7/29/2019 Roca1

1/50

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

7/29/2019 Roca1

2/50

The Problem of Unwinding the Long Double Helix( 1953 ... )

7/29/2019 Roca1

3/50

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

( Vinograd, 1960s )

Linear ( free ends )

form Icompact & hard to denature

form IIless compact & denaturable

7/29/2019 Roca1

4/50

form I form II

nick

7/29/2019 Roca1

5/50

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, 37C )

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

7/29/2019 Roca1

6/50

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 5Lk0 = 3.5Lkm= 3 o 4

G K (Lk) 2Calculate 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

7/29/2019 Roca1

7/50

Torsional energy (Lk) generates topoisomers of different shape

7/29/2019 Roca1

8/50

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

7/29/2019 Roca1

9/50

Interconversion between Tw and Wr

Being Lk constant :

Tw = - Wr

Tw > Wr

GLOBAL Tw and Wr = Local Tw and Wr

7/29/2019 Roca1

10/50

Lk = Tw + WrTOPOLOGY 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

7/29/2019 Roca1

11/50

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

7/29/2019 Roca1

12/50

7/29/2019 Roca1

13/50

Lk = Tw + Wr

Natural partition

by torsional energy

~ 30% Tw

~ 70% Wr

PHYSICAL DNA

Solenoid vs Plectoneme folding

7/29/2019 Roca1

14/50

Lk = Tw + WrTOPOLOGY 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

7/29/2019 Roca1

15/50

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

Z-DNA

Cruciform H-DNA

Tw

Tw

Wr

7/29/2019 Roca1

16/50

Lk = Tw + WrTOPOLOGY 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

7/29/2019 Roca1

17/50

Effect of DNA intercalators

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

7/29/2019 Roca1

18/50

- +

Wr < 0 Wr > 0Wr ~ 0

7/29/2019 Roca1

19/50

Intercalators

Tw Wr

Grove binder

Tw Wr

7/29/2019 Roca1

20/50

JRB

( - )

+ intercalator

R

( + )

R

7/29/2019 Roca1

21/50

-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

7/29/2019 Roca1

22/50

Lk = Tw + WrTOPOLOGY 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

7/29/2019 Roca1

23/50

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

NUCLEOSOMAL DNA TOPOLOGY and the Linking Number Paradox

DNAse I, Hydroxy radical

AA/TT periodics

X-tall structuresh ~ 10.2

However,

7/29/2019 Roca1

24/50

Solutions :

Geometry of linker regions

h is not uniform and fluctuates

Average Wr ~ -1.5

NUCLEOSOMAL DNA TOPOLOGY and the Linking Number Paradox

(-) (+)open

7/29/2019 Roca1

25/50

Dynamics of site juxtaposition in supercoiled DNA

Huang, Schlick, and Vologodskii (2005)

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

7/29/2019 Roca1

26/50

Random walks

Strong interactions

Bio - tunning

Weak & Transient Interactions

Directed walks

7/29/2019 Roca1

27/50

Lk = Tw + WrTOPOLOGY 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

7/29/2019 Roca1

28/50

7/29/2019 Roca1

29/50

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

DNA TOPOISOMERASES

1.

2.

Break and rejoin DNA strands by

means of a trans-estherificationreaction, during which a covalentphospho-tyrosyl intermediate isform.

7/29/2019 Roca1

30/50

DNA TOPOISOMERASE FAMILIES

Type-1A

Type-1B

Type-2

7/29/2019 Roca1

31/50

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

D. radiodurans TopIB (Topoisomerase I)

TOP3 (Topoisomerase III)

7/29/2019 Roca1

32/50

Type-1B Topoisomerases

Y

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

Topoisomerase I

(vacciniavirus)

N C

Y

80-110 kDa

N C 36 kDa

Tyrosin

Recombinases

7/29/2019 Roca1

33/50

Type-1B Mechanism

Reactions

No ATP required

T 2 T i

7/29/2019 Roca1

34/50

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

7/29/2019 Roca1

35/50

Type-2 MechanismReactions

7/29/2019 Roca1

36/50

GYRASE : A type - 2 topoisomerase that reduces Lk

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

Reactions

7/29/2019 Roca1

37/50

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

7/29/2019 Roca1

38/50

ReactionsMechanism Type 1A

No ATP required

ssDNA

7/29/2019 Roca1

39/50

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

Topoisomerase

+

Helicase (ATP)

7/29/2019 Roca1

40/50

7/29/2019 Roca1

41/50

in vivo

ROLES OF TOPOISOMERASES

7/29/2019 Roca1

42/50

( + )( - )

DNA transcription

Topo I

Topo IV Gyrase

E. Coli

~ - 0.06( chromatin ? )

7/29/2019 Roca1

43/50

( + )( - )

DNA transcription

Topo I

Topo II

S. cerevisiae

~ - 0.05( chromatin )

7/29/2019 Roca1

44/50

7/29/2019 Roca1

45/50

Catenates of sister duplexes

Fork Collision

7/29/2019 Roca1

46/50

Topo IV Gyrase

Topo I

7/29/2019 Roca1

47/50

Topo II

Topo I

7/29/2019 Roca1

48/50

Relaxation of Supercoiled DNA in the Chromatin Context

7/29/2019 Roca1

49/50

Figure adapted from Bancaud et al (2006)

Relaxation of Supercoiled DNA in the Chromatin Context

7/29/2019 Roca1

50/50