1. DNA: Structure, Dynamics and Recognition Les Houches 2004 L2: Introductory DNA biophysics and biology

2. STRUCTURE DETERMINATION

3. X-RAY DIFFRACTION  X-ray ≈ 1 Å ≈ atomic separation  requires crystals  phase problem (homologous structures, or heavy atom doping)

4. Crystallographic resolution - Resolution limit = /2.Sin  max - R-factor =  [|F obs | - |F cal |]/|F obs | (0.15-0.25 implies good agreement) 1.2 Å 2 Å 3 Å

5. Crystal packing effects Doucet et al. Nature 337, 1989, 190

6. Crystallographic curvature DiGabriele et al. PNAS 86, 1989, 1816

7.  Can excite atoms with nuclear spins, 1 H, 13 C, 15 N, 31 P  Relaxation leads to RF emissions which depend on the local environment  1D spectra of macromolecules suffer from overlapping signals NMR SPECTROSCOPY

8. COSY (COrrelation SpectroscopY) - covalently coupled atoms NOESY (Nuclear Overhauser Effect) - through space coupling 2D NMR SPECTRA

9. Sequential Resonance Assignments “Biomolecular NMR Spectroscopy” J.N.S. Evans (1995).

10.  identify residues in contact (>5 Å)  model structure using distance + torsional constraints and known valence geometry  check quality by reconstructing NMR spectrum  a range of structures generally fit the data (accounting for flexibility)  not easy to define resolution  problems of crystallisation are replaced with problems of solubility and size  may need isotopic labelling STRUCTURE FROM NMR DATA

11. OTHER SPECTROSCOPIC TECHNIQUES

12. Absorption Spectroscopy  Simple inexpensive technique  Optical density of sample compared to buffer solution  IR - molecular vibrations,  UV - electronic transitions  Macromolecules give broad spectra formed of many overlapping transitions

13.  More disorder  more absorption (e.g. diamonds)  ds DNA  ss DNA more absorption Absorption Spectroscopy UV

14.  Raman scattering gives acces to vibrations without water peak  can identify percentages of sugar puckers, glycosidic conformations,... Absorption Spectroscopy IR

15. Circular dichroism (CD)  Measures the difference in absorption between left- and right-handed circularly polarized light (ellipticity)  Sensitive to molecular chirality  ms resolution  simple experiments poly(dG-dC).poly(dG-dC) 0.2 M NaCl 3.0 M NaCl Pohl & Jovin J. Mol. Biol. 67, 1972, 675

16. Neutron scattering spectroscopy  Access to dynamics in ps  ns timescale  Vibrational density of states  Needs a lot of material and a reactor  H/D exchange for selective studies Sokolov et al. J. Biol. Phys. 27, 2001, 313 DNA/D 2 O Slow relaxation in solvent > 210 K

17. FRET - fluorescent resonance energy transfer  varies as r -6  detection ≈ 5-10 Å

18. Still to come.... Hydrogen exchange Single molecule experiments HN3 imino proton S S

19. STABILIZATION OF THE DOUBLE HELIX

20. Biological energy scale Chemical bondsC-H105kcal.mol -1 C=C172 Ionic hydrationNa + -93 Ca 2+ -373 Hydrogen bondsO…H-5(in vacuum) Protein folding~ 2-10(in solution) Protein-DNA binding~ 5-20 (~200 Å 2 contact)

21. Helix  Coil

22. UV melting curve for a bacterial DNA sample T m = T at which 50% of DNA is melted

23. T m increases with GC content

24. DNA energetics - I Stabilising factors :Base pairing (hydrogen bonds) Base stacking (hydrophobic) Ion binding (electrostatics) Solvation entropy Destabilising factors :Phosphate repulsion (electrostatics) Solvation enthalpy (electrostatics/ LJ) DNA strand entropy

25. Pairing in vacuum : Yanson, et. al. 18 (1979) 1149 Bases  H CG-21.0 AU-14.5 Pairing in chloroform : Kyoguku et al. BBA 179 (1969) 10 Bases  H CG-10.0  -11.5 AU-6.2 AA-4.0 Stacking in water (stronger than pairing) : T’so 1974 Bases  H AA-6.5 UU-2.7 TT-2.4 Base pairing and stacking

26. Separating a GC basepair in water Stofer et al. J. Am. Chem. Soc. 121, 1999, 9503

27. DNA energetics - II Breslauer empirical equation for ss  ds : (Biochemistry 83, 3748, 1986)  Gp = (  g i +  g sym ) +  k  g k Stack  g k GG-3.1 AA-1.9 G G A A T T C CGA-1.6 C C T T A A G GCG-3.6 GC-3.1  Gp = (5.0 + 0.4) - 2 x 3.1 TG-1.9 - 2 x 1.9 - 2 x 1.6 - 1.5AG-1.6 AT-1.5 GT-1.3  Gp = -9.3 Kcal/molTA-0.9  Gexp = -9.4 Kcal/mol

28. DNA energetics -III s1 :CGCATGAGTACGC Vesnaver and Breslauer PNAS 88, 3569, 1991 s2 :GCGTACTCATGCG dsss(h)ss(r) Kcal/molds  ss(r)s1(h  r)s2(h  r)Sum  G20.00.51.41.9  H117.029.127.256.3 T  S97.028.625.854.4

30. DNA TRANSCRIPTION

31. Biological time scale Bond vibrations1 fs(10 -15 s) Sugar repuckering1 ps(10 -12 s) DNA bending 1 ns(10 -9 s) Domain movement1  s(10 -6 s) Base pair opening1 ms(10 -3 s) Transcription20 ms / nucleotide Replication 1 ms / nucleotide Protein synthesis6.5 ms / amino acid Protein folding~ 10 s

