Supercoiling of DNA

1
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

2
(No Transcript)
3
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

4
(No Transcript)
5
Supercoiling of DNA

• 2. Numerical expression for degree of
  supercoiling
• A. Equation LkTwWr
• B. Llinking 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
  densityDLk/Lk0

6
(No Transcript)
7
(No Transcript)
8
Supercoiling of DNA

• 2. Numerical expression for degree of
  supercoiling
• A. Equation LkTwWr
• B. Llinking 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
  densityDLk/Lk0

9
(No Transcript)
10
(No Transcript)
11
Supercoiling of DNA

• 2. Numerical expression for degree of
  supercoiling
• A. Equation LkTwWr
• B. Llinking 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
  densityDLk/Lk0

12
(No Transcript)
13
(No Transcript)
14
(No Transcript)
15
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

16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
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

20
(No Transcript)
21
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

22
(No Transcript)
23
(No Transcript)
24
Supercoiling of DNA

• 4. Topoisomerases are required to relieve
  torsional strain
• A. Topoisomerases I
• breaks only one strand
• B. Topoisomerase II
• breaks both strands

25
(No Transcript)
26
(No Transcript)
27
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

28
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.

29
(No Transcript)