STRUCTURAL AND BIOCHEMICAL STUDIES OF ALKYLPURINE DNA GLYCOSYLASE ALKD By Emily Holtzman Rubinson 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 May, 2011 Nashville, Tennessee Approved: Professor Fredrick P. Guengerich Professor Lawrence J. Marnett Professor Richard Armstrong Professor D. Borden Lacy Professor Brandt F. Eichman For Mom and Dad, thank you for all your love and support ii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Brandt Eichman, not only for his unending guidance, support, and encouragement but also for taking the time to teach me how to be a passionate scientist both at the bench and away from it. I am also grateful for the members of the Eichman laboratory especially Audrey Metz, who taught me everything I ever wanted to know about the “art” of crystallography and who initiated me into the world of a formerly unknown glycosylase, AlkD. Also, Briana Greer, a friend and confidant without whom I would have never successfully cloned anything. The past and present members of the lab were not only trusted colleagues but also friends and I thank them all for their help and camaraderie over the years. I also want to thank my committee members, Drs. Fred Guengerich, Larry Marnett, Richard Armstrong, and Borden Lacy for providing advice and insight into my research progress as well as my career development and I want to acknowledge the Toxicology training grant for funding my research for the past four years. I would also like to acknowledge the faculty and staff with Chemical and Physical Biology program including Lindsay Meyers for all her help during my time at Vanderbilt. I would also like to thank my family, intermediate and beyond, especially my parents and my brothers Paul, Max, and Claude, for their love and support. I know with them in my corner I can accomplish anything. And finally, I want to thank my friends for keeping me sane throughout this process. I will carry great memories of my time at Vanderbilt and in Nashville with me forever. iii TABLE OF CONTENTS Page DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................... xii Chapter 1. INTRODUCTION ...................................................................................................1 Overview of DNA Damage .....................................................................................2 Endogenous DNA Damage ..........................................................................2 Exogenous DNA Damage ............................................................................6 DNA Repair of Single-stranded Adducts ................................................................8 Direct Damage Reversal ..............................................................................9 Base Excision Repair .................................................................................10 Nucleotide Excision Repair .......................................................................13 Mismatch Repair ........................................................................................14 DNA Glycosylases .................................................................................................15 Recognition of DNA Damage ....................................................................16 Substrate Specificity and Catalytic Mechanism of DNA Glycosylases ....18 Structural Superfamilies of DNA Glycosylases ....................................................19 T4 Endonuclease V Superfamily ...............................................................19 Uracil DNA Glycosylase Superfamily .......................................................20 Helix-Hairpin-Helix Superfamily ..............................................................23 Alkyladenine DNA Glycosylase Superfamily ...........................................26 Helix-Two Turn-Helix Superfamily ..........................................................26 Tandem Helical Repeat Superfamily .........................................................28 Alkylation Damage Response ................................................................................28 The Adaptive Response to Alkylation Damage .........................................29 Alkylpurine DNA Glycosylases ................................................................31 Structural and Biochemical Highlights of Selected Alkylpurine DNA Glycosylases ..............................................................................................32 Scope of this Work.................................................................................................39 II. A NEW PROTEIN ARCHITECTURE FOR PROCESSING ALKYLATION DAMAGED DNA: THE CRYSTAL STRUCTURE OF DNA iv GLYCOSYLASE ALKD.......................................................................................41 Summary ................................................................................................................41 Introduction ............................................................................................................42 Results ....................................................................................................................43 The Structure of AlkD ...............................................................................43 Putative Active Site....................................................................................47 The Variant HEAT Motif ...........................................................................50 A DNA Binding Model .............................................................................52 Discussion ..............................................................................................................56 Materials and Methods ...........................................................................................59 AlkD Purification and Crystallization .......................................................59 X-ray Data Collection, Phasing, and Structure Refinement ......................60 Glycosylase Activity Assay .......................................................................62 DNA Binding Assay ..................................................................................62 Acknowledgements ................................................................................................63 III. AN UNPRECEDENTED NUCLEIC ACID CAPTURE MECHANISM FOR EXCISION OF DNA DAMAGE ..........................................................................64 Summary ................................................................................................................64 Introduction ............................................................................................................65 Results ....................................................................................................................67 A New Architecture for Binding Nucleic Acids ........................................67 A Novel Lesion Capture Mechanism .........................................................70 AlkD Traps and Restructures Destabilized Base Pairs ..............................75 Base Excision by Solvent Exposure .........................................................78 Discussion ..............................................................................................................80 Methods Summary .................................................................................................83 Preparation of 3-deaza-3-methyladenine ..................................................83 AlkD/DNA Crystal Structure Determination ............................................83 Biochemical Assays ..................................................................................84 Acknowledgements ................................................................................................84 Author Contributions .............................................................................................85 Author Information ................................................................................................85 Methods..................................................................................................................85 AlkD Purification and Crystallization ......................................................85 X-ray Data Collection, Phasing, and Structure Refinement .....................86 Enzyme Activity .......................................................................................88 POB Adduct Excision ...............................................................................89 DNA Binding ............................................................................................91 IV. COMPREHENSIVE ANALYSIS OF DNA BINDING AND RECOGNITION BY ALKD..................................................................................93 Summary ................................................................................................................93