supercoiling of dna
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Supercoiling of DNA1. Topology
A. Right handed supercoiling = negative supercoiling (underwinding)B. Left handed supercoiling = positive supercoilingC. Relaxed state is with no bendsD. DNA must be constrained: plasmid DNA or by proteinsE. Unraveling the DNA at one position changes the superhelicity -F. Topology only defined for continuous deformation - no strand breakage
Supercoiling of DNA1. Topology
A. 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 by proteinsE. 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 supercoilingA. Equation Lk=Tw+WrB. 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 supercoilingE. specific linking difference or superhelical density=Lk/Lk0
Supercoiling of DNA
2. Numerical expression for degree of supercoilingA. Equation Lk=Tw+WrB. 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 supercoilingE. specific linking difference or superhelical density=Lk/Lk0
Supercoiling of DNA
2. Numerical expression for degree of supercoilingA. Equation Lk=Tw+WrB. 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 supercoilingE. specific linking difference or superhelical density=Lk/Lk0
Supercoiling of DNA1. Topology
A. 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 by proteinsE. 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 electrophoresisi. 1 dimensional ii. 2 dimensional
B. Density sedimentation
Supercoiling of DNA
4. Topoisomerases are required to relieve torsional strainA. Topoisomerases I :
breaks only one strand B. Topoisomerase II :
breaks both strands
Supercoiling of DNA
4. Topoisomerases are required to relieve torsional strainA. Topoisomerases I - breaks only one strand
i. 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 DNAv. introduces a change of increments of 1 in writhe
Supercoiling of DNA
4. Topoisomerases are required to relieve torsional strainB. Topoisomerase II - breaks both strands
i. 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
in writhe.
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