DNA: Structure, Dynamics DNA: Structure, Dynamics and Recognitionand Recognition

Les Houches 2004

L4: DNA deformation

BASE PAIR OPENING

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 2.5 ms / nucleotide

Protein synthesis 6.5 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

GC 15-25 ms

AT 5-10 ms

C G C A A G A A G C G

* * 4 1 1 23 4 5 4 * *

A4T4 versus T4A4

T T T T A A A A

1 17 19 4 4 19 17 1

A A A A T T T T

60 100 100 65 65 100 100 60

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 [Vi(q)] i =1

Pi(q)

Nw

ni exp [Fi(q)Vi(q)] i =1

Nw

Fi(q) kT ln Pi(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 (Å2)

T

G

Base movements are coupled

Adenine (°)

T

hym

ine

(°)

Thymine (°)

A

deni

ne (

°)

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 methyltransferaseKlimašauskas et al. Cell 76 (1994) 357

Minor groove Major groove

-200° opening

+160° opening

Backbone rearrangements

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 – L0) / L0 = L / L0

typically ~ -0.06 (1 crossing less per 17 turns)

Linking number (L or Lk) – a topological constant

Twist (T) versus Writhe (W)

Low force High 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'

3'

5'

5'

5' 3'

3' 5'

Major

Minor

DNA: global and local 3'3' stretching

TBP induced deformation

X-ray Model

Magnetic twisting control

DIG : AntiDIG

Biotin : Streptavidin

N S

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