DNA elasticity and effects on Type II topoisomerases 公开
Shao, Qing (2011)
Published
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
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