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Supervisors Foreword; Acknowledgment; Contents; Abbreviations; Summary; Chapter 1: Introduction; 1.1 Protein-DNA Interactions; 1.2 Sequence-Dependent DNA Deformability and Its Role in Target Recognition; 1.2.1 Free Energy Cost for Local Deformation of DNA; 1.2.2 Sequence-Dependent Base-Pair-Opening Rate Measured by NMR Imino Proton Exchange; 1.2.3 How Do Site-Specific Proteins Search for Their Target Sites on Genomic DNA?; 1.2.4 How Do Site-Specific Proteins Recognize Their Target Sites?; 1.2.4.1 Direct Versus Indirect Readout; 1.2.4.2 Induced-Fit Mechanism
1.2.5 Conformational Capture or Protein-Induced DNA Bending1.2.6 Measurements of DNA Binding and Bending Kinetics; 1.2.7 Competition Between 1D Diffusion and Binding-Site Recognition: The ``Speed-Stability ́́Paradox; 1.3 Experimental Techniques to Study Dynamics of Protein-DNA Interactions; 1.3.1 Laser Temperature-Jump Spectroscopy; 1.4 Thesis Overview; 1.4.1 DNA Bending Dynamics IHF-DNA Interaction; 1.4.2 Lesion Recognition in DNA by XPC Protein; 1.4.3 Recognition of Mismatches in DNA by MutS Protein; References; Chapter 2: Methods; 2.1 Equilibrium Measurements
2.2 Laser Temperature Jump Technique2.2.1 Laser Temperature Jump Spectrometer; 2.2.2 Theoretical Estimation of the Size of the T-Jump; 2.2.3 Photo-Acoustic Effects and Cavitation; 2.2.4 Estimation of Temperature Jump Using Reference Sample in a T-Jump Experiment; 2.2.5 T-Jump Recovery Kinetics; 2.2.6 Discrete Single- Or Double-Exponential Decay Convoluted with T-Jump Recovery; 2.2.7 Acquisition and Matching of Relaxation Traces Measured Over Different Time Scales; 2.2.8 Maximum Entropy Analysis; 2.3 Equilibrium FRET Measurements; 2.3.1 FRET Determination Using the Donor Emission
2.3.2 FRET Determination from Acceptor Emission2.3.3 Following are the FRET Pairs Used in This Thesis; 2.4 Nucleotide Analogue 2-Aminopurine (2AP); 2.5 Fraction of Protein and DNA in Complex at Equilibrium; 2.6 KD Measurements from Equilibrium FRET; 2.6.1 Conventional Titration Experiments; 2.6.2 Salt Titration Experiments [27]; References; Chapter 3: Integration Host Factor (IHF)-DNA Interaction; 3.1 Introduction; 3.1.1 Integration Host Factor; 3.1.2 IHF Binds to the Minor Groove on DNA and Recognizes Its Specific Site Via Indirect Readout; 3.1.3 Structure of IHF-H Complex
3.1.4 Background of IHF/H Interaction Dynamics3.1.5 Binding-Site Recognition Versus Protein Diffusional Search; 3.2 Materials and Method; 3.3 Results; 3.3.1 DNA-Bending Kinetics in the IHF-H Complex are Biphasic; 3.3.2 The Slow Phase Occurs on the Same Time Scale as Spontaneous bp Opening at a Kink Site; 3.3.3 Introducing Mismatches at the Site of the Kinks Affects the Slow Phase But Not the Fast Phase; 3.3.4 DNA-Bending Rates in the Slow Phase of IHF-TT8AT Complex Reflect Enhanced Base-Pair-Opening Rates in Mismatched DNA
1.2.5 Conformational Capture or Protein-Induced DNA Bending1.2.6 Measurements of DNA Binding and Bending Kinetics; 1.2.7 Competition Between 1D Diffusion and Binding-Site Recognition: The ``Speed-Stability ́́Paradox; 1.3 Experimental Techniques to Study Dynamics of Protein-DNA Interactions; 1.3.1 Laser Temperature-Jump Spectroscopy; 1.4 Thesis Overview; 1.4.1 DNA Bending Dynamics IHF-DNA Interaction; 1.4.2 Lesion Recognition in DNA by XPC Protein; 1.4.3 Recognition of Mismatches in DNA by MutS Protein; References; Chapter 2: Methods; 2.1 Equilibrium Measurements
2.2 Laser Temperature Jump Technique2.2.1 Laser Temperature Jump Spectrometer; 2.2.2 Theoretical Estimation of the Size of the T-Jump; 2.2.3 Photo-Acoustic Effects and Cavitation; 2.2.4 Estimation of Temperature Jump Using Reference Sample in a T-Jump Experiment; 2.2.5 T-Jump Recovery Kinetics; 2.2.6 Discrete Single- Or Double-Exponential Decay Convoluted with T-Jump Recovery; 2.2.7 Acquisition and Matching of Relaxation Traces Measured Over Different Time Scales; 2.2.8 Maximum Entropy Analysis; 2.3 Equilibrium FRET Measurements; 2.3.1 FRET Determination Using the Donor Emission
2.3.2 FRET Determination from Acceptor Emission2.3.3 Following are the FRET Pairs Used in This Thesis; 2.4 Nucleotide Analogue 2-Aminopurine (2AP); 2.5 Fraction of Protein and DNA in Complex at Equilibrium; 2.6 KD Measurements from Equilibrium FRET; 2.6.1 Conventional Titration Experiments; 2.6.2 Salt Titration Experiments [27]; References; Chapter 3: Integration Host Factor (IHF)-DNA Interaction; 3.1 Introduction; 3.1.1 Integration Host Factor; 3.1.2 IHF Binds to the Minor Groove on DNA and Recognizes Its Specific Site Via Indirect Readout; 3.1.3 Structure of IHF-H Complex
3.1.4 Background of IHF/H Interaction Dynamics3.1.5 Binding-Site Recognition Versus Protein Diffusional Search; 3.2 Materials and Method; 3.3 Results; 3.3.1 DNA-Bending Kinetics in the IHF-H Complex are Biphasic; 3.3.2 The Slow Phase Occurs on the Same Time Scale as Spontaneous bp Opening at a Kink Site; 3.3.3 Introducing Mismatches at the Site of the Kinks Affects the Slow Phase But Not the Fast Phase; 3.3.4 DNA-Bending Rates in the Slow Phase of IHF-TT8AT Complex Reflect Enhanced Base-Pair-Opening Rates in Mismatched DNA