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DNA: Structure, Dynamics and Recognition. L2: Introductory DNA biophysics and biology. Les Houches 2004. STRUCTURE DETERMINATION. X-ray l ≈ 1 Å ≈ atomic separation requires crystals phase problem (homologous structures, or heavy atom doping). X-RAY DIFFRACTION. 1.2 Å. 2 Å. 3 Å. - PowerPoint PPT Presentation
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DNA: Structure, Dynamics DNA: Structure, Dynamics and Recognitionand Recognition
Les Houches 2004
L2: Introductory DNA biophysics and biology
STRUCTURE DETERMINATION
X-RAY DIFFRACTION
X-ray ≈ 1 Å ≈ atomic separation
requires crystals
phase problem (homologous structures, or heavy atom doping)
Crystallographic resolution
- Resolution limit = /2.Sin max
- R-factor = [|Fobs| - |Fcal|]/|Fobs| (0.15-0.25 implies good agreement)
1.2 Å
2 Å
3 Å
Crystal packing effects
Doucet et al. Nature 337, 1989, 190
Crystallographic curvature
DiGabriele et al. PNAS 86, 1989, 1816
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
NMR SPECTROSCOPY
COSY (COrrelation SpectroscopY) - covalently coupled atoms
NOESY (Nuclear Overhauser Effect) - through space coupling
2D NMR SPECTRA
Sequential Resonance Assignments
“Biomolecular NMR Spectroscopy” J.N.S. Evans (1995).
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
OTHER SPECTROSCOPIC TECHNIQUES
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
More disorder more absorption (e.g. diamonds) ds DNA ss DNA more absorption
Absorption SpectroscopyUV
Raman scattering gives acces to vibrations without water peak can identify percentages of sugar puckers, glycosidic conformations, ...
Absorption SpectroscopyIR
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
Neutron scattering spectroscopy
Access to dynamics in psns timescale
Vibrational density of states
Needs a lot of material and a reactor
H/D exchange for selective studies0
40
80
120
Wet, 75% rh
5K 150K 210K 250K 315K
inte
nsity
[ct
s]
0 2 4 6
0
40
80
120
[GHz]
Dry, 11% rh
Sokolov et al. J. Biol. Phys. 27, 2001, 313
DNA/D2O
Slow relaxation in solvent > 210 K
FRET- fluorescent resonance energy transfer
varies as r -6
detection ≈ 5-10 Å
Still to come ....
Hydrogen exchange
Single molecule experiments
HN3 imino proton
S S
STABILIZATION OF THE DOUBLE HELIX
Biological energy scale
Chemical bonds C-H 105 kcal.mol-1C=C 172
Ionic hydration Na+ -93Ca2+ -373
Hydrogen bonds O…H -5 (in vacuum) Protein folding ~ 2-10 (in solution) Protein-DNA binding ~ 5-20 (~200 Å2 contact)
Helix Coil
UV melting curve for a bacterial DNA sample
Tm= T at which 50% of DNA is melted
Tm increases with GC content
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
Pairing in vacuum : Yanson, et. al. 18 (1979) 1149
Bases HCG -21.0AU -14.5
Pairing in chloroform : Kyoguku et al. BBA 179 (1969) 10
Bases HCG -10.0 -11.5AU -6.2AA -4.0
Stacking in water (stronger than pairing) : T’so 1974
Bases HAA -6.5UU -2.7TT -2.4
Base pairing and stacking
Separating a GC basepair in water
Stofer et al. J. Am. Chem. Soc. 121, 1999, 9503
DNA energetics - II
Breslauer empirical equation for ss ds :(Biochemistry 83, 3748, 1986) Gp = (gi + gsym) + k gk Stack gk
GG -3.1AA -1.9
G G A A T T C C GA -1.6C C T T A A G G CG -3.6
GC -3.1Gp = (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 -1.5GT -1.3
Gp = -9.3 Kcal/mol TA -0.9Gexp = -9.4 Kcal/mol
DNA energetics -III
s1 : CGCATGAGTACGC Vesnaver and Breslauer PNAS 88, 3569, 1991s2 : GCGTACTCATGCG
ds ss(h) ss(r)
Kcal/mol ds ss(r) s1(hr) s2(hr) Sum G 20.0 0.5 1.4 1.9H 117.0 29.1 27.2 56.3TS 97.0 28.6 25.8 54.4
DNA TRANSCRIPTION
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
CENTRAL DOGMA
DNA RNA
PROTEIN
DNA polymerase
RNA polymerase
ReverseTranscriptase
RNA replicase
TRANSCRIPTION
TRANSLATION
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)
DNA mRNA
RNA polymerase
snRNP
Splice outintrons
NTPs
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)
Prokaryote transcription - initiation
factor associates with -10 (TATA box) and -35
RNA polymerase binds
Bubble forms at -103
RNA polymerase
E.Coli. pol II, resolution ≈ 2.8Å
Cramer et al. Science 292, 2001, 1863
Prokaryote transcription - elongation
form ≈ 10 bp RNA-DNA hybrid
5'-end of RNA dissociates
factor dissociates and recycles
3' 5'
5'
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
EukaryoteTranscriptosome
DNA REPLICATION
DNA Replication
+
Semiconservative
E.coli ≈ 1000 bp.s-1
Replication is bidirectional
Prokaryotes have a single origin of replication
(AT-rich repeats)
DNA Replication
DNA Replication
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 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
DNA Polymerase variations
Bacteriophage T7 T. gorgorianus
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
3-component "ring"-type DNA polymerase
-subunit of E.Coli polymerase III
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
DNA REPAIR
Origins of damage
Polymerase errors
Endogenous damage - oxidation - depurination
Exogenous damage - radiation - chemical adducts
“Error-prone” DNA repair
Spontaneous damage
oxidation
hydrolysis
methylation
Mispairing induced by oxidative damage
Adenine deamination
UV radiation can create pyrimidine dimers
Damage by covalently bound carcinogens
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
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
Mechanism of MMR
CH3 CH35'3' 5'
3'
Initiation
CH3 CH35'3' 5'
3'CH3 CH3
5'3' 5'
3'
MutS MutL MutH MutS MutL MutH
Excision
CH3 CH35'3' 5'
3'CH3 CH3
5'3' 5'
3'
UvrD + RecJ or ExoVIIUvrD + ExoI or ExoX or ExoVII
ResynthesisCH3 CH3
5'
3' 5'
3'CH3 CH3
5'
3' 5'
3'
PolIII + ligase PolIII + ligase
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
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
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
Recognition and binding
UvrA finds lesion
Incision
3’ and 5’ nicks by UvrBC
Excision and repair
Helicase releases short fragment
E. Coli system
Complex human system
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
Model of Pol I action