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DNA TOPOLOGY DNA supercoiling DNA topoisomerases Topo II DNA preferences DNA knots Topo II mechanics DNA relaxation in vivo INTRODUCTION TO KEY EXPERIMENTS ON Lecture 1 Lecture 2 Lecture 3

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DNA TOPOLOGY

DNA supercoiling

DNA topoisomerases

Topo II DNA preferences

DNA knots

Topo II mechanics

DNA relaxation in vivo

INTRODUCTION TO

KEY EXPERIMENTS ON

Lecture 1

Lecture 2

Lecture 3

The Problem of Unwinding the “Long” Double Helix

( 1953 ... )

Polyoma viral DNA sediments into 2 forms:

Electron Microscopy: Both forms are circular !

Circular ( no free ends )

Discovery of Circular DNA and Supercoiled DNA Molecules

Linear ( free ends )

form I

compact & hard to denature

form II

less compact & denaturable

form I form II

nick

Measure of DNA “supercoiling” : Linking Number (Lk)

Lk = + 4

N = number of base pairs

h = helical repeat : average bp / turn ( h ~ 10.5 at 0.2M NaCl, pH 7, 37°C )

Lko = Lk of relaxed DNA ( minimal torsional energy ) = N / h

DextroLk = links between 2 closed curves in space:

When DNA is supercoiled ( has torsional energy ) : Lk = Lk - Lk0

Specific Linking Difference or Supercoiling Density : = Lk / Lk0

Lk0 = Lower energy Lk in a “given conditions”

Since N / h may not be an Integer

Then, Lkm = Close Integer to LKo

3’ 5’

5’ 3’

Lk0 = 4

Lkm = 4

3’ 3’

5’ 5’

Lk0 = 3.5

Lkm= 3 o 4

G K ( Lk) 2

Calculate the Free Energy of DNA Supercoiling

Calculate the Helical repeat of DNA in solution

h 10.5

Thermal fluctuation of DNA creates a normal distribution of Lk values

Agarose gel electrophoresis

Torsional energy ( Lk) generates topoisomers of different shape

DNA deformations driven by torsional energy ( Lk)

Wr

Tw

Wr ( Writhe ) Deviations from planarity of the DNA axis

Tw ( Twist ) Strand turnnig around the DNA axis

Lk = Tw + Wr

James White (1969)

Topology Geometry

Interconversion between Tw and Wr

Being Lk constant :

Tw = - Wr

Tw <<< Wr

Tw >>> Wr

GLOBAL Tw and Wr = Local Tw and Wr

Lk = Tw + Wr TOPOLOGY TOPOGRAPHY

Strand Break

&

Passage

TOPOISOMERASES

DNA INTERACTIONS

- Intercalators

- Grove binders

- Benders

- Unwinders

- Trackers

- .....

B-DNA TRANSITIONS

- Cruciforms, Z-DNA, H-DNA …

DNA PHYSICS

- Bending rigidity

- Torsional rigidity

- Efective diameter

DNA behaves like a stiff rod :

--> tendency to maximize base stacking

--> mutual interphosphate repulsion

Sequence

x

Thermal motion

-- Bend & twist rigidity

-- Intrinsic bend / twist

-- Induced bend / twist

Bending Rigidity :

Persistence length (P) is a measure of resistance to lateral bending

For B-DNA, P ~ 50 nm (150 bp)

Torsional Rigidity :

The distance between two DNA sites to become insensitive to torsional

phasing is ~ 2000 bp

Effective DNA Diameter :

Contributions of water ions to charge repulsion between duplexes

Lk = Tw + Wr

“Natural partition”

by torsional energy

~ 30% Tw

~ 70% Wr

PHYSICAL DNA

Solenoid vs Plectoneme folding

Lk = Tw + Wr TOPOLOGY GEOMETRY

Strand Break

&

Passage

TOPOISOMERASES

DNA INTERACTIONS

- Intercalators

- Grove binders

- Benders

- Unwinding

- Tracking

- .....

B-DNA TRANSITIONS

- Cruciforms, Z-DNA, H-DNA …

DNA PHYSICS

- Bending rigidity

- Torsional rigidity

- Efective diameter

B-DNA TRANSITIONS generated by torsional energy ( < 0 )

Z-DNA

Cruciform H-DNA

Tw

Tw

Wr

Lk = Tw + Wr TOPOLOGY GEOMETRY

Strand Break

&

Passage

TOPOISOMERASES

DNA INTERACTIONS

- Intercalators

- Grove binders

- Benders

- Unwinders

- Trackers

- .....

B-DNA TRANSITIONS

- Cruciforms, Z-DNA, H-DNA …

DNA PHYSICS

- Bending rigidity

- Torsional rigidity

- Efective diameter

Effect of DNA intercalators

Intercalators reduce Tw, therefore increase Wr in closed-circular molecules

- +

Wr < 0 Wr > 0Wr ~ 0

Intercalators

Tw Wr

Grove binder

Tw Wr

( - )

+ intercalator

R

( + )

R

-16

-17

B-DNA transitions revealed by 2D electrophoresis

Altered migration in the FIRST dimension :

Torsional energy drives a B-DNA transition

Normal migration in the SECOND dimension :

Intercalator stabilizes torsional energy

&

the transition reverts

Z-DNA H-DNA Cruciform

Lk = Tw + Wr TOPOLOGY GEOMETRY

Strand Break

&

Passage

TOPOISOMERASES

DNA INTERACTIONS

- Intercalators

- Grove binders

- Benders

- Unwinders

- Trackers

- .....

