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DNA TOPOLOGY

INTRODUCTION TO

DNA supercoiling DNA topoisomerases

Lecture 1

KEY EXPERIMENTS ON

Topo II DNA preferences DNA knots Topo II mechanics DNA relaxation in vivo
Lecture 3 Lecture 2

DNA TRANSPORT PREFERENCES OF TYPE-2 TOPOISOMERASES

(+)

?
Eukaryotic TOPO II G::T Bacterial GYRASE G:T

(-)

Bacterial TOPO IV

Roca (2003)

The DNA wrapping domain of bacterial GYRASE has a pin-wheel structure

From Corbett et al (2004)

Does TOPO II

BEND the interacting DNA ?

NO

?

MONOMER circle

LIG

DIMER circle

Trigueros et al. (2004)

Does TOPO II alter the TWIST of the interacting DNA ?

NO

Trigueros et al. (2004)

However ..., TOPO II is sensitive to DNA supercoiling HANDEDNESS
SUPERCOIL REMOVAL:

!!!

DECATENATION :

Roca (2002)

DNA CATENATION :

DNA KNOTTING :

Roca (2002)

With ( -- ) supercoiled, TOPO II is more prone to decatenate With ( + ) supercoiled, TOPO II is more prone to catenate

WHY ?

Could TOPO II be interacting with 3 DNA segments ?

Yes

Trigueros et al. (2004)

Where is the third DNA segment ?

- The dimeric enzyme may recognize simultaneously two pT segments -

Roca (2002)

Why a 3 segment interaction can affect DNA transport preferences ?
Supercoiling handedness DETERMINES the TOPO II orientation relative to the DNA contour

( + ) & ( -- ) SUPERCOILS are EQUALLY removed ( -- ) (+) SUPERCOILS favour DECATENATION SUPERCOILS favour CATENATION
Roca (2002) Trigueros et al. (2004)

Biological relevance ... ?

(R)

( -- )

?

TRIPLE NODES may “ORIENTATE” the TOPOISOMERASE TO UNLINK DOMAINS without RELAXING SUPERCOILING

DNA TOPOLOGY
INTRODUCTION TO

DNA supercoiling DNA topoisomerases

Lecture 1

KEY EXPERIMENTS ON

Topo II DNA preferences DNA knots
Lecture 2

Topo II mechanics DNA relaxation in vivo

Lecture 3

DNA Knots

Topoisomerases Recombinases

nick

nick

KNOT COMPLEXITY KNOT TYPES

( number of irreducible crossings )

( primes & composites ) 01

-

-

+ +
41

+ 31

52

51

+3#-3

61

62

63

3

#

3

mirror

... etc ...

NATURAL FOLDING of DNA
The persistence length of DNA (p) is so small compared with its total length (L), that its natural low energy conformation is already substantially condensed: dimensions of (p.L)1/2

10 3 bp 10 4 bp 10 5 bp

A 2 m “DNA molecule” would naturally fold in dimensions under 500 m

KNOTTING PROBABILITY of DNA in FREE SOLUTION

Upon cyclization of a 10 kb linear DNA molecule:

>
> 96 % 2%

>
< 0.2 %

Informs about physical parameters of DNA

Rybenkov, V. V., Cozzarelli, N. R., and Vologodskii, A. V. (1993). Probability of DNA Knotting and the Effective Diameter of the DNA Double Helix. Proc. Natl. Acad. Sci. USA. 90, 5037-5311.

In 1982, DNA extracted from a bacterial virus (phage P4) was found to be cyclisated and highly knotted
Knotting probability ~ 1

P4 genome is a 10 kb linear DNA molecule with cos ends DNA cyclisation normally occurs after DNA is injected inside the bacteria

DNA

[DNA] > 500 mg/ml

What is the COMPLEXITY of DNA KNOTS in PHAGE P4 ?
At low voltage, DNA migration relates linearly to the number of knot crossings
Stasiak, A., Katrich, V., Bednar, J., Michoud, D., and Dubochet, J. (1996). Electrophoretic mobility of knots. Nature 384, 122.

Unknotting by TOPO II

0 1 2 3 4

min

Arsuaga, J., Vazquez, M., Tigueros, S., Sumners, D. W., & Roca, J. (2002) Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids. Proc. Natl. Acad. Sci. USA 99, 5373-5377.

Computer simulations of random polygons confined to spherical volumes demonstrate that Knotting probability and Knot complexity increase by the effect of confinement

Arsuaga et al (2002)

What is the DISTRIBUTION of KNOT TYPES in PHAGE P4 DNA ?
A two-dimensional gel electrophoresis, combining low and high voltages, separates knotted from supercoiled and linear DNA molecules and also knot types of the same number of crossings

KNOT SPECTRA

Trigueros et al. (2001) Arsuaga et al. (2004)

Simulations of Electrophoretic Collisions of DNA knots with Gel Obstacles
C. Weber, P. De Los Rios, G. Dietler, and A. Stasiak (2006)

EM by Stasiak

AFM by Valle & Dietler

Could knots capture information on the long range folding of DNA ?

Random Walk Non-random Walk

Random KNOT types Specific KNOT types

Predicted DISTRIBUTION for random DNA packaging

The packaging of DNA is not random

Experimental KNOT DISTRIBUTION in Phage P4

Arsuaga et al. (2005)

The scarcity of knot 41 suggests a chiral packaging of the DNA

The knot 41 is achiral, its writhe is zero

+ 41

+
Wr = 0.00

+ + +
51R

+
Wr ~ + 6.26

+

Computed distributions of the knots 41, 51, 52 in ideal chains randomly confined to a sphere of fixed radius, when conformations with writhe below a given value are rejected.

Relative probability

N = 90

R=4

simulation

51

52

41
0 2 4 6 8

Writhe exclusion value experimental

Could knots capture information on the long range folding of DNA ?

Random Walk Non-random Walk

Random KNOT types Specific KNOT types

104 bp (~ 3

m)

108 bp (~ 30 cm) 10 m

40 nm

~

?