Breathing is not enough

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Breathing is not enough
Krystyna Zakrzewska Laboratoire de Biochimie
Théorique, UPR 9080 Institut de Biologie
Physico-Chimique 13, rue P. et M. Curie, 75005
Paris krystyna_at_ibpc.fr,
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Institut de Biologie Physico-Chimique
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The Central Dogma of molecular biology
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Transcription
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Transcription
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Transcription initiation complex
3
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DNA protein recognition
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Recognition pattern in the grooves
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Direct recognition
Sequence-specific because amino acid side chains
H-bond with DNA base pairs in major
groove. Structural basis well understood.
But
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Double helix ?
Dickerson dodecamer CGCGAATTCGCG
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DNA bending by transcription factors
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Examples of bent complexes
CAP
TBP
IHF
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Indirect recognition
Protein recognizes DNA structure Minor groove
features Role of water molecules DNA
flexibility May be sequence specific Sequence
determines structure
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DNA bending
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Alex Rich asymmetric neutralisation
Mirzabekov and Rich PNAS 76, 1979, 1118
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Jim Maher bending by neutralisation
Strauss and Maher, Science 266, 1994, 1829
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Distribution of Hydrogen Bond Types in
DNA-Protein ComplexesMandel-Gutfeund et al,
J.Mol.Biol.,1995
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Modifying Charges of GCN4
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Catabolite Activator Protein (CAP) DNA Complex
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Methodology

• All-atom representation
• Internal/helicoidal variables (JUMNA)
• AMBER force field
• Continuum solvent e(r), GB
• Normal mode analysis (LIGAND)

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Force field used
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CAP phosphate neutralisation pattern
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CAP binding
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Phosphate Neutralisation Study
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DNA oligomers from the complexes studied
Red dots salt bridges Green dots negative
contacts
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DNA bending in protein DNA complexes-conclusion
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Molecular dynamics simulations
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Molecular dynamics
Time integration of Newton's equation of
motion F ma -dE/dr m dr2/dt2 Taylor
expansion r(t dt) r(t) dt dr(t)/dt dt2/2
d2r(t)/dt2 Fastest movements O(10-15 s)
r
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Periodic boundary conditions
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Equilibration
Temp (K)
Time (ns)
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MD snapshots
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MD time series- sugar phase- groove width
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Hha1 methyltransferaseKlimaauskas et al. Cell
76 (1994) 357
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Enzymatic base chemistry
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Base opening lifetimes
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Minor groove ?? Major groove
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Ion analysis and problems
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Simulation protocol

• 50 ns simulation time
• CCATGCGCTGAC dodecamer,
• the target sequence for HhaI
• cytosine-methyltransferase
• AMBER 6/7, Parm99
• 22 Na ions, 22 K ions
• 5000 TIP3P H2O
• PME
• Integration time 2fs, SHAKE

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Most frequently visited zones
K
Na
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Tight ion binding
Na
K
C C A T G C G
C T G A C G G T A
C G C G A C T
G 1 2 3 4 5 6
7 8 9 10 11 12
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Mtf binding site twistNa-red, K blue
deg
C C A T G C G C T G A C
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Mtf binding site twist_8Na-red, K blue
deg
time (ns)
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Backbone torsion angles
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ag transition
a
g
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a/g transitions in Na dynamics
red g green-a
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Correlation of a/g to Na binding to phosphates
red g green-g
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Acknowledgements
Emmanuel Giudice Raphaël Gurlie Tap Ha
Duong Richard Lavery Péter Varnai
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Recent experimental data
Stacked-Unstacked Equilibrium at the Nick Site of
DNA E. Protozanova, P. Yakovchuk M.D.
Frank-Kamenetskii, JMB to be published
DG stack(5-3) (kcal/mole) TA -0.19 AT
-1.34 TT -1.11 TC -1.43 TG -0.55
GT -1.81 CC -1.44 CT -1.03 CG
-0.91 GC -2.17
DGHB (kcal/mole) AT 0.07 GC -0.60
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Cells are crowded
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Biological time scale
Bond vibrations 1 fs (10-15 s) Sugar
repuckering 1 ps (10-12 s) DNA bending 1
ns (10-9 s) Domain movement 1 ?s (10-6 s) Base
pair opening 1 ms (10-3 s) Transcription 2.5 ms
/ nucleotide Protein synthesis 6.5 ms / amino
acid Protein folding 10 s RNA lifetime 300 s
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JUMNA JUnction Minimization of Nucleic Acids
dihedral pseudorotation helicoidal
10- to 100-fold less variables
R. Lavery et al. Comput. Phys. Comm. 91 (1995)
135-158