Dynamics and mechanism of DNA-bending proteins in binding site recognition : doctoral thesis accepted by University of Illinois at Chicago, Chicago, Illinois, USA

Using a novel approach that combines high temporal resolution of the laser T-jump technique with unique sets of fluorescent probes, this study unveils previously unresolved DNA dynamics during search and recognition by an architectural DNA bending protein and two DNA damage recognition proteins. Many cellular processes involve special proteins that bind to specific DNA sites with high affinity. How these proteins recognize their sites while rapidly searching amidst ~3 billion nonspecific sites in genomic DNA remains an outstanding puzzle. Structural studies show that proteins severely deform DNA at specific sites and indicate that DNA deformability is a key factor in site-specific recognition. However, the dynamics of DNA deformations have been difficult to capture, thus obscuring our understanding of recognition mechanisms. The experiments presented in this thesis uncover, for the first time, rapid (~100-500 microseconds) DNA unwinding/bending attributed to nonspecific interrogation, prior to slower (~5-50 milliseconds) DNA kinking/bending/nucleotide-flipping during recognition. These results help illuminate how a searching protein interrogates DNA deformability and eventually “stumbles” upon its target site. Submillisecond interrogation may promote preferential stalling of the rapidly scanning protein at cognate sites, thus enabling site-recognition. Such multi-step search-interrogation-recognition processes through dynamic conformational changes may well be common to the recognition mechanisms for diverse DNA-binding proteins. Supervisor ́s Foreword 6 Acknowledgment 10 Contents 12 Abbreviations 16 Summary 17 Chapter 1: Introduction 22 1.1 Protein-DNA Interactions 22 1.2 Sequence-Dependent DNA Deformability and Its Role in Target Recognition 23 1.2.1 Free Energy Cost for Local Deformation of DNA 25 1.2.2 Sequence-Dependent Base-Pair-Opening Rate Measured by NMR Imino Proton Exchange 27 1.2.3 How Do Site-Specific Proteins Search for Their Target Sites on Genomic DNA? 28 1.2.4 How Do Site-Specific Proteins Recognize Their Target Sites? 29 1.2.4.1 Direct Versus Indirect Readout 29 1.2.4.2 Induced-Fit Mechanism 30 1.2.5 Conformational Capture or Protein-Induced DNA Bending 31 1.2.6 Measurements of DNA Binding and Bending Kinetics 31 1.2.7 Competition Between 1D Diffusion and Binding-Site Recognition: The ``Speed-Stability ́ ́ Paradox 32 1.3 Experimental Techniques to Study Dynamics of Protein-DNA Interactions 33 1.3.1 Laser Temperature-Jump Spectroscopy 34 1.4 Thesis Overview 35 1.4.1 DNA Bending Dynamics IHF-DNA Interaction 35 1.4.2 Lesion Recognition in DNA by XPC Protein 36 1.4.3 Recognition of Mismatches in DNA by MutS Protein 37 References 38 Chapter 2: Methods 44 2.1 Equilibrium Measurements 44 2.2 Laser Temperature Jump Technique 44 2.2.1 Laser Temperature Jump Spectrometer 46 2.2.2 Theoretical Estimation of the Size of the T-Jump 48 2.2.3 Photo-Acoustic Effects and Cavitation 49 2.2.4 Estimation of Temperature Jump Using Reference Sample in a T-Jump Experiment 49 2.2.5 T-Jump Recovery