DNA: Structure, Dynamics and Recognition

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DNA Structure, Dynamics and Recognition
L2 Introductory DNA biophysics and biology
Les Houches 2004
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STRUCTURE DETERMINATION
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X-RAY DIFFRACTION

• X-ray l 1 Å atomic separation
• requires crystals
• phase problem (homologous structures, or heavy
  atom doping)

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Crystallographic resolution
1.2 Å
2 Å
3 Å
- Resolution limit l/2.Sin qmax - R-factor S
Fobs - Fcal/Fobs (0.15-0.25 implies
good agreement)
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Crystal packing effects
Doucet et al. Nature 337, 1989, 190
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Crystallographic curvature
DiGabriele et al. PNAS 86, 1989, 1816
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NMR SPECTROSCOPY

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

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2D NMR SPECTRA
COSY (COrrelation SpectroscopY) - covalently
coupled atoms NOESY (Nuclear Overhauser Effect) -
through space coupling
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Sequential Resonance Assignments
Biomolecular NMR Spectroscopy J.N.S. Evans
(1995).
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STRUCTURE FROM NMR DATA

• identify residues in contact (gt5 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

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OTHER SPECTROSCOPIC TECHNIQUES
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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

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Absorption Spectroscopy
UV

• More disorder? more absorption (e.g. diamonds)
• ds DNA ? ss DNA more absorption

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Absorption Spectroscopy
IR

• Raman scattering gives acces to vibrations
  without water peak
• can identify percentages of sugar puckers,
  glycosidic conformations, ...

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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
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Neutron scattering spectroscopy
DNA/D2O

• 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

Slow relaxation in solvent gt 210 K
Sokolov et al. J. Biol. Phys. 27, 2001, 313
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FRET - fluorescent resonance energy transfer

• varies as r -6
• detection 5-10 Å

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HN3 imino proton
Still to come .... Hydrogen exchange Single
molecule experiments
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STABILIZATION OF THE DOUBLE HELIX
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Biological energy scale
Chemical bonds C-H 105 kcal.mol-1 CC 172   
 Ionic hydration Na -93 Ca2 -373    Hydrogen
bonds OH -5 (in vacuum)     Protein folding
2-10 (in solution)    Protein-DNA binding
5-20 (200 Å2 contact)
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Helix ? Coil
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• UV melting curve for a bacterial DNA sample

Tm T at which 50 of DNA is melted
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• Tm increases with GC content

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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
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Base pairing and stacking
Pairing in vacuum Yanson, et. al. 18 (1979)
1149   Bases DH CG -21.0 AU -14.5   Pairing in
chloroform Kyoguku et al. BBA 179 (1969)
10   Bases DH CG -10.0 ? -11.5 AU -6.2 AA -4.0
  Stacking in water (stronger than pairing)
Tso 1974 Bases DH AA -6.5 UU -2.7 TT -2.4  
     
 
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Separating a GC basepair in water
Stofer et al. J. Am. Chem. Soc. 121, 1999, 9503
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DNA energetics - II
Breslauer empirical equation for ss ? ds
(Biochemistry 83, 3748, 1986)   DGp (Dgi
Dgsym) Sk Dgk Stack Dgk   GG -3.1 AA -1.9 G
G A A T T C C GA -1.6 C C T T A A G
G CG -3.6 GC -3.1 DGp (5.0 0.4) - 2 x 3.1
TG -1.9 - 2 x 1.9 - 2 x 1.6 -
1.5 AG -1.6 AT -1.5 GT -1.3 DGp -9.3
Kcal/mol TA -0.9 DGexp -9.4 Kcal/mol
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DNA energetics -III
s1 CGCATGAGTACGC Vesnaver and Breslauer
PNAS 88, 3569, 1991 s2 GCGTACTCATGCG   ds ss(h)
ss(r)
Kcal/mol ds ? ss(r) s1(h?r) s2(h?r) Sum   DG 20.0
0.5 1.4 1.9 DH 117.0 29.1 27.2 56.3 TDS 97.0 28.6
25.8 54.4
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• ... and now for something completely different ...

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DNA TRANSCRIPTION
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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 20 ms /
nucleotide Replication 1 ms / nucleotide Protein
synthesis 6.5 ms / amino acid Protein folding
10 s
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CENTRAL DOGMA
TRANSCRIPTION
TRANSLATION
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DNA Transcription
NTPs
snRNP

• 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)

Splice out introns
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Transcription Factors (TAFs)

• 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

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• s factor associates with -10 (TATA box) and -35
• RNA polymerase binds
• Bubble forms at -10?3

Prokaryote transcription - initiation
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RNA polymerase
E.Coli. pol II, resolution 2.8Å
Cramer et al. Science 292, 2001, 1863
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5'
3'
5'

• form 10 bp RNA-DNA hybrid
• 5'-end of RNA dissociates
• s factor dissociates and recycles

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

Prokaryote transcription - termination
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EukaryoteTranscriptosome
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DNA REPLICATION
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DNA Replication
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Semiconservative

• E.coli 1000 bp.s-1
• Replication is bidirectional
• Prokaryotes have a single origin of replication
• (AT-rich repeats)

DNA Replication
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• DNA polymerase I requires NTPs , Mg2 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

DNA Replication
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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

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DNA Polymerase variations
Bacteriophage T7 T. gorgorianus
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• Processivity is very variable ( 10 ? 105)
• Fidelity 10-6-10-7 (primer plays an important
  role)
• DNA polymerases can proofread (increases fidelity
  by 103)
• Incorrect nucleotide stalls polymerase and leads
  to 3'?5' exonuclease excision

DNA Replication
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3-component "ring"-type DNA polymerase
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b-subunit of E.Coli polymerase III
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• 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
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DNA REPAIR
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Origins of damage

• Polymerase errors
• Endogenous damage - oxidation - depurination
• Exogenous damage - radiation - chemical
  adducts
• Error-prone DNA repair

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Spontaneous damage
oxidation
hydrolysis
methylation
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Mispairing induced by oxidative damage
Adenine deamination
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UV radiation can create pyrimidine dimers
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Damage by covalently bound carcinogens
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Damage control

• 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

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Mismatch repair

• Post-replication mismatch repair system
• Similar in prokaryotes and eukaryotes
• MMR improve spontaneous mutation rates by up to
  103
• Defects can lead to cancer in humans
• Also processes mispairs occurring during
  recombination

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Mechanism of MMR
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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

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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

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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

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

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Model of Pol I action