A Biochemical Characterization of the DNA Glycosylase DEMETER By Sonja Claudia Brooks Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemical and Physical Biology August, 2014 Nashville, Tennessee Approved: Walter Chazin Carmelo Rizzo Martin Egli Neil Osheroff Brandt Eichman For Braden, Thank you for all your love and support ii ACKNOWLEDGMENTS This work was supported through a National Science Foundation Graduate Research Fellowship grant, a position on the National Institutes of Health Molecular Biophysics Training Program, and funding from the National Institutes of Health. I am grateful for the continuous support and guidance from my advisor, Brandt Eichman. His excitement and dedication to his research projects has always been inspiring. I would also like to thank my committee members for their direction throughout my dissertation. In addition, the support of my lab members over the last few years has been invaluable. I especially thank Emily Rubinson and Suraj Adhikary, who taught me the ins and outs of the lab and structural biology, while we were becoming friends. I am forever grateful for current lab members and friends Briana Greer and Claire Cato, for always offering their help when needed and having plenty of fun in the process. Finally, I thank my family and friends for their continued support. My parents have always encouraged me to pursue my dreams, and for that I am forever grateful. My friends in Nashville have made this experience fun, and I will fondly remember our adventures here. I would not be here without the love and support of my fiancé Braden, who encouraged me from qualifying exam to defense. iii TABLE OF CONTENTS Page DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii LIST OF ABBREVIATIONS ............................................................................................ ix Chapter I. Introduction ..............................................................................................................1 5mC is an Epigenetic Mark......................................................................................1 Mechanism of Transcriptional Repression ..............................................................5 DNA Methyltransferases .........................................................................................6 Dysregulation of Methylation Leads to Disease ....................................................11 Environmental Epigenetics ....................................................................................13 Base Excision Repair .............................................................................................14 Passive DNA Demethylation .................................................................................17 Active DNA Demethylation ..................................................................................17 5mC Oxidation Derivatives ...................................................................................18 Active DNA Demethylation in Plants....................................................................24 Scope of this Work.................................................................................................27 II. Structural Mechanisms of DNA Glycosylases ......................................................29 Introduction ............................................................................................................29 Oxidative Damage .................................................................................................33 Alkylation Damage ................................................................................................35 AAG ...........................................................................................................37 Helix Hairpin Helix Superfamily ...............................................................42 Uracil/Thymine/5mC .............................................................................................51 A Possible Role of BER in DNA demethylation .......................................53 Structural Insight into TDG Function ........................................................54 MBD4 ........................................................................................................62 DME/ROS1 ................................................................................................63 iv III. 5-methylcytosine Recognition by Arabidopsis thaliana DNA Glycosylases DEMETER and DML3 ..........................................................................................66 Introduction ............................................................................................................66 Materials and Methods ...........................................................................................70 Protein Expression and Purification...........................................................70 Glycosylase Activity Assay .......................................................................71 DNA Binding .............................................................................................72 DME Homology Model .............................................................................73 Results ....................................................................................................................73 A Model for DNA Binding by DME .........................................................73 Specificity of DME and DML3 for oxidized 5mC ....................................79 Active Site Residues Confer Specificity for Substrate DNA.....................83 Discussion ..............................................................................................................86 IV. Discussion and Future Directions ..........................................................................92 DNA Glycosylases Regulate DNA Methylation ...................................................92 DME is a Multi-Domain Glycosylase: the Role of Domain B ..............................94 DME Paralogs Have Distinct Functions ................................................................95 DME Unstructured Regions of Unknown Function ..............................................97 Role of the Iron-Sulfur Cluster ..............................................................................99 Epigenetics and Human Health ............................................................................101 State of the Field and Future Directions ..............................................................101 Appendix I .......................................................................................................................104 Appendix II ......................................................................................................................110 REFRENCES ...................................................................................................................113 v LIST OF TABLES Table Page 1. DNA binding and 5mC excision activities for DME point mutants ............................77 2. Rates of excision of oxidized 5mC derivatives by DME and DML3 ..........................79 3. Substrate specificity of DME .......................................................................................81 4. Effects of predicted DME active site residues on nucleobase excision .......................86 5. Thermal denaturation of 5mC oxidation derivatives .................................................106 A1. DME, DML3, and DMLa Constructs .......................................................................110 vi LIST OF FIGURES Figure Page 1. DNA methyltransferase mechanism ...........................................................................8 2. Base Excision Repair (BER) Pathway ......................................................................16 3. 5mC derivatives ........................................................................................................20 4. Common DNA lesions ..............................................................................................30 5. Chemical reaction catalyzed by DNA glycosylases .................................................31 6. DNA glycosylase structural superfamilies ................................................................33 7. Alkylpurine DNA glycosylases ................................................................................38 8. Binding of εA and εC to AAG ..................................................................................41 9. Yeast 3-methyladenine DNA glycosylases MAG and Mag1 ...................................47 10. Protein-DNA contacts within the TDG active site ...................................................56 11. Comparison of TDG and MBD4 contacts to the strand opposite the lesion .............59 12. SUMO1 modified TDG creates steric clash with DNA............................................61 13. DME paralogs and constructs ...................................................................................69