B-DNA TRANSITIONS

- Cruciforms, Z-DNA, H-DNA …

DNA PHYSICS

- Bending rigidity

- Torsional rigidity

- Efective diameter

Each nucleosome estabilises Lk ~ – 1.0

Nucleosome ~ 1.8 levo DNA turns ( Wr ~ -1.8 )

Then, DNA must be overtwisted ( Tw ~ + 0 8 ) such that average h ~ 10.0

AA/TT periodics

X-tall structures

h ~ 10.2

However,

Solutions :

h is not uniform and fluctuates

Average Wr ~ -1.5

(-) (+)open

Dynamics of site juxtaposition in supercoiled DNA

Huang, Schlick, and Vologodskii (2005)

Supercoiling does not correspondingly increase the rate of juxtaposition between any sites

Random walks

Strong interactions

Bio - tunning

Weak & Transient Interactions

Directed walks

Lk = Tw + Wr TOPOLOGY GEOMETRY

Strand Break

&

Passage

TOPOISOMERASES

DNA INTERACTIONS

- Intercalators

- Grove binders

- Benders

- Unwinders

- Trackers

- .....

B-DNA TRANSITIONS

- Cruciforms, Z-DNA, H-DNA …

DNA PHYSICS

- Bending rigidity

- Torsional rigidity

- Efective diameter

Allow the passage of anotherstrand, or strands, of DNA acrossthe transient break.

DNA TOPOISOMERASES

1.

2.

Break and rejoin DNA strands bymeans of a trans-estherificationreaction, during which a covalentphospho-tyrosyl intermediate isform.

DNA TOPOISOMERASE FAMILIES

Type-1A

Type-1B

Type-2

Type 1A Type 1B Type 2A Type 2B

Gene (Protein) Gene (Protein) (Protein)Gene Gene (Protein)

TopRG (Gyrase Reverse)

H. sapiens

D. melanogaster

C. elegans

S. pombe

S. cerevisiae

A. thaliana

EUKARYA

TOP3a

TOP3b

TOP3a

TOP3b

TOP3a

TOP3b

TOP3

TOP3

(Topoisomerase III )

(Topoisomerase III )

(Topoisomerase III )

(Topoisomerase III )

(Topoisomerase III )

(Topoisomerase III )

(Topoisomerase III)

(Topoisomerase III)

TOP1

TOP1

TOP1

TOP1

TOP1

TOP1

TOP2a

TOP2b

TOP2

TOP2

TOP2

TOP2

TOP2

(Topoisomerase I)

(Topoisomerase I)

(Topoisomerase I)

(Topoisomerase I)

(Topoisomerase I)

(Topoisomerase I)

(Topoisomerase II)

(Topoisomerase II)

(Topoisomerase II)

(Topoisomerase II)

(Topoisomerase II)

(Topoisomerase II )

(Topoisomerase II )

VIRUS

BACTERIA

ARCHEA

Phage T4

Poxvirus

E. coli

H. pylori

TopA

TopB

(Topoisomerase I)

(Topoisomerase III)

TopA (Topoisomerase I)

TopA

TopB

(Topoisomerase I)

(Topoisomerase III)

TopA (Topoisomerase I)

(Topoisomerase IV)

(Topoisomerase IV)

(Gyrase)

(Gyrase)

(Gyrase)

(Gyrase like)(( Topoisomerase V ))

GyrA + GyrB

GyrA + GyrB

GyrA + GyrB

GyrA + GyrB

ParC + ParE

ParC + ParE

TOP1 (Topoisomerase I)

(Topoisomerase II)

TopVIA + TopVIB(Topoisomerase VI)

Genes (39+52+60)

TOPOISOMERASES

TOP3 (Topoisomerase III)

Type-1B Topoisomerases

Y

Topoisomerase I(H. sapiens )(S. cerevisiae)

Topoisomerase I(vaccinia virus)

N C

Y

80-110 kDa

N C 36 kDa

Tyrosin

Recombinases

Type-1B Mechanism

Reactions

No ATP required

ATP

Y

Type - 2 Topoisomerases

(GyrB) (GyrA)

N

ATP

N

N

Topoisomerase II(S. cerevisiae ,..,..,..)

Topoisomerase IV(E. coli ,..,..,.. )

Gyrase(E. coli ,..,..)

(Top2)

(ParE)

C

C

C

Y

(ParC)

dim 170 kDa x 2

Type-2 Mechanism Reactions

GYRASE : A type - 2 topoisomerase that reduces Lk

(+)(+) (-) (-)

Reactions

Type-1A Topoisomerases

Topoisomerase I (E. coli)

Topoisomerase III (E. coli)

Topoisomerase III (S. cerevisiae)

“Reverse Gyrase” (M. janaschii)

Y

H e l i c a s e

N C

97 kDa

Y

ReactionsMechanism Type 1A

No ATP required

ssDNA

Reverse GYRASE : A type-1A topoisomerase that increases Lk

Topoisomerase

+

Helicase (ATP)

in vivo

ROLES OF TOPOISOMERASES

( + )( - )

DNA transcription

Topo I

Topo IV Gyrase

E. Coli

~ - 0.06

( chromatin ? )

( + )( - )

DNA transcription

Topo I

Topo II

S. cerevisiae

~ - 0.05

( chromatin )

Catenates of sister duplexes

Fork Collision

Topo IV Gyrase

Topo II

Topo I

Figure adapted from Bancaud et al (2006)

Relaxation of Supercoiled DNA in the Chromatin Context