DNA: Structure, Dynamics and Recognition

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DNA Structure, Dynamics and Recognition
L5 Recognizing DNA
Les Houches 2004
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DNA-BINDING LIGANDS
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DNA-binding ligand famillies

• Covalent
• Intercalators
• Groove binding
• Coordination

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Covalent Binding Ligands
Nitrogen mustard
Mitomycin
Anthramycin
Acetylaminofluorene
Psoralen
Sibiromycin
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Anthramycin
- binds to a guanine amino group in the minor
groove
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Intercalators
9-Aminoacridine
Proflavine
Ethidium
Acridine orange
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Proflavine
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Daunomycin
- leukemia therapy
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Ditercalinium
- a bisintercalator - inhibits DNA repair
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Groove binding ligands
Distamycin
Netropsin
Hoechst 33258
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Netropsin complex with DNA
- minor groove - AT specific
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DNA-binding ligand "kits"
Linkers
Rings
Ends
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NMR structure of 21 complex of distamycin
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Linked polyamide ligands
Cyclic
H-pin
Hairpin
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Polyamide 2-1DNA complex
ImImPyPy-b-Dp
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Inhibition of gene transcription using polyamides
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Cl
NH3
Pt
Cl
NH3
- shielding from excision repair
Coordination ligands cisPt
http//web.mit.edu/chemistry/lippardlab/lab.html
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DNA-BINDING PROTEINS
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DNA binding families

• Transcription factors (CAP, 434, Cro, )
• Architectural (histones, HMG, )
• Enzymatic (nucleases, methyltransferases, )
• Replication factors
• Repair enzymes

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• ?-helices generally interact with the major
  groove of B-DNA (12Å wide), while ?-sheets can
  bind to the minor groove (6Å wide)
• Protein binding to DNA often deforms the double
  helix (bending, untwisting, ...). Base pairing is
  usually not destroyed, but major structural
  changes can occur
• Complexes are stabilized by arginine or lysine
  side chains binding to the DNA phosphate groups
  (salt bridges)

General features
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"Direct" versus "Indirect"

• DIRECT RECOGNITION
• Formation of hydrogen bonds between
• between the amino acid side chains of the
  protein and the DNA bases
• INDIRECT RECOGNITION
• Detection of sequence dependent changes in DNA
  structure or flexibility (bending, twisting,
  groove opening, )

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Switching genes on and off in phages
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Induce lysogenic or lytic phase
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Transcriptosome
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DNA bound to TBPTF11ATF11B
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Transcription factors often have a modular
structure
NH3
COO

-
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RECOGNITION MECHANISMS
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Coding with four bases A, C, G, T
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Energetics
Stabilising factors Hydrogen bonding (base-aa,
backbone-aa)   Partial stacking (aromatic side
chains)   Salt bridges (phos-arg/lys) Ion
release   Solvation entropy      Destabilising
factors DNA / protein deformation   DNA /
protein entropy Solvation enthalpy
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Recognizing base pairs
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Recognition major versus minor groove
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Recognition sites in the major groove
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HELIX-TURN-HELIX MOTIF
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HTH characteristics
Prokaryotes - homodimers - bind to palindromic
sequences - contact DNA through recognition helix
(RH) - orientation of RH can vary significantly
Eukaryotes - monomers - bind to non-palindromic
sequences - orientation of RH less variable
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DNA binding domain (DBD) 434 repressor
8-4-8 aa
Recognition helicies
Dimerisationdomain
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Different dimerisation domains
Cro
l repressor
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DNA binding 434 repressor, HTH motif
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DNA binding 434 Cro, HTH motif
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Position of the recognition helices
HTH RMSD 0.6 Å
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CAP dimer
DBD
ATP cofactor
Activation domain
Dimerisation domain
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CAP dimer bound to DNA
50 of curvature
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trp repressor cofactor binding control
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trp repressor
Recognizes ACTAGTTAACTAGT No direct H-bonds to
bases Several water bridged bonds
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Engineering HTH protein specificity
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Eukaryote ? Prokaryote
Long recognition helix
Homeodomain l repressor Trp
repressor
Monomeric
Dimeric
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Engrailed homeodomain
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ZINC FINGERS
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C2/H2 zinc finger composition
30 aa Largest eukaryote family Monomeric with
repeated fingers - TF111A 9, Sp1 3
C2/H2 consensus
-Tyr/Phe-X-Cys-X2-4-Cys-X-Tyr/Phe-X5-Leu-X2-His-X3
-4-His-
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Zif268 3 fingers bound to 10 bp
b-hairpin / turn / a-helix
3 bp per finger
2xCys 2xHis
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C4 zinc finger composition
Homo- or heterodimers Nuclear receptors
(progesterone, estrogen, )
C4 consensus
-Cys-X2-Cys-X13-Cys-X2-Cys-X15-Cys-X5-Cys-X9-Cys-X
2-Cys-
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Glucocorticoid Receptor
4xCys
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Hormones regulate gene expression
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Gal4 - binuclear zinc cluster
1.5 turns
6xCys
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LEUCINE ZIPPERS
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Leucine zipper hydrophobic stabilisation
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GCN4 (bZIP) basic leucine zipper
½ turn
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Max (b-HLH-Zip) helix-loop-helix
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b-SHEETS
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Met repressor
Anti-parallel b-sheet
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Met repressor - b-sheet in major groove
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IHF-DNA complex
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TATA BOX BINDING PROTEIN
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Tata-box binding protein (TBP)
b-sheet in minor groove
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TBP-DNA complex
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Sex determining Region on chromosome Y
HMG box (3 a-helices) b-sheet in minor groove DNA
bending (70) to initiate transcription,
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A WORD ON PROTEIN INDUCED BENDING
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Bending and transcription
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Nucleosome schematic view
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Nucleosome dinucleotide distributions
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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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ENZYMATIC PROTEINS
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Hha I methyltransferase
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EcoRI endonuclease
Recognises GAATTC
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Action of EcoRI
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Cutting DNA with restriction enzymes
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Crystal Form IV of EcoRV DNA complexHorton
Perona, PNAS, 97, 5729 (2000)
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Conformation change in EcoRV
Apo
Non-specific
Specific