DNA elasticity and effects on Type II topoisomerases Público

Abstract

DNA elasticity and effects on Type II topoisomerases
By Qing Shao

The exceptional stiffness of DNA is routinely attributed to base pair stacking, hydrogen bonding, and electrostatic repulsion between neighboring, negatively charged phosphates. Understanding how these factors contribute to the stiffness and mechanical properties of DNA is very important to understand how cells use counterions, sequence variety as well as enzymes to manage DNA. Using Magnetic Tweezers to twist and stretch single DNA molecules, experiments were performed to probe the parameters of DNA stiffness. Experiments showed that diaminopurine (DAP) substitution for adenine, which adds an additional hydrogen bond to A:T base pairs, stiffens DNA by about 40% without significantly changing the buckling transition point. Moreover, DAP-substituted DNA fragments showed different behavior under extreme unwinding at high tension indicating sequence dependent DNA deformation and right to left hand transition. Furthermore, adding low molecular weight polycations such as spermine or spermidine to the solution appeared to soften DNA and promote plectoneme formation at lower values of torsion.

Bending a stiff polymer like DNA requires considerable energy and could represent the rate limiting step in enzymatically catalyzed processes that modify the topology of DNA. For example the activity of type II topoisomerases that catalyze DNA decatenation and unwinding which is essential for cell division might be altered. A recently published crystal structure shows that, during the catalytic cycle, a yeast type II topoisomerase can bend a 34 base pair DNA segment by up to 150 degrees. Bacterial gyrase, another type II topoisomerase, can wrap an approximately 100 bp DNA segment into a tight 180 degree turn. To test whether or not DNA stiffness modifies topoisomerase activity, DAP DNA has been used as a substrate for topoisomerase II-mediated relaxation of plectonemes introduced in single molecules using Magnetic Tweezers. The overall rate of relaxation of plectonemes by recombinant human topoisomerase II alpha and E. coli gyrase decreased on the stiffer DNA. In addition the binding affinity as well as the ability of E. coli gyrase to wrap DNA also decreased. These dynamic measurements of DNA supercoil relaxation by type II topisomerases support the idea that DNA is significantly deformed in the rate-determining step.

DNA elasticity and effects on Type II topoisomerases
By
Qing Shao
B.S., Shanghai Jiao Tong University,
China 2005
Advisor: David Dunlap, Ph.D.
Advisor: Laura Finzi, Ph.D
A dissertation submitted to the Faculty of the
James T. Laney Graduate School Studies of Emory University
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
in Physics
2011

Table of Contents

Table of Contents
Chapter 1 Introduction...1

1.1 Motivation and hypothesis...2
1.2 DNA primary and secondary structure...4

1.2.1 DNA double helical structure is stable...4
1.2.2 Polymorphism of DNA structure: B-DNA, A-DNA and Z-DNA...5

1.3 DNA behavior under tension and torsion...6

1.3.1 Stretching and twisting single DNA molecule...6
1.3.2 DNA phase transition under extreme tension and torsion...8

1.4 Why is DNA a stiff molecule?...10

1.4.1 Definition of DNA stiffness and experimental methods to study it...10
1.4.2 The origins of DNA stiffness...11
1.4.3 DNA stiffness affects DNA-protein interaction...13

1.5 Natural polyamines (spermine and spermidine)...13

1.5.1 DNA supercoiling regulates DNA topology, DNA replication and transcription...14
1.5.2 Polyamine can interact with DNA electrostatically...15
1.5.3 Spermine and spermidine affect transcription modifying the level of DNA supercoiling...16
1.5.4 Single molecule studies of DNA condensation by polyamines...17

1.6 Type II DNA topoisomerases...17

1.6.1 Structure and function of type II topoisomerases...18
1.6.2 Type II topoisomerases can bend DNA during the enzymatic cycle...19

1.7 Experimental techniques...20

1.7.1 Principle and development of Magnetic Tweezers...20
1.7.2 Principle and application of EMSA...22

Chapter 2 Material and Methods...23

2.1 Magnetic Tweezers instrumentation...24

2.1.1 Hardware of Magnetic Tweezers Microscope...24
2.1.2 software of Magnetic Tweezers Microscope: controller interfaces...26
2.1.3 User manual of the software of Magnetic Tweezers Microscope...28

2.2 Design, produce and label DNA fragments...31

2.2.1 DNA fragments for Magnetic Tweezers experiments...31
2.2.2 DNA fragments for EMSA...32

2.3 Visualize and quantify DNA and protein bands...33

2.3.1 Agarose gel electrophoresis...33
2.3.2 Non-denaturing (native) polyacrylamide gel electrophoresis...34
2.3.3 Use spectrophotometer to measure DNA concentration...35

2.4 Flow chamber design and preparation...37
2.5 Experimental procedures...39

2.5.1 Selecting a full length, dsDNA tether in Magnetic Tweezers...39
2.5.2 Polyamines affect DNA supercoilings...40
2.5.3 Unwinding and rewinding normal DNA and DAP DNA...41
2.5.4 DNA and type II topoisomerases interactions...42

