An Investigation of the Supercoiling Activities of E. coli and Salmonella Gyrases Using Magnetic Tweezers Público
Fountain, Chandler (2012)
Abstract
Abstract
An Investigation of the Supercoiling Activities of E. coli
and Salmonella Gyrases Using Magnetic Tweezers
Eukaryotic and prokaryotic organisms possess members of the topoisomerase family of enzymes in order to regulate the level of DNA supercoiling throughout their life cycle. DNA supercoiling is regulated in a dynamic fashion and is known to play a role in the expression of genes, site-specific recombination, DNA condensation, and the segregation of chromosomes. K. Champion and N. P. Higgins showed that replacing the gyrB subunit of gyrase in E. coli with that of Salmonella resulted in death of the bacteria. The goal of this study was to explore the differences between E. coli gyrase and Salmonella gyrase using single molecule experiments. Magnetic tweezers were used to investigate the relative relaxation rates of (+) supercoils, frequency of pausing, duration of pausing, and relative introduction rates of (-) supercoils. It was found that Salmonella gyrase relaxed (+) supercoils at a slightly slower rate than E. coli gyrase. Furthermore, Salmonella gyrase was observed to pause for smaller durations, but at a higher frequency than E. coli gyrase. It was also found that Salmonella gyrase relaxed (-) supercoils at a higher rate than E. coli gyrase. Additionally, Salmonella gyrase introduced a slightly larger number of supercoils in each supercoiling event.
Table of Contents
TABLE OF CONTENTS
1.Introduction...1
1.1Motivation...1
1.2DNA and its interaction with protein...2
1.3DNA Supercoiling - Terminology and importance...3
1.4DNA Gyrase...5
1.5Magnetic Tweezers...8
2.Materials and Methods...11
2.1DNA Construct...11
2.1.1 Main Fragment (ApaI-NgoMIV
Digest)...11
2.1.2 Main Fragment (XmaI-PciI Digest)...14
2.1.3 Tail Fragments (for the ApaI-NgoMIV Main Fragment)...15
2.1.4 Tail Fragments (for the XmaI-PciI Main Fragment)...17
2.1.5 Ligation of Main Fragment and Tails...17
2.2 Magnetic Tweezers Chamber
Preparation...18
2.3 Magnetic Tweezers Experiment...21
2.3.1 Chapeau curves for extension vs.
turns...24
2.3.2 Force vs. extension curves for force values..25
2.3.3 Difference in two experiment styles...26
2.3.4 (+) Supercoil realaxation experiment...26
2.3.5 Force modulation experiment...30
3. Results...32
3.1 (+) Supercoil relaxation results...32
3.2 Force modulated activity of gyrase...36
4. Discussion...39
5. Conclusion...46
Appendix...48
A1 Plot Trace File...48
A2 Rate Calibration...48
Bibliography...49
List of Figures
Figure 1.1: (A) Gene map with primary domains in different
colors for three representative type IIa Topoisomerases (b) S.
Cerevisiae gyrase structure with colored domains (E.
coli gyrase in inset). The C-terminal domain is not shown.
[11]...6
Figure 1.2: (A) model of Type IIA Topoisomerase (B) Reaction
mechanism: G-segment binds to dna gate (step 1). T-segment captured
after binding of ATP (step 2). hydrolysis of ATP and release of
Pi causes DNA gate to open for strand passage (step 3).
Remaining hydrolysis products released while G-segment is
religated, T-segment is released, and ATP gate is reset (step 4).
[13]...7
Figure 1.3: Magnetic tweezer setup...9
Figure 1.4: Manipulation of tension and twist on a DNA molecule
using magnetic tweezers...9
Figure 2.1: A Plasmid map of pUC18-nuB104 showing the primers used
to amplify the ApaI-NgoMIV main fragment...12
Figure 2.2: Restriction digest map of the plasmid pUC18-nuB104 used
to produce the XmaI-PciI main fragment...14
Figure 2.3: Magnetic tweezers chamber...19
Figure 2.4: Magnetic tweezers instrument...22
Figure 2.5: Low force chapeau curve...24
Figure 2.6: High force chapeau curve...25
Figure 2.7: Example trace for (+) relaxation...29
Figure 3.1: Relaxation rates for E. coli gyrase vs.
Salmonella gyrase. (a) Raw data points with mean and
standard deviation bars. (b) Box and whisker plots showing sample
minimum, lower quartile, median, upper quartile, and sample
maximum...34
Figure 3.2: Number of pauses observed per relaxation
event...35
Figure 3.3: Cumulative distribution of the duration of pauses for
(+) supercoil relaxation by E. coli and Salmonella
gyrase...36
Figure 3.4: Rates of introduction of (-) supercoils by E.
coli and Salmonella gyrase. (a) Raw data points with
mean and standard deviation bars. (b) Box and whisker plots showing
sample minimum, lower quartile, median, upper quartile, and sample
maximum...37
Figure 3.5: Number of (-) turns introduced by E. coli and
Salmonella gyrases in each complete supercoiling event. (a)
Raw data points with mean and standard deviation bars. (b) Box and
whisker plots showing sample minimum, lower quartile, median, upper
quartile, and sample maximum...38
Figure 4.1: Amino acid sequence alignment of gyrA. Light
gray represents dissimilarities, red represents negatively charged
residues, and green
represents positively charged residues...42
Figure 4.2: Amino acid sequence alignment of gyrB. Light
gray represents dissimilarities, red represents negatively charged
residues, and green
represents positively charged residues...43
List of Tables
Table 2.1: PCR protocol for main fragment...13
Table 2.2: Main fragment double digestion...14
Table 2.3: PCR protocol for biotin labeled tail...16
Table 2.4: PCR protocol for digoxigenin labeled tail...16
Table 2.5: Main fragment and tail ligation protocol...18
Table 2.6: Buffers and chemicals for MT chamber
preparation...18
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