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Florida State University Libraries Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2017 Insights into the Complex Formation between Nucleoside Diphosphate Kinase and a Highly Polymorphic DNA G- Quadruplex Mykhailo Kopylov Follow this and additional works at the DigiNole: FSU's Digital Repository. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES INSIGHTS INTO THE COMPLEX FORMATION BETWEEN NUCLEOSIDE DIPHOSPHATE KINASE AND A HIGHLY POLYMORPHIC DNA G-QUADRUPLEX By MYKHAILO KOPYLOV A Dissertation submitted to the Department of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2017 Mykhailo Kopylov defended this dissertation on October 16, 2017. The members of the supervisory committee were: M. Elizabeth Stroupe Professor Directing Dissertation Karen M. McGinnis University Representative Hank W. Bass Committee Member Wei Yang Committee Member Hong Li Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements. ii This dissertation is dedicated to my beloved wife, Elina. Thank you for the endless support, motivation, encouragement, patience and understanding. This would not be possible without you. iii ACKNOWLEDGEMENTS First I all I would like to thank my PI, Beth Stroupe. Thank you for all your support and guidance throughout the years. Thank you for allowing a lot of independence for my projects, while keeping me on track and getting necessary experiments done. I would like to thank all of my committie members: Hank Bass, Hong Li, Wei Yang and Karen McGinnis for their invaluable input and critique during my committee meetings. I would like to acknowledge all of the IMB staff, but especially Soma Sundaram, Claudius Mundoma, Joan Hare and Jana Sevcikova for all their help with multiple projects. My huge thanks and warmest regards to Lyn Kittle, for making Tallahasse a home away from home. I would also like to acknowledge my friends and colleagues with whom I had an honor to travel along this fun journey towards my PhD degree (in no particular order): Joe Pennington, Trevia Jackson, Travis Hand, John Spear, Matt Johnson, James McGivern, Isabel Askenasy, Alex Noble, Cory Hearn and many others. Finally, huge thanks to my parents Galina and Serhiy Kopylov, who were always very supportive and engaged in my research. iv TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... viii LIST OF TABLES .......................................................................................................................... x ABSTRACT ................................................................................................................................... xi CHAPTER 1. INTRODUCTION ................................................................................................... 1 1.1 Preface .............................................................................................................................. 1 1.2 Discovery of G4s .............................................................................................................. 1 1.3 G-quadruplex structures and properties ........................................................................... 3 1.4 G4 distribution in genomes .............................................................................................. 6 1.5 Protein G4-interactions .................................................................................................... 8 1.6 Methods for in-vitro G4 investigation ............................................................................ 10 1.7 G4 distribution in the maize genome ............................................................................. 11 CHAPTER 2. THE MAIZE (ZEA MAYS L.) NUCLEOSIDE DIPHOSPHATE KINASE1 (ZMNDPK1) GENE ENCODES A HUMAN NM23-H2 HOMOLOG THAT BINDS AND STABILIZES G-QUADRUPLEX DNA ...................................................................................... 15 2.1 Introduction .................................................................................................................... 15 2.2 Experimental Procedures................................................................................................ 17 2.2.1 Phage library screen ................................................................................................ 17 2.2.2 Protein expression and purification ........................................................................ 18 2.2.3 Point variant generation and purification ................................................................ 19 2.2.4 Structure determination ........................................................................................... 19 2.2.5 Maize extract pulldown........................................................................................... 19 2.2.6 Kd and stoichiometry determination ....................................................................... 20 2.2.7 Competition experiments ........................................................................................ 21 2.2.8 Activity assays ........................................................................................................ 21 2.2.9 G4 DNA folding experiments ................................................................................. 22 2.2.10 Additional methods ................................................................................................. 22 2.3 Results ............................................................................................................................ 24 2.3.1 The ZmNDPK1 gene encodes a protein with G4-binding activity ......................... 24 2.3.2 Structure determination of ZmNDPK1 ................................................................... 25 2.3.3 Native and recombinant ZmNDPK1 bind to G4 element hex4_A5U .................... 26 2.3.4 ZmNDPK1 and NM23-H2 have different G4 binding properties .......................... 26 2.3.5 ZmNDPK1 binding to G4 DNA is specific and they bind with defined stoichiometry........................................................................................................... 27 2.3.6 ZmNDPK1 G4 binding, nucleotide binding, and nucleoside kinase activity ......... 28 v 2.3.7 Lys149 is important for G4 binding........................................................................ 29 2.3.8 ZmNDPK1 binds to folded G4 DNA...................................................................... 30 2.4 Discussion ...................................................................................................................... 31 2.4.1 G4s in maize ........................................................................................................... 31 2.4.2 Binding mode of G4 to NDPK................................................................................ 32 2.4.3 Structural basis for G4 binding by NDPKs............................................................. 33 2.4.4 Implications for in vivo activity .............................................................................. 34 2.4.5 Conclusions ............................................................................................................. 35 CHAPTER 3. BULGED AND CANONICAL G-QUADRUPLEX CONFORMATIONS FORMED BY A SINGLE G-RICH DNA SEQUENCE CO-EXIST IN SOLUTION AND DETERMINE PROTEIN BINDING SPECIFICITY ................................................................... 50 3.1 Introduction .................................................................................................................... 50 3.2 Materials and methods ................................................................................................... 52 3.2.1 Oligonucleotide and protein preparation ................................................................ 52 3.2.2 Absorption spectrophotometry................................................................................ 52 3.2.3 Circular dichroism spectrophotometry ................................................................... 52 3.2.4 Dimethyl sulfide footprinting ................................................................................. 52 3.2.5 Nitrocellulose filter binding assays for ZmNDPK1/G4 DNA binding affinity analysis .................................................................................................................... 53 3.2.6 Analytical ultracentrifugation ................................................................................. 53 3.2.7 Fluorescence Resonance Energy Transfer (FRET)................................................. 54 3.2.8 Electron microscopy ............................................................................................... 54 3.3 Results ............................................................................................................................ 55 3.3.1 hex4_A5U adopts a G4 conformation in the presence of cations ........................... 55 3.3.2 hex4_A5U oligonucleotide is not limited to a single G4 conformation ................. 56 3.3.3 Locked hex4_A5U variants form G4s with distinct properties .............................. 57 3.3.4 ZmNDPK1 requires two consecutive G-tracts with a single one base loop for efficient binding .....................................................................................................
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