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