4 Repair is nick-directed .......................................................................................... 45 2.3.5 Yeast RPA and bacterial SSB cannot substitute for human RPA in enhancing repair ..................................................................................................................... 48 2.3.6 Inhibition of slipped-DNA repair by high concentrations of aRPA ..................... 50 2.3.7 RPA is limiting for the repair of nick-in-repeat slipped-DNAs, this effect is independent of MutSβ ........................................................................................... 53 2.3.8 FEN1 is not required for slipped-DNA repair ...................................................... 56 2.3.9 RPA and aRPA bind to and melt slipped-DNAs differently ................................ 59 2.4 Conclusions and Discussion ............................................................................................. 63 2.4.1 Nick-in-repeat slip-outs have greater structural heterogeneity than nick-in- flank slip-outs ........................................................................................................ 63 2.4.2 Nick-in-repeat slip-outs are better repaired .......................................................... 63 2.4.3 RPA and aRPA can enhance slipped-DNA repair ................................................ 65 2.4.4 Inhibition of slipped-DNA repair by high concentrations of aRPA ..................... 67 2.4.5 Repair of large slipped-DNAs is independent of MMR and BER ........................ 68 2.4.6 MMR does not require RPA ................................................................................. 69 2.4.7 RPA and aRPA bind to and denature slipped-DNAs differently .......................... 69 v 2.4.8 aRPA function ....................................................................................................... 70 3 Summary and future directions ................................................................................................ 72 3.1 Thesis summary ...............................................................................................................