Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition

Abstract

"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. 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.