DNA supercoiling



Jerome Vinograd, 1965
•sedimentation equilibrium experiments with viral DNA
•circular DNAs can exist in two distinct forms differing in buoyant density
•  supercoiled (compact ) and relaxed (loose)
http://caltech.discoverygarden.ca/islandora/object/ct1%3A5579/datastream/JPG/view
http://openi.nlm.nih.gov/imgs/512/394/2853108/2853108_gkp1161f1.png
http://www.biotechniques.com/multimedia/archive/00003/BTN_A_000112902_O_F00_3774b.jpg
electron microscopy
AFM
agarose gel elfo

Double helical DNA behaves like a rubber rod
•some torsional and bending elasticity
•„shape memory“
•tendency to keep B-conformation
•tendency to keep the axis straight
•Interplay between twisting and bending deformations
•
http://www.maths.uq.edu.au/%7Einfinity/Infinity7/images/supercoiling.gif

Superhelicity is a property of DNA without free ends
•circular duplex DNA (plasmids)
•
•linear molecules with constrained (anchored) ends (chromatin loops)
•
•linear and circular nicked DNAs are inherently relaxed
•
•plectonemic and toroidal supercoils
•
•superhelicity absorbed in nucleosomes and other protein-DNA complexes
•
http://openi.nlm.nih.gov/imgs/512/29/545790/545790_gb-2004-5-12-252-2.png
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-18/18_21.jpg

Lk= Tw+Wr
How many times a strand is crossing a strand
Number of crossing points in duplex DNA (=number of double helix turns)
Number of superturns
relaxed DNA: Lk = N/10.5 = Lk0, where N=number of base pairs
equation Lk= Tw+Wr is valid even for relaxed DNA;
when relaxed DNA lies on a plane, Wr=0, Lk0=Tw= N/10.5
(definition of relaxed DNA; Wr may be ≠0)
supercoiled DNA: Lk ≠ Lk0
(definition of supercoiled DNA)
negatively scDNA: Lk < Lk0 (linking deficit)
positively scDNA:  Lk > Lk0 (linking extent)
Lk - Lk0 = DLk (superhelicity level)
DLk/Lk0 = s (superhelix density: superhelicity level normalized on DNA molecule size)
DLk= DTw+DWr
Lk cannot be changed without interrupting at least one DNA strand

http://chemistry.umeche.maine.edu/CHY431/Nucleic/Supercoil3.gif
relaxed
positively supercoiled
(with linking excess)
duplex
globally untwisted
duplex
locally untwisted
duplex
twisted normally, supercoils formed
In real situation, superhelicity distributed between
DTw (locally or globally)
and DWr
negatively supercoiled
(with linking deficit)
positive superhelical stress forces the right-handed double helix
to close
negative superhelical stress forces the right-handed double helix
to open

Open local structures
•formed in appropriate sequence motifs
•characterized by locally reduced twist, compared to B-DNA
•paranemic: possible to form/abolish without mutual rotation of opposite strands
http://www.mls.sci.hiroshima-u.ac.jp/smg/education/gene-jpeg/cruciform.jpg
http://www.fasebj.org/content/22/6/1625/F1.large.jpg
Left-handed (Z-form) duplex
Quadruplexes

cruciform DNA
inverted repeat (sequences with dyad symmetry)
Open local structures in negatively supercoiled DNA
unpaired bases
local reduction of twist
partial relaxation of negative superturns
(cca 1 superturn per 10 bp of cruciform struture)

Open local structures in negatively supercoiled DNA
Intramolecular triplex
homoPu•homoPy segment with mirror symmetry
TAT (H*-DNA) – stabilized by Mg2+; C+GC (H-DNA) – stabilized in weakly acidic media)
T
T
T
T
T
T
A
A
A
A
A
A
A
local reduction of twist
partial relaxation of negative superturns
(cca 1 superturn per 10 bp of cruciform struture)

