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9. Beskrive forekomsten og funktionen af supercoiling af DNA: positive og negative supercoils (superhelix, supertwist), samt funktionen af topoisomeraser (type I og II). Beskrive L = T + W.
Devlin, s.60- s.62
Devlin, s.64 - fig.2.43
Devlin, s.65 - c.c. 2.7
Stryer, s.125, fig.5.18
Stryer, s.754-7
 

The DNA in our cells can be found in the following forms:

relaxed DNA - no manipulation with the DNA molecule. No super helical turns. This form of DNA has a greatly reduced activity in number of crucial biological processes, fx. replication, translation and recombination.

supercoiling - coiling of the axis of the double helix. Can be created by either unwinding or overwinding the DNA molecule. Supercoling can hinder or favor the capacity of the double helix to unwind and thereby affect the interactions between DNA and other molecules.

• negative supercoling - a right-handed coil is assigned a negative number. It is the biologically active form of DNA. Most naturally occurring DNA molecules are negatively supercoiled. In essence, negative supercoiling prepares DNA for processes requiring separation of the DNA strands, such as replication or transcription.
  The negatively supercoiled DNA is more unstable because it is unwinded (twisted one time less), and the hydrogen bonds between the base pairs are more easily broken.
  - On the site of replication, DNA is unwinded.
  - It also appears when the DNA wraps itself around the histones.

• positive supercoiling - a left-handed coil is assigned a positive number. It makes strand separation more difficult and isn’t biologically active, because the DNA is overwinded (twisted one time extra) and is extremely stable. During replication, overwinding appears after the replication site as a result of unwinding of the helix on the replication site. These positive supercoils must be removed if DNA replication is to continue.
  - before and after the replication fork, the DNA is positively supercoiled

L = T + W

L - linking number - number of times that a strand of DNA winds in the right-handed direction around the helix axis, when the axis is constrained to lie in plane.   

(antallet af gange en DNA streng snor sig i højre handed retning rundt om den anden DNA-streng)

T -twisting number - measure of helical winding of the DNA strands around each other

(antallet af dobbelt helix snoringer)

W - writing number - measure of the coiling of the axis of the double helix

(antallet af supercoils)

Topoisomers - DNA molecules differing only in linking number. Topoisomers of DNA can be interconverted only by cutting one or both DNA strands and then rejoining them.

Topoisomerases - enzymes that catalyze the interconversion of topoisomers of DNA. These enzymes alter the linking number of DNA by catalyzing a 3-step process:

1. The cleavage of one or both strands of DNA. Topoisomerases form a transient break in the DNA backbone and then resealing it. The break is not formed by hydrolysis of the sugar-phosphate backbone, but by a transesterification reaction that creates a phosphate-enzyme bond as a transient intermediate. 

2. The passage of a segment of DNA through the break

3. The releasing of the DNA. Rejoining the backbone phosphodiester displaces the enzyme. 

There is a difference between topoisomerase in prokaryotes and eukaryotes, which is EXTREMLY confusing and difficult to remember.

Topoisomerase I

• catalyze the relaxation of supercoiled DNA, (in prokaryotes removes negative supercoils, in human cells removes both positive and negative supercoils)

• cleaves only one strand of DNA

• does not use ATP, because this is a thermodynamically favorable process

Topoisomerase II

• adds negative supercoils to relaxed or supercoiled DNA

• cleaves both strands of DNA

• uses ATP

• found only in prokaryotes, the human topoisomerase has a different function

The degree of supercoiling of DNA is determined by the opposing actions of the two enzymes. Negative supercoils are introduced by topoisomerase II and relaxed by topoisomerase I. The amounts of these enzymes and their activities are regulated to maintain an appropriate degree of negative supercoling.

In treatment of cancer, both topoisomerase I and II can be targeted, by interfering with the enzyme-catalyzed rejoining of the two DNA strands. This may result in degradation of DNA, introduction of mutations or inhibition of translation or replication.

Man kan måske finde en bedre forklaring på human topoisomerase-funktionen på:
http://www.smi.stanford.edu/projects/helix/geis/documentaries/topo.html

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