DNA: Structure, Dynamics and 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 =  [|F obs | - |F cal |]/|F obs | ( 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, 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

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 Spectroscopy UV

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

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 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

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

Helix  Coil

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

T m 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  H CG-21.0 AU-14.5 Pairing in chloroform : Kyoguku et al. BBA 179 (1969) 10 Bases  H CG-10.0  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

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 = (  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 = ( ) - 2 x 3.1 TG x x AG-1.6 AT-1.5 GT-1.3  Gp = -9.3 Kcal/molTA-0.9  Gexp = -9.4 Kcal/mol

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  G  H T  S

DNA TRANSCRIPTION

Biological time scale Bond vibrations1 fs( s) Sugar repuckering1 ps( 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

CENTRAL DOGMA DNARNA PROTEIN DNA polymerase RNA polymerase Reverse Transcriptase 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) DNAmRNA RNA polymerase snRNP Splice out introns 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 -10  3

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'

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

Eukaryote Transcriptosome

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 polymerase I requires NTPs, Mg 2+ and primer  Works in the 5'  3' direction  Leads to "Okazaki" fragments ( 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  ≈ 10 5 )  Fidelity ≈ (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

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 10 3  Defects can lead to cancer in humans  Also processes mispairs occurring during recombination

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

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