DNA: Structure, Dynamics and Recognition Les Houches 2004 L4: DNA deformation

BASE PAIR OPENING

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) Transcription2.5 ms / nucleotide Protein synthesis6.5 ms / amino acid Protein folding~ 10 s RNA lifetime~ 300 s

Enzymatic base chemistry

Adenine-Thymine base pair HN3 imino proton S S

Guanine-Cytosine base pair HN1 imino proton S S

Base opening lifetimes GC15-25 ms AT 5-10 ms C G C A A G A A G C G * * * *

A 4 T 4 versus T 4 A 4 T T T T A A A A A A A A T T T T Leroy et al. Biochemistry 27, 1988, 8894 Base pair lifetimes (ms) 15°C

B-DNA - 2ns dynamic trajectory

Free energy calculations using restrained opening Guidice et al. ChemPhysChem 2, 2001, 673 Varnai & Lavery J. Am. Chem. Soc. 124, 2002, 7272

WHAM FREE ENERGY PROFILE BIASED PROBABILITY HISTOGRAM N(q) W(q) Reaction coordinate (q) Nw  P* i (q) exp [  V i (q)] i =1 P i (q)   Nw  n i exp  [F i (q)  V i (q)] i =1 Nw F i (q)   kT ln  P i (q) i =1

B-DNA oligonucleotide studied CTCTCTCTCTCTC GAGAGAGAGAGAG

Extraction d’une base de l’ADN

Closed AT pair

Adenine –50° (minor)

Adenine –100° (minor)

Adenine +50° (major)

Adenine +100° (major)

Free energy curves for base opening

Imino proton accessibility (Å 2 ) T G

Base movements are coupled  Adenine (°)  Thymine (°)  Adenine (°)

Sequence effects on opening: A-tracts  T A-tract T Ref

Bending amplitude (°)  < -50° -50° <  < +50°  > +50° G T

A word of warning!

BASE FLIPPING

Hha1 methyltransferase Klimašauskas et al. Cell 76 (1994) 357

Minor groove  Major groove

-200° opening +160° opening

Backbone rearrangements

SUPERCOILING

DNA supercoiling (circular plasmid)

DNA supercoiling L = linking number = number of strand crossings T = twist = number of turns of double helix W = writhe = number of helix crossovers L = T + W  = supercoiling density = (L – L 0 ) / L 0 =  L / L 0 typically  ~ (1 crossing less per 17 turns)

Linking number (L or L k ) – a topological constant

Twist (T) versus Writhe (W) Low forceHigh force

L = T + W

Interwound and toroidal forms of a negatively supercoiled plasmid L.H. R.H.

Ethidium bromide intercalates into DNA and reduces its twist by ~26°

Effect of an intercalator on a negatively supercoiled plasmid

Topoisomerases Topoisomerase I- single strand cuts - releases negative supercoiling Topoisomerase II- double strand cuts (eukaryotes)- releases negative supercoiling Topo II (gyrase)- generates negative supercoiling (prokaryotes)- consumes ATP Reverse gyrase- generates positive supercoiling (thermophiles)

Topoisomerase I – single strand cuts

Topoisomerase II – double strand cuts

Topo II (gyrase) DNA wrapping

DNA packed on nucleosomes

Nucleosome – schematic view

EXTREME DEFORMATIONS

DNA stretching Cluzel et al. Science 271, 1996, 792

70 pN phase transition

S-DNA: fibre and ribbon forms

Fibre diffraction of stretched DNA Greenall et al. J. Mol. Biol. 2001, 305, 669 Rise ~ 5.6 Å Helix spacing ~ 13 Å

TBP-DNA complex

DNA: local stretching 3' 5' 3' 5' Major Minor

DNA: global and local 3'3' stretching

TBP induced deformation X-rayModel

Magnetic twisting control DIG : AntiDIG Biotin : Streptavidin NS Strick et al. Biophys. J. 74, 1998, 2016 Allemand et al. Proc. Natl. Acad. Sci. (USA) 95, 1998, 14152

Twisted DNA forms plectonemes

DNA twisting under tension > 3 pN < 0.3 pN

Simulation of DNA twisting

Simulating twisting