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Supercoiling of DNA. Topology A. Right handed supercoiling = negative supercoiling (underwinding) B. Left handed supercoiling = positive supercoiling C. Relaxed state is with no bends D. DNA must be constrained: plasmid DNA or by proteins

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Supercoiling of dna
Supercoiling of DNA

  • Topology

    A. Right handed supercoiling = negative supercoiling (underwinding)

    B. Left handed supercoiling = positive 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. Topology only defined for continuous deformation - no strand breakage


Supercoiling of dna1
Supercoiling of DNA

  • Topology

    A. Right handed supercoiling = negative supercoiling (underwinding)

    B. Left handed supercoiling = positive 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. Topology only defined for continuous deformation - no strand breakage


Supercoiling of dna2
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=DLk/Lk0


Supercoiling of dna3
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=DLk/Lk0


Supercoiling of dna4
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=DLk/Lk0


Supercoiling of dna5
Supercoiling of DNA

  • Topology

    A. Right handed supercoiling = negative supercoiling (underwinding)

    B. Left handed supercoiling = positive 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. Topology only defined for continuous deformation - no strand breakage


Supercoiling of dna6
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 dna7
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 dna8
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 dna9
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 dna10
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.