32. CENTRAL DOGMA DNARNA PROTEIN DNA polymerase RNA polymerase Reverse Transcriptase RNA replicase TRANSCRIPTION TRANSLATION

33. DNA Transcription  Regulation by transcription factor binding  Initiation (at a promoter site)  Formation of a transcription bubble  Elongation (3'  5' on template strand, ≈ 50 s -1 )  Termination (at termination signal)  Many RNA polymerases can function on 1 gene (parallel processing) DNAmRNA RNA polymerase snRNP Splice out introns NTPs

34.  Activators: specific DNA-binding proteins that activate transcription  Repressors: specific DNA-binding proteins that repress transcription  Some regulatory proteins can work as both activators and repressors for different genes  TAF sites are more difficult to locate than genes  Nucleosome positioning influences gene transcription Transcription Factors (TAFs)

35. Prokaryote transcription - initiation   factor associates with -10 (TATA box) and -35  RNA polymerase binds  Bubble forms at -10  3

36. RNA polymerase E.Coli. pol II, resolution ≈ 2.8Å Cramer et al. Science 292, 2001, 1863

37. Prokaryote transcription - elongation  form ≈ 10 bp RNA-DNA hybrid  5'-end of RNA dissociates   factor dissociates and recycles 3' 5'

38. Prokaryote transcription - termination  inverted repeat preceding A-rich region  hairpin formation competes with RNA-DNA hybrid  RNA transcript dissociates  Can also involve RNA- binding protein Rho

40. Eukaryote Transcriptosome

41. DNA REPLICATION

42. DNA Replication

43. + Semiconservative  E.coli ≈ 1000 bp.s -1  Replication is bidirectional  Prokaryotes have a single origin of replication (AT-rich repeats) DNA Replication

44.  DNA polymerase I requires NTPs, Mg 2+ and primer  Works in the 5'  3' direction  Leads to "Okazaki" fragments (10-1000 bp)  Initially these fragments are ≈ 10nt RNA primers  Fragments are finally joined together by a ligase

45. DNA polymerases features  Right hand: “palm”, “fingers”, “thumb”  Palm  phosphoryl transfer  Fingers  template and incoming nucleoside triphosphate  Thumb  DNA positioning, processivity and translocation  Some have 3'  5' exonuclease “proofreading” second domain

46. DNA Polymerase variations Bacteriophage T7 T. gorgorianus

47.  Processivity is very variable (≈ 10  ≈ 10 5 )  Fidelity ≈ 10 -6 -10 -7 (primer plays an important role)  DNA polymerases can proofread (increases fidelity by ≈ 10 3 )  Incorrect nucleotide stalls polymerase and leads to 3'  5' exonuclease excision DNA Replication

48. 3-component "ring"-type DNA polymerase

49.  -subunit of E.Coli polymerase III

50. Replication also requires:  DNA Helicase - hexameric, unwinds DNA, uses ATP  SSB - single-stranded DNA binding protein, stops ss re-annealing or behind degraded  Gyrase (Topo II) - relaxes + ve supercoiling ahead of replication fork  More complex in eukaryotes (telomeres, nucleosomes,...) DNA Replication

51. DNA REPAIR

52. Origins of damage  Polymerase errors  Endogenous damage - oxidation - depurination  Exogenous damage - radiation - chemical adducts  “Error-prone” DNA repair

53. Spontaneous damage oxidation hydrolysis methylation

54. Mispairing induced by oxidative damage Adenine deamination

55. UV radiation can create pyrimidine dimers

56. Damage by covalently bound carcinogens

57.  Endogenous errors: polymerase base selection, proofreading, mismatch repair  Endogenous/exogenous damage: base excision repair, nucleotide excision repair, (recombination, polymerase bypass)  Recombination and polymerase bypass do not remove damage but remove its block to replication. Polymerase bypass is itself often mutagenic  Apoptosis Damage control

58. Mismatch repair  Post-replication mismatch repair system  Similar in prokaryotes and eukaryotes  MMR improve spontaneous mutation rates by up to 10 3  Defects can lead to cancer in humans  Also processes mispairs occurring during recombination

59. Mechanism of MMR CH 3 3 5' 3'5' 3' Initiation CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' MutS MutL MutH Excision CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' UvrD + RecJ or ExoVIIUvrD + ExoI or ExoX or ExoVII Resynthesis CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' PolIII + ligase

60. MutS bound to DNA  Recognizes all base substitutions excepts CC  Recognizes short frameshift loops  Recognizes "new" strand by lack of methylation  DNA kinked by 60°  Opens up minor groove

61. Base excision repair  Repair of modified bases, uracil misincorporation, oxidative damage  DNA glycosylases identify lesion, flip out base and create an abasic site  AP endonucleases incise phosphodiesterase backbone adjacent to AP site  AP nucleotide removed by exonuclease/dRPase and patch refilled by DNA synthesis and ligation

62. Nucleotide excision repair  Recognizes bulky lesions that block DNA replication (covalently bound carcinogens, pyrimidine photodimers  Incision on both sides of lesion  Patch excised, resynthesized and ligated  Can be coupled to transcription  Defects can lead to skin cancer

63. Recognition and binding UvrA finds lesion Incision 3’ and 5’ nicks by UvrBC Excision and repair Helicase releases short fragment E. Coli system

64. Complex human system

65. Lesion bypass polymerization  Replication-blocking lesions are difficult to repair in ss DNA  “Bypass” polymerases can overcome this problem  Error-prone, dissociative (1 nt per binding)  No 3'  5' proofreading ability  Highly regulated as a function of DNA damage

67. Model of Pol I action