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Supercoiling of DNA

1. 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 DNA
1. 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 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. 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 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.