Kinetics 51 2.2.6 Discrete Single- Or Double-Exponential Decay Convoluted with T-Jump Recovery 53 2.2.7 Acquisition and Matching of Relaxation Traces Measured Over Different Time Scales 54 2.2.8 Maximum Entropy Analysis 54 2.3 Equilibrium FRET Measurements 57 2.3.1 FRET Determination Using the Donor Emission 57 2.3.2 FRET Determination from Acceptor Emission 57 2.3.3 Following are the FRET Pairs Used in This Thesis 58 2.4 Nucleotide Analogue 2-Aminopurine (2AP) 63 2.5 Fraction of Protein and DNA in Complex at Equilibrium 64 2.6 KD Measurements from Equilibrium FRET 65 2.6.1 Conventional Titration Experiments 65 2.6.2 Salt Titration Experiments [27] 65 References 67 Chapter 3: Integration Host Factor (IHF)-DNA Interaction 69 3.1 Introduction 69 3.1.1 Integration Host Factor 69 3.1.2 IHF Binds to the Minor Groove on DNA and Recognizes Its Specific Site Via Indirect Readout 70 3.1.3 Structure of IHF-H Complex 72 3.1.4 Background of IHF/H Interaction Dynamics 74 3.1.5 Binding-Site Recognition Versus Protein Diffusional Search 76 3.2 Materials and Method 77 3.3 Results 78 3.3.1 DNA-Bending Kinetics in the IHF-H Complex are Biphasic 78 3.3.2 The Slow Phase Occurs on the Same Time Scale as Spontaneous bp Opening at a Kink Site 78 3.3.3 Introducing Mismatches at the Site of the Kinks Affects the Slow Phase But Not the Fast Phase 80 3.3.4 DNA-Bending Rates in the Slow Phase of IHF-TT8AT Complex Reflect Enhanced Base-Pair-Opening Rates in Mismatched DNA 83 3.3.5 DNA Modifications Away from the Kink Sites Have No Effect on Either of the Two Rates 88 3.3.6 Two Plausible Scenarios for Biphasic Relaxation Kinetics 89 3.3.7 Salt-Dependence of the Fast and Slow Components 89 3.3.8 Protein Mutations Distal to the Kink Sites Affect Affinity and Bending Rate of Slow Phase 95 3.3.9 Control Experiments to Rule Out Contributions to the Relaxation Kinetics from Dye Dynamics or Dye Interactions with Prot... 97 3.4 Discussion 104 3.4.1 Nonspecific Search and Specific Recognition by IHF 104 3.4.2 Nonspecific Binding of DNA by IHF and Its Structurally Homologous Cousin HU 105 3.4.3 Nonspecific Binding Facilitates 1-D Diffusion on DNA 106 3.5 Concluding Remarks 107 References 107 Chapter 4: Lesion Recognition by XPC (Rad4) Protein 111 4.1 Introduction 111 4.1.1 Nucleotide Excision Repair 111 4.1.2 Experimental Design 115 4.2 Method 118 4.2.1 Preparation of Double-Stranded DNA Substrates 118 4.2.2 Preparation of Rad4-Rad23 Complexes 118 4.2.3 Duplex Melting Temperatures of Mismatched and Undamaged/Matched DNA 118 4.2.3.1 Method 1 119 4.2.3.2 Method 2 119 4.2.4 Apparent Binding Affinities (Kd,app) Determined by Electrophoretic Mobility Shift Assays 120 4.2.5 Equilibrium FRET Temperature Scan Experiments with tC/tCnitro Probes 120 4.2.6 Acquisition and Analyses of T-Jump Relaxation Traces 121 4.2.6.1 T-Jump Recovery Kinetics 121 4.2.6.2 Maximum Entropy Method for Analyzing T-Jump Relaxation Rates 121 4.2.6.3 Single-Exponential Decay Convoluted with T-Jump Recovery 123 4.2.6.4 Double-Exponential Decay Convoluted with T-Jump Recovery 123 4.2.6.5 MEM Versus Discrete-Exponential Analysis 123 4.2.6.6 Amplitude Analysis 124 4.3 Results 125 4.3.1 Kinetics of Rad4 (Wild Type) Induced