A Single Molecule Study of Two Bacteriophage Epigenetic Switches Open Access
Wang, Haowei (2011)
Published
Abstract
Epigenetic switches allow organisms to evolve into different
states by activating/repressing different sets of genes without
mutations of the underlying DNA sequence. The study of epigenetic
switches is very important to understand the mechanism of human
development, the origin of cancer, mental illness and fundamental
processes such as gene regulation.
The coliphage λ epigenetic switch, which allows switching from
lysogeny to lysis, has been studied for more than 50 years as a
paradigm, and has recently received renewed attention. Atomic force
microscopy (AFM) was used here to show that the λ repressor
oligomerizes on DNA, primarily as a dodecamer, to secure a DNA
loop, which is the basis of the λ switch. This study also
provides support for the idea that specifically bound repressor
stabilizes adjacent, non-specifically bound repressor molecules,
which confers robustness to the switch.
186 is a member of a different coliphage family. One of the major
differences between the two coliphage families is that lambda
phages can be induced to switch from the lysogenic to the lytic
state by UV radiation, but most coliphages of P2 family, to which
186 belongs, cannot. Interaction between coliphage 186 repressor
and DNA is characterized by AFM and tethered particle microscopy
(TPM). To expedite analysis of the AFM data, MatLab codes were
written to automate the laborious, manual tracing procedures. The
programs automatically recognize DNA segments and protein particles
in an image, in order to measure the DNA length and position of
bound particles as well as their height, diameter and volume.
Application of these algorithms greatly improved the efficiency of
AFM analysis. It was showed that 186 CI dimers form heptameric
wheels, which induce DNA wrapping and different kinds of DNA
looping producing various conformations of nucleoprotein complexes.
Information about the dynamics of DNA wrapping and looping on 186
CI particles was also obtained by TPM.
Table of Contents
Table of Contents
Chapter 1
Introduction
................................................................................................1
§1.1 Epigenetic switches
...............................................................................1
§1.2 Prophage and the λ epigenetic switch
.......................................................3
§1.3 The 186 prophage
.................................................................................6
§1.3.1 Transcriptional interference
..................................................................6
§1.3.2 Coliphage 186
....................................................................................8
§1.3.3 Chromatin and DNA wrapping
...............................................................10
Chapter 2 AFM Studies of λ Repressor Oligomers Securing
DNA Loops
§2.1 Background
.........................................................................................12
§2.2 Material and method
.............................................................................15
§2.2.1 Material
...........................................................................................15
§2.2.2 DNA contour length on mica surface
......................................................17
§2.3 Result and discussion
............................................................................19
§2.3.1 DNA contour length measured by AFM
...................................................19
§2.3.2 Specific binding to operator sites
..........................................................21
§2.3.3 Weak affinity for the OR3 operator site
..................................................23
§2.3.4 Multiple operators may recruit dimers
.....................................................24
§2.3.5 Loop equilibrium
.................................................................................26
§2.3.6 Volume calibration
..............................................................................27
§2.3.7 Loop closures are prevalently dodecamers
..............................................30
§2.3.8 Alternative loop closures
.....................................................................33
§2.3.9 Conclusion
........................................................................................35
Chapter 3 AFM and TPM Studies of DNA wrapping and looping of
phage 186
§3.1 Background and introduction
...................................................................37
§3.2 Material and method
..............................................................................39
§3.2.1 AFM sample preparation
.......................................................................39
§3.2.2 TPM sample preparation
.......................................................................40
§3.2.3 Measurement of wheel diameter
............................................................41
§3.3 Result and discussion
.............................................................................45
§3.3.1 Conformation of basic model
.................................................................45
§3.3.1.1 The 186 repressor and its assembling
...................................................45
§3.3.1.2 CI regulated mechanism
....................................................................50
§3.3.2 Pseudo site on FL
...............................................................................55
§3.3.3 Asymmetric DNA wrapping on pR region
...................................................57
§3.3.4 DNA wrapping/unwrapping
....................................................................58
§3.3.5 TPM study of DNA wrapping and looping
..................................................58
§3.3.6 DNA looping
.......................................................................................62
§3.3.7 Other CI binding forms and non-specific binding
........................................70
Chapter 4 Automated DNA segmentation and protein recognition
from AFM images
§4.1
Background.…………………………………………....................................……….……………71
§4.2 Method and
algorithm……………………………………………………..................................73
§4.2.1
Filtering…………………………………………………………........................................…..73
§4.2.2 Threshold and
segmentation……………………………………….................................74
§4.2.3 Thinning and selection of DNA
skeleton…………………………..............................77
§4.2.4 DNA length estimation
………………………..................................………………...….80
§4.3 Application and
programming………………………….............................………………..…82
§4.3.1 DNA tracing
…………………………………………..….......................................………..82
§4.3.2 Masking and interactive modifying
…………..…………...........................…………...83
§4.3.3 DNA contour length
measuring……………………..………………...............................87
§4.3.4 Automatic particle analysis on DNA or surface
…..………….......................………90
§4.3.5 Auto-analysis of DNA protein interaction
……………...…………….........................94
§4.3.6 Data
conversion…………………………………………………........................................
96
§4.4 Discussion
………………………………………….........................................…………………96
References
………………………………………………...……………………..................................…97
Apendices
…………………......................................………………………………………………….110
Appendix A AFM studies of λ repressor oligomers securing DNA
loops .....................111
Appendix B The lambda bacteriophage epigenetic switch: new insight
from
single-molecule microscopy
................……………………………………….....................…...119
Appendix C A missing link between transcription factors and
nucleosomes:
the bacteriophage 186 CI repressor wraps and loops DNA
……….............................139
Appendix D Matlab code for protein particle measurement and
simulation ................148
Appendix E Matlab code for automated tracing and DNA protein
analysis programs
155
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