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Supercoiling of DNA

1.

Topology

A. Right handed supercoiling = negative supercoiling (underwinding)

B.

C.

Left handed supercoiling = positive supercoiling

Relaxed state is with no bends

D.

proteins

E.

DNA must be constrained: plasmid DNA or by

Unraveling the DNA at one position changes the

superhelicity -

F.

- no strand breakage

Topology only defined for continuous deformation

Supercoiling of DNA

1.

Topology

A.

(underwinding)

B.

C.

Right handed supercoiling = negative supercoiling

Left handed supercoiling = positive supercoiling

Relaxed state is with no bends

D.

proteins

E.

DNA must be constrained: plasmid DNA or by

Unraveling the DNA at one position changes the

superhelicity -

F.

- no strand breakage

Topology only defined for continuous deformation

Supercoiling of DNA

2. Numerical expression for degree of supercoiling A. Equation Lk=Tw+Wr

B. L:linking number, # of times that one DNA

strand winds about the others strands, is always an integer

C.

T: twist,# of revolutions about the duplex helix

D.

W: writhe, # of turns of the duplex axis about

the superhelical axis by definition the measure of the degree of supercoiling E. specific linking difference or superhelical

density=Lk/Lk0

Supercoiling of DNA

2. Numerical expression for degree of supercoiling A. Equation Lk=Tw+Wr

B. L:linking number, # of times that one DNA

strand winds about the others strands, is always an integer

C.

T: twist,# of revolutions about the duplex helix

D.

W: writhe, # of turns of the duplex axis about

the superhelical axis by definition the measure of the degree of supercoiling

E. specific linking difference or superhelical

density=Lk/Lk0

Supercoiling of DNA

2. Numerical expression for degree of supercoiling

A. Equation Lk=Tw+Wr

B. L:linking number, # of times that one DNA

strand winds about the others strands, is always an integer

C. T: twist,# of revolutions about the duplex helix

D. W: writhe, # of turns of the duplex axis about

the superhelical axis by definition the measure of the degree of supercoiling E. specific linking difference or superhelical

density=Lk/Lk0

Supercoiling of DNA

1.

Topology

A.

(underwinding)

B. Left handed supercoiling = positive supercoiling

Right handed supercoiling = negative supercoiling

C. Relaxed state is with no bends

D. DNA must be constrained: plasmid DNA or by

proteins

E.

Unraveling the DNA at one position changes the

superhelicity -

F.

- no strand breakage

Topology only defined for continuous deformation

Supercoiling of DNA

3. DNA compaction requires special form of supercoiling A. Interwound: supercoiling of DNA in solution B. Toroidal- tight left handed turns, packing of DNA both forms are interconvertible

Supercoiling of DNA

4. Methods for measuring supercoiling - based on how compact the DNA is

A. Gel electrophoresis

i. 1 dimensional

ii. 2 dimensional

B. Density sedimentation

Supercoiling of DNA

4. Topoisomerases are required to relieve torsional strain

A. Topoisomerases I :

breaks only one strand B. Topoisomerase II :

breaks both strands

Supercoiling of DNA

4. Topoisomerases are required to relieve torsional strain

A. Topoisomerases I - breaks only one strand

i. monomeric protein

ii. after nicking DNA the 5'-PO4 is covalently linked to

enzyme (prokaryotes)

or the 3' end is linked to the enzyme (eukaryotes)

iii. evidence is the formation of catenates

iv. E. coli Topo I relaxes negatively supercoiled DNA

v. introduces a change of increments of 1 in writhe

Supercoiling of DNA

4. Topoisomerases are required to relieve torsional strain

B. Topoisomerase II - breaks both strands

i. supercoils DNA at the expense of ATP hydrolysis

ii. two subunits: (alpha)2 and (beta)2

iii. becomes covalently linked to the alpha subunit

iv. relaxes both negative and positively

supercoiled DNA

v. introduces a change in increments of 2 in

writhe.