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DNA: Structure, Dynamics and Recognition

DNA: Structure, Dynamics and Recognition. L5: Recognizing DNA. Les Houches 2004. DNA-BINDING LIGANDS. Covalent Intercalators Groove binding Coordination. DNA-binding ligand famillies. Nitrogen mustard. Mitomycin. Anthramycin. Acetylaminofluorene. Covalent Binding Ligands.

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DNA: Structure, Dynamics and Recognition

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  1. DNA: Structure, Dynamics and Recognition L5: Recognizing DNA Les Houches 2004

  2. DNA-BINDING LIGANDS

  3. Covalent • Intercalators • Groove binding • Coordination DNA-binding ligand famillies

  4. Nitrogen mustard Mitomycin Anthramycin Acetylaminofluorene Covalent Binding Ligands Psoralen Sibiromycin

  5. - binds to a guanine amino group in the minor groove Anthramycin

  6. 9-Aminoacridine Proflavine Intercalators Ethidium Acridine orange

  7. Proflavine

  8. - leukemia therapy Daunomycin

  9. - a bisintercalator - inhibits DNA repair Ditercalinium

  10. Distamycin Netropsin Hoechst 33258 Groove binding ligands

  11. - minor groove - AT specific Netropsin complex with DNA

  12. Linkers Rings Ends DNA-binding ligand "kits"

  13. NMR structure of 2:1 complex of distamycin

  14. Cyclic H-pin Hairpin Linked polyamide ligands

  15. ImImPyPy-b-Dp Polyamide 2-1DNA complex

  16. Inhibition of gene transcription using polyamides

  17. NH3 Cl Pt NH3 Cl - shielding from excision repair Coordination ligands: cisPt http://web.mit.edu/chemistry/lippardlab/lab.html

  18. DNA-BINDING PROTEINS

  19. Transcription factors (CAP, 434, Cro, …) Architectural (histones, HMG, …) Enzymatic (nucleases, methyltransferases, …) Replication factors Repair enzymes DNA binding families

  20. -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

  21. 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, …) "Direct" versus "Indirect"

  22. Switching genes on and off in phages

  23. Induce lysogenic or lytic phase

  24. Transcriptosome

  25. DNA bound to TBP+TF11A+TF11B

  26. DNA Binding Domain (DBD) TransActivation Domain (TAD) or Repression Domain Oligomerization Domain : nuclear localization signal : Ubiquitin-mediated degradation signal : flexible linker NH3 COO + - Transcription factors often have a modular structure

  27. RECOGNITION MECHANISMS

  28. 1 4 11 4.2x106 2 16 12 1.7x107 3 64 13 6.7x107 4 256 14 2.7x108 5 1024 15 1.1x109 6 4096 7 16384 8 65536 9 262144 10 1.0x106 TAFS Drugs Nucleases Coding with four bases: A, C, G, T

  29. 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 Energetics

  30. A T T A G C C G Recognizing base pairs

  31. Recognition: major versus minor groove

  32. Recognition sites in the major groove

  33. HELIX-TURN-HELIX MOTIF

  34. 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 HTH characteristics

  35. 8-4-8 aa “Recognition” helicies Dimerisationdomain DNA binding domain (DBD) 434 repressor

  36. Cro l repressor Different dimerisation domains

  37. DNA binding: 434 repressor, HTH motif

  38. DNA binding: 434 Cro, HTH motif

  39. HTH RMSD = 0.6 Å Position of the recognition helices

  40. DBD ATP cofactor Activation domain Dimerisation domain CAP dimer

  41. 50° of curvature CAP dimer bound to DNA

  42. trp repressor – cofactor binding control

  43. Recognizes: ACTAGTTAACTAGT No direct H-bonds to bases Several water bridged bonds trp repressor

  44. Engineering HTH protein specificity

  45. Long recognition helix Homeodomain l repressor Trp repressor Monomeric Dimeric Eukaryote ← Prokaryote

  46. Engrailed homeodomain

  47. ZINC FINGERS

  48. ~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- C2/H2 zinc finger composition

  49. b-hairpin / turn / a-helix ≈3 bp per finger 2xCys + 2xHis Zif268 – 3 fingers bound to 10 bp

  50. Homo- or heterodimers Nuclear receptors (progesterone, estrogen, …) C4 consensus: -Cys-X2-Cys-X13-Cys-X2-Cys-X15-Cys-X5-Cys-X9-Cys-X2-Cys- C4 zinc finger composition

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