super coiling
Post on 30-May-2018
227 Views
Preview:
TRANSCRIPT
-
8/14/2019 Super Coiling
1/29
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
-
8/14/2019 Super Coiling
2/29
-
8/14/2019 Super Coiling
3/29
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
-
8/14/2019 Super Coiling
4/29
-
8/14/2019 Super Coiling
5/29
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
-
8/14/2019 Super Coiling
6/29
-
8/14/2019 Super Coiling
7/29
-
8/14/2019 Super Coiling
8/29
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
-
8/14/2019 Super Coiling
9/29
-
8/14/2019 Super Coiling
10/29
-
8/14/2019 Super Coiling
11/29
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
-
8/14/2019 Super Coiling
12/29
-
8/14/2019 Super Coiling
13/29
-
8/14/2019 Super Coiling
14/29
-
8/14/2019 Super Coiling
15/29
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
-
8/14/2019 Super Coiling
16/29
-
8/14/2019 Super Coiling
17/29
-
8/14/2019 Super Coiling
18/29
-
8/14/2019 Super Coiling
19/29
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
-
8/14/2019 Super Coiling
20/29
-
8/14/2019 Super Coiling
21/29
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
-
8/14/2019 Super Coiling
22/29
-
8/14/2019 Super Coiling
23/29
-
8/14/2019 Super Coiling
24/29
Supercoiling of DNA
4. Topoisomerases are required to relieve
torsional strainA. Topoisomerases I :
breaks only one strandB. Topoisomerase II :
breaks both strands
-
8/14/2019 Super Coiling
25/29
-
8/14/2019 Super Coiling
26/29
-
8/14/2019 Super Coiling
27/29
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
-
8/14/2019 Super Coiling
28/29
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.
-
8/14/2019 Super Coiling
29/29
top related