Super Coiling

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Supercoiling of DNA

1. TopologyA. Right handed supercoiling = negative supercoiling(underwinding)B. Left handed supercoiling = positive supercoilingC. Relaxed state is with no bends

D. DNA must be constrained: plasmid DNA or byproteinsE. Unraveling the DNA at one position changes thesuperhelicity -

F. Topology only defined for continuous deformation- no strand breakage


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Supercoiling of DNA

1. TopologyA. Right handed supercoiling = negative supercoiling

(underwinding)B. Left handed supercoiling = positive supercoilingC. Relaxed state is with no bends




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Supercoiling of DNA

2. Numerical expression for degree of supercoilingA. Equation Lk=Tw+WrB. L:linking number, # of times that one DNAstrand winds about the others strands, is always aninteger

C. T: twist,# of revolutions about the duplex helixD. W: writhe, # of turns of the duplex axis aboutthe superhelical axis

by definition the measure of the degree ofsupercoilingE. specific linking difference or superhelical

density=

Lk/Lk0


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Supercoiling of DNA




density=

Lk/Lk0


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Supercoiling of DNA




density=

Lk/Lk0


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Supercoiling of DNA

1. TopologyA. Right handed supercoiling = negative supercoiling

(underwinding)B. Left handed supercoiling = positive supercoilingC. Relaxed state is with no bends




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Supercoiling of DNA

3. DNA compaction requires special form

of supercoilingA. Interwound: supercoiling of DNA

in solutionB. Toroidal- tight left handed turns,packing of DNA

both forms are interconvertible


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Supercoiling of DNA

4. Methods for measuring supercoiling -

based on how compact the DNA isA. Gel electrophoresis

i. 1 dimensionalii. 2 dimensional

B. Density sedimentation


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Supercoiling of DNA

4. Topoisomerases are required to relieve

torsional strainA. Topoisomerases I :

breaks only one strandB. Topoisomerase II :

breaks both strands


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Supercoiling of DNA

4. Topoisomerases are required to relieve torsionalstrain

A. Topoisomerases I - breaks only one strandi. monomeric proteinii. 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 catenatesiv. E. coli Topo I relaxes negatively supercoiled DNA

v. introduces a change of increments of 1 in writhe


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Supercoiling of DNA

4. Topoisomerases are required to relieve torsionalstrain

B. Topoisomerase II - breaks both strandsi. supercoils DNA at the expense of ATP

hydrolysis

ii. two subunits: (alpha)2 and (beta)2iii. becomes covalently linked to the alpha subunitiv. relaxes both negative and positively

supercoiled DNAv. introduces a change in increments of 2 inwrithe.


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Super Coiling

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