Probing the Causes of Disease: Single Molecule Studies of Dancing DNA

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Probing the Causes of Disease Single Molecule
Studies of Dancing DNA
For all Please pick up a rubber band from the
back of the room and keep it Clean! For those in
the main lecture hall Please pick up a response
keypad in the back of the room

• Seth Blumberg
• University of Michigan
• Physics Department / Medical School
• www.umich.edu/sblumber

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Probing the Causes of Disease Single Molecule
Studies of Dancing DNA

• Seth Blumberg
• University of Michigan
• Physics Department / Medical School
• www.umich.edu/sblumber

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50 Years of DNA
http//web.math.fsu.edu
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50 Years of DNA
www.simr.org.uk
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50 Years of DNA
"It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material. Watson Crick
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Quiz Time!

• What happens when I heat steel and
• rubber?
• They both contract
• They both expand
• Steel contracts, rubber expands
• Steel expands, rubber contracts

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Material Properties
Metal
Rubber
polymer.bu.edu/trunfio
http//www.cmmp.ucl.ac.uk
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Dancing DNA
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Disorder is Favorable
Improbable
Probable
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To create order,work must be done
Pressure
Tension
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Stretching DNA
Carlos Bustamante Research Group, UC Berkeley
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Force-Extension Relation for DNA
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Quiz Time!

• What happens when I twist this cylinder by
• three clockwise revolutions and then by
• three counter-clockwise revolutions?
• The dye will reform a straight line
• The dye will swirl around in in a cyclic pattern
• The dye will be well mixed
• The dye will change color

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Low Reynolds Number Laminar Flow
V velocity R characteristic distance r
density h viscosity For bacteria, Re .00003
www.zoology.ubc.ca/courses/bio332
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Protein-DNA Interactions

• The magic of moleclar biology

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Introduction to DNA Transcription Translation
DNA
mRNA
Protein
water.usgs.gov/nrp/proj.bib/microbiology
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Protein-Mediated DNA Looping

• Green Protein
• Red DNA binding sites
• Blue DNA loop (rigid rod model)

Theoretical Biophysics Group Beckman Institute
University of Illinois
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Mechanical Effects on Loop Formation
Linear
Torsional
Linear Tension
Torsional Tension
Loop Formation Rates
Operator separation
Helical Operator Alignment
TranscriptionalRepression
Capture Volume
Protein Flexibility
DNA Stiffness
Supercoiling
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Quiz Time!

• Which Equation best describes our results?

CENSORED
CENSORED
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Our Experimental Mission

• Can we turn looping on and off by pulling on the
  DNA substrate?

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Our Experimental Mission

• Can we turn transcription on and off by pulling
  on the DNA substrate?

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Optical Tweezers
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Quiz Time!

• How do optical tweezers work?
• Focused light gets bent by the target and this
  pulls target into place
• Light ionizes the target and charge repulsion
  creates a force
• High intensity light results in the creation of a
  positron and electron which then reposition the
  target
• Two photons wrap around the target, thus
  squeezing it back in place

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Tools Optical Tweezers

• Ray optics approximation
• Microsphere acts as a lens and changes the
  momentum of passing photons
• Conservation of momentum requires restoring force
  on microsphere

Microscope objective
Focal plane
Polystyrene microsphere
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Tying a Knot into a DNA Molecule
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Fluorescence
Color change is caused by photons with different
energies
www.pharm.uky.edu
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Total Internal Reflection Fluorescence Microscopy

• DNA construct is labeled at one end with a
  fluorescent marker
• Construct is tethered to a glass surface
• Evanescent wave excites fluorophores
• Measure loop formation rates by monitoring
  intensity of the emitted light

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Data Acquisition
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Force-free Loop Formation in DNA
Looping Protein Added
Motion (nanometers)
Loops ?
Time (seconds)
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DNA Acquisition
A delicate process .
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Lab Tour
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Thanks to Ari Gajraj
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Gene Regulation and Cancer
www.nobel.se/medicine/laureates/2001
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Acknowledgements

• Saturday Morning Physics Support
• Dr. M. Lois Tiffany and other donors
• Prof. David Gerdes Carol Rabuck
• Lecture Preparation
• Demolab
• Warren Smith, Harminder Sandhu, Mark Kennedy
• Slide Preparation
• Prof. Chris Meiners
• Ben Leisfeld, Ari Gajraj Raj Nambiar

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My Last slide

• Physics plays an important role in biology,
  medicine the well-being of society
• Physics is interesting fun!
• You do not have to be a genius to understand the
  way things work
• But learning is a lifelong experience
• Ive learned a lot from you Thanks for your
  support!

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Probing the Causes of Disease Single Molecule
Studies of Dancing DNA

• Seth Blumberg
• University of Michigan
• Physics Department / Medical School
• www.umich.edu/sblumber

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Microscope Optics
www.micro.magnet.fsu.edu
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Force Measurement by Video Analysis
Force-extension curve of a single l-phage DNA
molecule
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Theory Results
Normalized Loop Formation Rate
Tension (Femtonewtons)
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Protein-DNA Interactions
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The lac Operon