Isaac Wong

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Isaac Wong wongis_at_uky.edu or iwong3_at_email.uky.edu
Combs 124C (Office), 118-119 (Labs) 323-1216 (Lab)
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Specific Site Recognition needle in a haystack

• EcoR1 Restriction Site GAATTC
• Statistical frequency 1 in 46 or 1/4096
• For every statistical GAATTC sites, there are
  4096 nonspecific sites.
• If nonspecific binding is 4096 times weaker than
  specific binding, then GAATTC site statistically
  cannot be found

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• Sequence Specific Interactions
• Affinity vs. Specificity
• Beyond Lock and Key type recognition
• Adaptive Binding
• Indirect Readout
• Kinetics of target search
• Taking advantage of nonspecific sites
• Sliding
• Intersegmental transfer

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Affinity vs Specificity

• Affinity Keq or KD or ?G0
• Specificity Relative Affinity / Difference in
  ?G0
• Kspecific/Knonspecific
• ??G0

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Site-Specific Binding Includes a NON-specific
affinity component

• Any charge-charge interaction is NON-specific
• Major component of non-specific binding
• Any Binding to common features of DNA is
  NON-specific
• May show significant sequence effects or
  preferences

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Simple Model of Base-Specific Recognition

• Extension of Watson-Crick Code

Me
X
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(No Transcript)
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• Selectivity Beyond energies of H-bonds.
• Selectivity Beyond Binding energies
• Incorrect sequences are bound with moderate
  affinity BUT these are not catalytically
  productive
• Induced Fit Selectivity
• Counterion Release
• Correct 8
• Incorrect 6

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Induced Fit Selectivity
rate
Binding
Dramatic Effects beyond H-bonding
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• Interference Footprints
• Map positions where ethylation lowers/interferes
  with binding affinity
• Shows positions of strong, specific contacts
  between protein and DNA
• Gives complementary info from footprint protection

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Unbound DNA
Bound DNA



Cleave at ethylation site
Interference sites
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• Interference Footprints
• Positions where ethylation lowers binding
  affinity
• Shows positions of strong, specific contacts
  between protein and DNA

• Canonical Sequences shows 3 clamps (symmetric)
• Contact changes AWAY from the site of
  substitution
• Changing seq in ½-site lowers contacts on the
  incorrect half-site but INCREASES contacts in the
  correct ½-site!
• New clamp at the scissile phosphate
• Isosteric substitution retains symmetry.

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• Adaptive Binding
• Static Model of a recognition code is too
  simplistic
• Dynamic adaptation of binding contacts
• Dramatic changes in ALL contacts resulting from
  failure to make 1 contact (including loss of
  phosphate contacts)
• Even single-H-bond changes can lead to minimum
  rearrangement/adaptation
• Adaptation leads to formation of nonproductive
  complexes induced fit.

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• Induced-Fit
• Adaptation leads to formation of nonproductive
  complexes induced fit.

Eactive-DNAcorrect
E DNA
E-DNA
Einactive-DNAincorrect
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Indirect Readout

• Is there sequence information in the phosphate
  backbone?
• Can a protein scan the DNA OUTSIDE and detect
  sequences INSIDE?

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Clamps correspond to kinks in the backbone
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X-ray Structure of 5CGCGAATTCGCG

• Average dimensions agree with average
  Watson-Crick B-form DNA parameters
• Average 9.65 bp/turn, 3.33 Å/bp
• Large local deviations
• Twist angle ? of each bp measures the local
  bp/turn
• C G C G A A T T C G C G
• 9.4 9.1 10.8 9.6 9.6 11.2 10.0
  8.7 11.1 8.0 9.7

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Sequence-Sensitive Dynamics

• Hypothesis sequences affect dynamics of the
  backbone.
• Extreme examples
• poly-A tracts are stiff and bend DNA
• Some CGrich sequences prone to Z-DNA
• More subtle examples
• Many specific complexes are kinked
• Are proteins exploiting natural propensities of
  specific sequences to kink in a specific manner?

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hUDG Co-Crystal
DNA kinked by 3 Ser pinches
Parikh, S. S., Mol, C. D., Slupphaug, G.,
Bharati, S., Krokan, H. E., and Tainer, J. A.
(1998) Embo J 17, 5214-5226.
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Pull Before Push
U-Flipping
Fluorescence (Volts)
L-Insertion
90 nM pseudo-1st order component
Time (sec)
30 mM Tris-HCl pH 7.4, 1 mM EDTA, 5 (w/v)
glycerol, 25?C
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Pull-Push Mechanism

• Base-Flipping Prior to Leu-Insertion

2. Dynamic and Reversible Base-Flipping
3. Isomerization (Leu-Insertion)
a. Stabilization (door stop) b. Trigger for
Excision
Does it flip every base to check for U? Or
alternatively, indirect readout hypothesis Does
Serine pinches induces Uracil extrusion
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Using non-specific DNA to facilitate search

• Diffusion control 109 M-1s-1
• Observed 2nd-order on rate 18x faster
• DNA substrate 21-mer with 1 U, 20x more
  nonspecific bases than U.
• Facilitated Diffusion Hypothesis binding to ANY
  base speeds up target search

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Iso-energetic Rail

• Protein movement displaces c-ions ahead but
  c-ions re-condenses behind

K
K
K
K
K
K
K
K






K
K
K
K
K
K
K
K
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Search in 1-D instead of 3-D

• Large excess of nonspecific sites actually helps
  us
• Easy to find ANY DNA-segment
• follow the river to find the sea

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Modes of facilitated diffusion

• Sliding (limited to 2000 bp)
• Hopping microscopic dissociation re-association
  (transfer to nearby sites)
• Intersegmental transfer DNA looping facilitated
  transfer to distal sites

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Rate of dissociation of specific complex is DNA
length dependent
E DNon E-DNon EDSp
Nonspecific DNA catalyzes dissociation and
association
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Long and Short DNA each contain 1 EcoR1
site Mixed at t0 At different times after
mixing, MgCl2 added with trap DNA EcoR1 finds
sites on Long DNA faster than Short Kinetic
Effect at longer times, equilibrium is reached