Open local structures in negatively supercoiled DNA
Intramolecular quadruplexes
G:C-rich motifs
G-quadruplexes C-quadruplexes (i-motifs)
(K+-stabilized) (weakly acidic pH)

Open local structures in negatively supercoiled DNA
Lef-handed Z-DNA
(Pu-Py)n segment within negatively supercoiled DNA)
local reduction of twist
(to negative values)
partial relaxation of negative superturns
(cca 2 superturns per 14 bp of Z-DNA structure)

DNA supercoiling and nucleosome formation
0606-01.gif
http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageSer
vice/Articleimage/2014/CC/c4cc04789c/c4cc04789c-f1_hi-res.gif

DNA supercoiling and replication/transcription
•local untwisting of duplex in replication fork/transcription complex induces formation of
superturns
https://i.ytimg.com/vi/qj_eYWgQ90k/hqdefault.jpg https://i.ytimg.com/vi/yMKGzq5c8i4/hqdefault.jpg
(link to video)

DNA supercoiling and replication/transcription
•local untwisting of duplex in replication fork/transcription complex induces formation of
supercoils
http://www.nature.com/nrm/journal/v12/n12/images/nrm3228-f5.jpg

DNA supercoiling and replication/transcription
•local untwisting of duplex in replication fork/transcription complex induces formation of
supercoils
http://www.nature.com/nrm/journal/v12/n12/images/nrm3228-f4.jpg

Topoisomers
•molecules of circular duplex DNA differing in Lk value
http://www.udel.edu/chem/bahnson/chem645/websites/Sapra/pic14
separation of palsmid molecules differing in |Wr| in agarose gel
0
±1
±2
±3
etc.
bundle of unresolved topoisomers of high |Wr|
electron microscopy

Superhelicity and intercalation
•intercalators: planar ligands intercalating between base pairs in duplex DNA
•stacking interaction
http://online-media.uni-marburg.de/chemie/bioorganic/vorlesung1/grafik/k513image002.gif

http://cmgm.stanford.edu/biochem201/Slides/DNA%20Topology/073%20EB%20Intercalation.JPG
Superhelicity and intercalation
•characteristic changes in DNA conformation:
•extension in length
•untwisting
•

Superhelicity and intercalation
•increasing concentration of an intercalator:
•gradual relaxation of negative superturns
•formation of positive superturns
•intercalation reduces Lk0 value!
•(even in unconstrained relaxed DNA, number of double helix turns is reduced)
•
http://cmgm.stanford.edu/biochem/biochem201/Slides/DNA%20Topology/074%20S.D%3E%20by%20Sedimentation
.JPG
Lk < Lk0
Lk > Lk0
Lk = Lk0
Lk0 decreases, Lk constant!

Superhelicity and intercalation
•preparation of topoisomers:
•an intercalator is used to modulate superhelicity level
•topoisomerase removes superturns existing at the given intercalator concentration
•negative superturns which were absorbed by intercalation are restored after the intercalator
removal
•

2D electrophoresis of topoisomers
and detection of structural transitions
•open local structures are formed in scDNA with sufficiently negative superhelix density
•they absorb a part of the superhelical stress, which is reflected in reduction of Wr (number of
superturns)
•decrease of the negative superhelicity causes the open structures to disintegrate and B-DNA duplex
to reform
•negative superhelicity reduction can be attained by intercalation

2D electrophoresis of topoisomers
and detection of structural transitions
•topoisomers are prepared and separated in first dimension
•then the gel is soaked with chloroquine (CQ) to remove certain number of superturns (e.g., 4) and
second dimension is run
https://upload.wikimedia.org/wikipedia/commons/thumb/f/f1/Chloroquine.svg/220px-Chloroquine.svg.png
slowest in absence of CQ (Wr=0)
slowest in the presence of CQ (-Wr reduced by 4 to 0)
4 positive superturns in CQ
resolution of
± topoisomers unresolved without CQ
resolution of
negative topoisomers unresolved without CQ
Wr>0
Wr<0
without structural transition: C-shaped pattern

