DNA Supercoiling as a Regulatory Signal for the Lambda Repressor and An Integrated Calibration Method for the Molecular Fluorescence Force Probes Público

Abstract

In the first part of this dissertation, we studied the role of DNA supercoiling on the stability of the lambda repressor (cI) - mediated regulatory loop. DNA supercoiling senses environmental stress, modifies the accessibility of chromatin, and coordinates the transcription of genes. Therefore, it likely affects protein-mediated long-range DNA interactions, such as looping, which regulate transcription. cI maintains the quiescent stage of bacteriophage lambda in infected E. coli, by binding to specific sites and mediating a DNA loop that prevents over-expression of the repressor protein to preserve sensitivity to host conditions that trigger virulence. Here, we assessed how well the cI-mediated DNA loop topologically isolates the cI promoter and determined whether supercoiling enhances cI-mediated DNA looping. We demonstrated that supercoiling levels in cI-mediated DNA loops under conditions of DNA tension and twist were as high as -10 or +16%. Furthermore, supercoiling was essential for DNA looping under tension and lowered the free energy of loop formation by cI. Therefore, the lambda repressor system appears to utilize supercoiling for lysogeny maintenance; it encodes sensitivity to the overall cell health through supercoiling-enhanced looping and creates independent modules of distinct superhelicity.

In the second part of this dissertation, we developed a technique to calibrate fluorescence based probes that are used to measure cellular forces. Despite the importance of mechanotransduction, few methods are available to measure molecular forces in living systems. One of these methods is molecular tension - based fluorescence microscopy (MTFM), a technique that allows visualization of piconewton forces by flanking a flexible polymer by a fluorophore-quencher pair and detecting the fluorescence increase resulting from polymer extension under tension. To accurately calibrate MTFM probes, we integrated a magnetic tweezers system to a total-internal-reflection fluorescence (TIRF) microscope for single molecule fluorescence - force measurement, and designed a 4.4 micron lambda-DNA fragment to link to a surface-immobilized DNA hairpin probe. Preliminary results showed binary fluorescence changes which report on the opening and closing of the hairpin, and its force-extension data, in good agreement with a theoretical model, supporting the idea that this experimental setup could indeed be used to measure molecular forces with high accuracy.

Table of Contents

Part I: DNA supercoiling: A Regulatory Signal for the Lambda Repressor…………...…….1

Chapter 1. Introduction……………………………………………………………..………...2

1.1. DNA Supercoiling: A common DNA feature…………………...…………...…………...…..2

1.2. The importance of supercoiling: A messenger in gene regulation…………...……………….3

1.3. Lambda repressor-mediated loop and other protein-mediated DNA loops………...…………4

1.4. Magnetic tweezers (MT): The ideal method to stretch and twist polymers…………….....….8

1.5. DNA behavior under torsion and tension: Hat curves and force-extension curve……...…...10

1.6. Research Questions……………..…………………………………………...……..………..13

Chapter 2. Materials and Methods…………………………….…………………..………...14

2.1. Design of plasmid DNA constructs for supercoiling study in MT experiments………….....14

2.2. DNA manipulation for supercoiling studies…………………..……………………………..18

2.3. Data acquisition and analysis for supercoiling study: The change-point algorithm...............19

Chapter 3. Results and Discussion……………..….…...…………………….……………..20

3.1. cI-loops can constrain high levels of supercoiling…………………………………………..20

3.2. Loops alter effective DNA elasticity…………………………………………………...……24

3.3. Supercoiling influences the ability of lambda cI repressor to secure DNA loops………..….27

3.3.1. Looping requires (negative) supercoiling………………………………………………..27

3.3.2. Negative supercoiling lowers free energy of looping……..……………………………..29

3.3.3. Negative superhelical density offsets tension to favor looping……………………….....32

3.4. Discussion……………………………………………….…………………………………..34

Part II: A Combined Magnetic Tweezers (MT) and Single Molecule Fluorescence Resonance Efficiency Transfer (smFRET) Instrument to Calibrate Molecular Tension - Based Fluorescence Probes………………………………………………………………….….37

Chapter 4. Introduction…………………………………….………………………………..38

4.1. Cellular Force: Important yet poorly understood………………………….……...…………38

4.2. Molecular Tension - Based Fluorescence Microscopy (MTFM) and its probes………...…..38

4.3. Research Questions………………..……………………………………...…………………41

Chapter 5. Materials and Methods…………………………………………………………..43

5.1. Integration of magnetic tweezers (MT) into the fluorescence microscope………………….43

5.1.1. Set-up and illumination of the magnet tweezers................................................................44

5.1.2. Fluorescence microscopy system.......................................................................................47

5.1.3. Image acquisition and processing……………………………………………..…………47

5.2. Design of lambda DNA constructs for cellular force probe calibration combining MT-smFRET experiments………………………………….……………………………………..49

5.3. Functionalization of coverslip chambers (surface passivation)………….…………………..51

5.4. Functionalization of paramagnetic beads……………………….……………………..…….53

5.4.1. Preparing Beads………………………………………………………………………….53

5.4.2. Binding anti-digoxigenin…………………………………...……………………………53

5.4.3. DMP Crosslinking……………………………………………………………………….54

5.5. DNA manipulation: Calibration and force - extension measurement in MT-smFRET experiments………………………………………………..…………………………………55

5.6. Coupling single molecule fluorescence measurement in MT-smFRET experiments……….57

Chapter 6. Results and Discussion…….………………………………......………………..60

6.1. Determining fluorophore concentration: Statistics for a single molecule experiment…........60

6.2. Calibrating a DNA tether: Force - extension curve on the TIRF microscope………….……61

6.3. Proof of concept: Binary fluorescence change with the open and close of a DNA hairpin....63

6.4. Future work………………………………………………………...………………………..66

Chapter 7. Conclusions………………….…..………………………………………………68

References…………………………...……………………………………………………………69

Appendix 1 DNA-Nicking Gel Assay………………………………………………………..81

About this Dissertation

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School	Laney Graduate School
Department	Physics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Physics, Molecular Biology, Molecular Biophysics, General
Palabra Clave	instrumentation epigenetics molecular fluorescence force probe single molecule FRET lambda repressor biophysics DNA supercoiling magnetic tweezers calibration method transcriptional regulation
Committee Chair / Thesis Advisor	Finzi, Laura, Emory University Salaita, Khalid, Emory University
Committee Members	Weeks, Eric, Emory University Warncke, Kurt, Emory University Urazhdin, Sergei , Emory University

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