DNA-Opening Rate 125 4.3.1.1 Interaction Between Rad4 Mutant and DNA 129 4.3.1.2 Comparison of DNA-Opening Rate/Nucleotide-Flipping Rate with Imino Proton Exchange Measurements of Base-pair-Opening R... 130 4.3.1.3 Interplay Between Residence Time and Opening Time 135 4.3.2 tC and tCnitro FRET Pair as Probes for Sensing Changes in DNA Helical Structure 136 4.3.2.1 tC/tCnitro-Labeled TTT/TTT-Mismatched DNA (AN12) as a Model Lesion for Specific Binding 137 Differences Between Measured and Calculated FRET in the AN12-Rad4 Complex 142 4.3.2.2 Increase in Temperature Amplifies Rad4-Induced Opening Sensed by the Probes in AN12 143 4.3.2.3 tC/tCnitro-Sensed Dynamics in Rad4-Bound AN12 are Concurrent with 2AP-Sensed, Rad4-Induced Lesion-``Opening ́ ́ Dynamics 145 4.3.2.4 beta-Hairpin Mutants Exhibit Novel Sub-Millisecond Kinetics and Help Reveal the Multistep Nature of Lesion Recognition... 149 4.3.2.5 Fast Sub-Millisecond Kinetics are Also Observed with Wild-Type Rad4 on Nonspecific DNA Substrates: AN14/AN14u 152 4.3.3 DNA-Bending Dynamics Measured with Extrinsically Attached FRET Pair/AN7 159 4.3.3.1 DNA-Bending Rate in the Presence of Mutant Rad4 162 4.4 Discussion 163 4.4.1 Rad4/XPC-Induced Nucleotide-Flipping/Open Dynamics Measured with 2AP Probe 163 4.4.2 Rad4/XPC-Induced Helical Distortion Dynamics Measured Using tC/tCnitro 169 4.4.2.1 Slow Phase 169 4.4.2.2 Fast Phase 170 4.4.3 Rad4/XPC-Induced DNA-Bending Dynamics Measured Using TAMRA/Cy5 FRET Pair 173 4.5 Conclusion 174 References 174 Chapter 5: DNA Mismatch Repair 179 5.1 Introduction 179 5.1.1 Structural Studies on MutS Bound to Mismatched DNA 180 5.1.2 What Role Does the Intrinsic Flexibility of DNA Play in the Mismatch Recognition and Subsequent Repair? 183 5.1.3 Dynamics of DNA Binding and Bending by MutS 183 5.2 Results 184 5.2.1 Taq MutS Binding to Mismatch (T-Bulge) DNA as Probed by 2AP 184 5.2.1.1 Equilibrium Temperature-Dependent Change in 2AP Intensity for TDNA-Taq MutS Complex 186 5.2.1.2 T-Jump Measurements on TDNA-Taq MutS Complex with 2AP Probe 187 5.2.2 Taq MutS Binding to Mismatch (T-Bulge) DNA as Probed by FRET Pair 188 5.2.2.1 Temperature-Dependent FRET Change in T-Bulge (FRET) (TFT)-Taq MutS Complex 188 5.2.2.2 T-Jump Measurements on T-Bulge (FRET)-Taq MutS 191 5.2.2.3 Taq MutS-Induced DNA Kinking/Bending Rates Compared with Intrinsic bp-Opening Dynamics of Mismatched DNA 192 5.2.3 MutSα Binding to Mismatch (T-Bulge) DNA as Probed by 2AP (in DNA) and Trp (in MutS) 193 5.3 Discussion 196 5.4 Conclusion 197 References 198 Appendix A 201 Energetic Cost for Bending DNA 201 Sequence-Dependent DNA Elasticity 201 DNA Geometry 202 Appendix B 205 Equilibrium Perturbation Using Temperature Jump (T-Jump) 205 Temperature Jump Relaxation Kinetics 206 Relaxation Kinetics Obtained from a Master Equation (Referred from Ref. [14]) 207 Fluorescence Resonance Energy Transfer (FRET) 207 FRET Efficiency of tC-tCnitro 211 References 212 VITA 214 Front Matter....Pages i-xxi Introduction....Pages 1-22 Methods....Pages 23-47 Integration Host Factor (IHF)–DNA Interaction....Pages 49-90 Lesion Recognition by XPC (Rad4) Protein....Pages 91-158 DNA Mismatch Repair....Pages 159-180 Back Matter....Pages 181-199