2D electrophoresis of topoisomers
and detection of structural transitions
•topoisomers are prepared and separated in first dimension
•then the gel is soaked with chloroquine (CQ) to remove certain number of superturns (e.g., 4) and
second dimension is run
https://upload.wikimedia.org/wikipedia/commons/thumb/f/f1/Chloroquine.svg/220px-Chloroquine.svg.png
with structural transition: G-shaped pattern
these two molecules are not resolved
(Wr=-2 or Wr=-2 + another two superturns absorbed by cruciform)
superturns removed
cruciform removed, 2 superturns maintained
resolution of topoisomers with and without the transition

Chemical probing of non-B structures (to recall)
•the open local structures contain unpaired bases, unstacked base pairs or otherwise distorted
sites
•
•
•
•
•
•loops, junctions...
•increased chemical reactivity of the nucleobases
•
http://www.mls.sci.hiroshima-u.ac.jp/smg/education/gene-jpeg/cruciform.jpg
http://www.fasebj.org/content/22/6/1625/F1.large.jpg
Left-handed (Z-form) duplex
Quadruplexes

Chemicals selectively reacting with unpaired bases:
osmium tetroxide complexes
(Os,L)
(T, more slowly C)
chloroacetaldehyde
(CAA)
(A, C)
diethyl pyrocarbonate
(DEPC)
(A, G)

Using the Maxam-Gilbert technique, it is possible to determine with a high preciseness which
nucleotides are forming the local structure
Ø modification of supercoiled DNA
Ø restriction cleavage, radiactive labeling
Ø hot piperidine
Ø sequencing PAGE
the structure can be deduced from the modification pattern
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Single-strand selective enzymes
•only detection of a open structure, not identification at the sequence level
•often sufficient: evidence of formation of a expected structure
•nucleases S1, P1, mung bean... cleave ss DNA (or RNA)
•scDNA cleaved by S1, then restriction cleavage to map S1 celavage site
S1
S1
restriction site
restrictase
restrictase
http://www.nature.com/onc/journal/v23/n12/images/1207324f4.jpg
agarose elfo
distinct bands indicate site-specific cleavage by S1

Combination of chemical probes with S1 nuclease
•chemical probes work within wider range of conditions than enzymes
•modification of scDNA
•then restrictase cleavage
•chemical modification of bases in structure that existed in scDNA prevent formation of B-DNA
•then S1 cleavage in the modified site
S1
chemical modification
restriction site
restrictase
S1
http://www.nature.com/onc/journal/v23/n12/images/1207324f4.jpg
agarose elfo
distinct bands indicate site-specific modification

Topoisomerases
•enzymes relaxing (or introducing) superhelical stress in DNA: changing Lk
•solving the „knotty problem“ in replication, transcription
•
https://i.ytimg.com/vi/qj_eYWgQ90k/hqdefault.jpg
C:\Users\Uživatel\Documents\dokumenty\prezentace\knihy-Biochemie\přednášky VUT\nature01407-i1.0.jpg
(video)

Topoisomerase I
•creating and sealing a single-strand break
•only relaxation
•no ATP needed: transesterification, covalent binding of the enzyme to DNA (phosphoester of a Tyr
residue)
•relax either only negative superturns (E. coli topo I), or both positive and negative (topo I from
wheat germ)
•
Lk changed by 1 (one strand threaded through a ssb)

Topoisomerase II
•creating and sealing a double-strand break
•relaxing or introducing superhelicity (DNA gyrase)
•ATP consumption (conformational changes of the protein)
•
Lk changed by 2 (double helix threaded through a dsb)

Other processes catalyezd by topoisomerases
Topoisomerase I:
Topoisomerase II:
knotting/unknotting of ss circles
catenation/decatenation of nicked circles
circular duplex formation of two complementary ss circles (=relaxation of negatively scDNA!)
knotting/unknotting of duplex circles
catenation/decatenation of duplex circles

http://www.nature.com/nrm/journal/v12/n12/images/nrm3228-f4.jpg
Importance of decatenation activity in replication