Chapter 3 Effects of polyamines on DNA supercoiling...44

3.1 Outline...45
3.2 Experimental results and analysis...45

3.2.1 Spermine and Spermidine stabilize the right-handed B-DNA...49
3.2.2 Spermine and Spermidine promote plectoneme formation...51
3.2.3 Spermine and Spermidine shrink plectonemic supercoils...52

3.3 Calculations of the plectoneme condesation...53
3.4 Discussion...58

Chapter 4 Behavior of DAP DNA under tension and torsion...61

4.1 Outline...62
4.2 Chemical structure and properties of DAP DNA...62
4.3 DAP substituted DNA is stiffer...64
4.4 Effect of H bonds on the transition from right-handed to left-handed dsDNA...67

4.4.1 Structure transition of DNA under extensive untwisting at high tension...67
4.4.2 Effect of H bonds on right hand to left hand transition...70
4.4.3 Dynamics of left hand to right hand transition...73

Chapter 5 DNA stiffness affects Type II topoisomerases-DNA interaction and activity...75

5.1 Outline...76
5.2 DAP DNA affects the binding of E. coli gyrase to DNA...76

5.2.1 The binding affinity of E. coli gyrase was reduced on DAP DNA...76
5.2.2 The wrapping ability of E. coli gyrase was reduced for DAP DNA...78

5.3 The activity of E. coil gyrase on supercoiled DAP and normal DNA...81
5.4 The activity of recombinant human topoII alpha on supercoiled DAP and normal DNA...86
5.5 Conclusion...89

References...91

List of Figures
Chapter 1 Introduction

Figure 1.1 GC and AT base pairing and polymorphism of DNA (A, B and Z DNA)...5
Figure 1.2 Magnetic Tweezers setup (left) and DNA behavior under tension and torsion...7
Figure 1.3 Force-torque (a) and force-σ (b) phase diagram of DNA...9
Figure 1.4 Schematic illustrations of the origins of DNA stiffness...12
Figure 1.5 Schematic structure of the two polyamines...14
Figure 1.6 Type II topoisomerases structure and mechanism...18

Chapter 2 Material and Methods

Figure 2.1 Magnetic Tweezers Microscope instrumentation...24
Figure 2.2 Schematic structure of the magnets...25
Figure 2.3 Graphic interface of the Magnetic Tweezers Microscope software...27
Figure 2.4 DNA and protein visualized in agarose and polyacrylaimde gels...34
Figure 2.5 Spectrum of Cy5-labeled DNA...36
Figure 2.6 Two designs for flow chambers...38

Chapter 3 Effects of polyamines on DNA supercoiling

Figure 3.1 DNA extension vs. supercoiling density at different concentrations of spermine (left column) and spermidine (right column)...46
Figure 3.2 A qualitative sketch showing free energy competition between the plectonemic and stretched states of DNA in extension versus twisting experiments...48
Figure 3.3 Effect of polyamines concentration on the slope of hat curve for negative supercoiling...50
Figure 3.4 Effect of polyamine concentration at 1 pN and 0.6 pN on the observed transition points...52
Figure 3.5 Slopes of the positive supercoiling part of the hat curve...53
Figure 3.6 Persistence length (bending stiffness) vs. polyamine concentration...56
Figure 3.7 Writhe per helical turn in the plectonemic phase as a function of polyamine concentration...56
Figure 3.8 Radius of the plectoneme as a function of polyamine concentration...57

Chapter 4 Behavior of DAP DNA under tension and torsion

Figure 4.1 illustrations of A:T, G:C and DAP:T base pair...63
Figure 4.2 Normal DNA and DAP DNA behavior under tension and torsion...65
Figure 4.3 Buckling transition for normal DNA (a) and DAP DNA (b) at 1 pN...66
Figure 4.4 Extension vs. rotation turns data at different force for a single DNA molecule...68
Figure 4.5 Underwound single normal DNA (a) and DAP DNA (b) molecule at high force...70
Figure 4.6 Dynamic traces of left hand to right hand transition...73
Figure 4.7 Schematic illustration of DNA extension drop and recovery during rewinding...74

Chapter 5 DNA stiffness affects Type II topoisomerases-DNA interaction and activity

Figure 5.1 EMSA of gyrase binding to normal and DAP DNA...77
Figure 5.2 Schematic illustration of gyrase wrapping detected with Magnetic Tweezers...79
Figure 5.3 Gyrase wrapping assay on normal DNA and DAP DNA...80
Figure 5.4 Schematic illustration of procedure to monitor supercoiling relaxation with Magnetic Tweezers...82
Figure 5.5 Activity of gyrase on normal and DAP DNA...83
Figure 5.6 Mean pause time of gyrase at different ATP concentration for normal and DAP DNA...85
Figure 5.7 Activity of human topoisomerase II alpha on normal and DAP DNA...87
Figure 5.8 Mean pause time of human topo II alpha at different ATP concentration for normal and DAPDNA...89

About this Dissertation

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School	Laney Graduate School
Department	Physics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biophysics, General
Palavra-chave	Polyamines DAN elasticity Topoisomerases Magnetic Tweezers
Committee Chair / Thesis Advisor	Finzi, Laura, Emory University Dunlap, David, Emory University
Committee Members	Weeks, Eric, Emory University Warncke, Kurt, Emory University Family, Fereydoon, Emory University Rasnik, Ivan, Emory University

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