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Single-Molecule Studies of Rad4-Rad23 Reveal a Dynamic Dna Damage Recognition Process

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Single-Molecule Studies of Rad4-Rad23 Reveal a Dynamic Dna Damage Recognition Process SINGLE-MOLECULE STUDIES OF RAD4-RAD23 REVEAL A DYNAMIC DNA DAMAGE RECOGNITION PROCESS by Muwen Kong B.A. Physics, Knox College, 2008 M.S. Physics, Carnegie Mellon University, 2010 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2017 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Muwen Kong It was defended on June 30, 2017 and approved by Guillermo Romero, PhD., Associate Professor, Department of Pharmacology and Chemical Biology Marcel Bruchez, PhD., Associate Professor, Departments of Biological Sciences and Chemistry, Carnegie Mellon University Neil Kad, PhD., Senior Lecturer, School of Biosciences, University of Kent Patricia Opresko, PhD., Associate Professor, Department of Environmental and Occupational Health Dissertation Director: Bennett Van Houten, PhD., Professor, Department of Pharmacology and Chemical Biology ii Copyright © by Muwen Kong 2017 iii Single-Molecule Studies of Rad4-Rad23 Reveal a Dynamic DNA Damage Recognition Process Muwen Kong, PhD University of Pittsburgh, 2017 Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix- destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. As the first step towards productive repair, the human NER damage sensor XPC-RAD23B needs to efficiently locate sites of damage among billons of base pairs of undamaged DNA. In this dissertation, we investigated the dynamic protein-DNA interactions during the damage recognition step using a combination of fluorescence-based single-molecule DNA tightrope assays, atomic force microscopy, as well as cell survival and in vivo repair kinetics assays. We observed that quantum dot-labeled Rad4-Rad23, the yeast homolog of human XPC-RAD23B, formed nonmotile complexes on DNA or conducted a one-dimensional search via either random diffusion or constrained motion along DNA. Using atomic force microscopy, we studied binding of Rad4 lacking the β-hairpin domain 3 (BHD3) to damage-containing DNA and found that this structural motif is non-essential for damage-specific binding or DNA bending. Furthermore, we demonstrated that deletion of seven residues in the tip of β-hairpin in BHD3 increased Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance. Finally, we explore existing physical models and examples of subdiffusive motion, and discuss a model in which constrained motion by Rad4-Rad23 on DNA may be driven by conformational changes of the protein. iv TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ...................................................................................................................... x PREFACE ................................................................................................................................... xiii 1.0 INTRODUCTION ............................................................................................................ 1 1.1 NUCLEOTIDE EXCISION REPAIR......................................................................... 1 1.1.1 Overview .................................................................................................................... 2 1.1.2 Initiation of TC-NER ................................................................................................ 4 1.1.3 Initiation of GG-NER ............................................................................................... 5 1.1.4 Damage Verification and Assembly of Pre-Incision Complex .............................. 9 1.1.5 Excision, Repair Synthesis, and Ligation ............................................................. 11 1.2 DISEASES ASSOCIATED WITH NER .................................................................. 12 1.2.1 Xeroderma Pigmentosum (XP) .............................................................................. 12 1.2.2 Cockayne Syndrome, UV-Sensitive Syndrome, and Trichothiodystrophy ....... 13 1.3 DAMAGE RECOGNITION BY XPC-RAD23B AND RAD4-RAD23 .................. 14 1.3.1 XPC-RAD23B and Rad4-Rad23 ............................................................................ 14 1.3.2 Molecular Mechanism of Damage Recognition ................................................... 15 1.4 DIFFUSION................................................................................................................. 17 1.4.1 Introduction to Diffusion ........................................................................................ 17 1.4.1.1 Brownian Motion ............................................................................................ 17 1.4.1.2 Fickian Diffusion ............................................................................................. 18 v 1.4.1.3 Einstein’s Theory of Brownian Motion......................................................... 20 1.4.2 The Target Search Problem: Solving the Speed-Stability Paradox. .................. 22 1.4.2.1 Facilitated Diffusion........................................................................................ 22 1.4.2.2 Speed-Stability Paradox ................................................................................. 24 1.5 HYPOTHESES AND SCOPE ................................................................................... 26 2.0 MATERIALS AND METHODS ................................................................................... 28 2.1 DEFINED-LESION SUBSTRATES ......................................................................... 28 2.1.1 Incorporation of Site-Specific Lesion in pSCW01 Plasmid ................................ 28 2.1.2 Substrates for DNA Tightrope Assay .................................................................... 29 2.1.3 Substrates for Atomic Force Microscopy (AFM) ................................................ 30 2.2 UV-IRRADIATION OF λ-DNA ................................................................................ 31 2.3 SINGLE-MOLECULE DNA TIGHTROPE ASSAY .............................................. 31 2.4 ATOMIC FORCE MICROSCOPY .......................................................................... 34 2.5 CPD AND 6-4PP REPAIR KINETICS BY ANTIBODY SLOT BLOT ................ 35 2.6 AGAROSE GEL ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) 37 2.7 FLUORESCENCE ANISOTROPY .......................................................................... 38 2.8 ESTIMATION OF HYDRODYNAMIC RADII...................................................... 39 2.9 ESTIMATION OF THEORETICAL LIMIT OF DIFFUSION COEFFICIENT 39 2.10 CALCULATION OF ENERGY BARRIER TO FREE DIFFUSION ................... 40 2.11 ESTIMATION OF RESIDENCE TIME AT EACH BASE PAIR ......................... 41 2.12 ESTIMATION OF MINIMUM TARGET SITE ENERGY ................................... 41 2.13 ESTIMATION OF GENOME SEARCH TIME ..................................................... 42 vi 2.14 RAD4 MUTANT STRAIN CONSTRUCTION, UV SURVIVAL MEASUREMENTS, AND WESTERN BLOTTING .......................................................... 43 2.15 CPD REPAIR KINETICS BY T4 ENDO V DIGESTION ..................................... 44 2.16 ESTIMATION OF RATE OF PHOTOPRODUCT REMOVAL .......................... 45 3.0 RESULTS ........................................................................................................................ 47 3.1 RAD4-RAD23 UTILIZES A COMBINATION OF 3D AND 1D APPROACHES TO SEARCH FOR DAMAGE ON DNA .............................................................................. 49 3.2 SLIDING IS THE MAIN COMPONENT OF OBSERVED 1D DIFFUSION OF RAD4-RAD23 .......................................................................................................................... 52 3.3 RAD4-RAD23 EXHIBITS LESION-SPECIFIC DAMAGE RECOGNITION .... 56 3.4 TRUNCATIONS IN THE β-HAIRPIN DOMAIN 3 (BHD3) OF RAD4 INCREASE CONSTRAINED MOTION ................................................................................................... 58 3.5 RAD4 VARIANT LACKING β-HAIRPIN DOMAIN 3 (ΔBHD3) IS CAPABLE OF SPECIFIC BINDING AND DNA BENDING TO FL-dT-CONTAINING DNA FRAGMENTS ......................................................................................................................... 63 3.6 DELETIONS OF C-TERMINAL REGIONS IN RAD4 CONFER VARYING DEGREES OF UV SENSITIVITY AND REPAIR IN S. CEREVISIAE ........................... 66 3.7 DISCUSSION .............................................................................................................. 70 3.7.1 Alternative Damage Recognition Mechanism for Sub-Optimal Substrates through Constrained Motion by Rad4-Rad23 ................................................................. 71 3.7.2 BHD3-Independent DNA Bending as an Initial Quality Check by Rad4 .......... 72 3.7.3 A Dynamic Multi-Step Damage Recognition Model............................................ 73 3.8 CONCLUSION ........................................................................................................... 78 vii 3.9 ACKNOWLEDGEMENT .......................................................................................... 78 4.0 DISCUSSION .................................................................................................................
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