Nucleic acids and chromatin
Nucleic acids and chromatin

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Nucleic acids and chromatin

DNA topoisomerases

Topoisomerases create temporary strand breaks in DNA, thereby allowing the DNA to ‘swivel’ around the helical axis and releasing torsional strain within the area before resealing the break. With the cellular DNA in a supercoiled state, topoisomerases play a critical role in regulating both how tightly packed DNA is within the cell and the dynamic state of torsional energies during DNA processing. There are two classes of this enzyme: topoisomerase I (topo I) functions by breaking just one strand of the DNA, whilst the action of topoisomerase II (topo II) results in a double-strand break. DNA gyrase is a prokaryotic type II topoisomerase that utilises ATP to introduce twist in the bacterial chromosome.

SAQ 15

What effect will topoisomerase action have on supercoiling?

Answer

DNA twisting results in alterations to supercoiling within the helix. Thus untwisting as a result of topoisomerase action modifies the degree of supercoiling in DNA.

Whilst both type I and type II topoisomerases cause regional relaxation or increased DNA supercoiling, topo II also has other functions. Since topo II breaks both strands of the DNA, simultaneous passage of both strands through a break in the duplex chain is possible. Thus topo II has the ability to knot and unknot DNA as well as to catenate (interlink) and decatenate circular double-stranded DNA, as shown in Figure 13.

Figure 13
Figure 13 The functions of topoisomerase II in decatenating, relaxing and unknotting DNA.

SAQ 16

Why is this activity essential during replication of the circular bacterial chromosome?

Answer

After replication of the circular chromosome, the two daughter chains will be interlinked and will require separation before cell division can occur.

Topoisomerases are often highly expressed in rapidly proliferating cells and have been found to be present at very high levels in cervical, breast, colon and lung cancer cells.

SAQ 17

Why do you think high levels of topoisomerase would be advantageous to cancer cells? (Hint: think back to changes to the DNA helix that could occur during replication, similar to those in transcription in Figure 12.)

Answer

Rapidly growing and dividing cells, such as cancer cells, have a high level of replication and transcription. High topoisomerase activity would facilitate this DNA processing.

It is perhaps not surprising, therefore, that topoisomerases are targets for some cancer chemotherapy agents. Several derivatives of the drug camptothecin, which is a powerful topo I inhibitor, have been found to be effective against some human colon and ovarian cancers. Several topo II inhibitors have also been used successfully. As already mentioned, topo II breaks the deoxyribose-phosphate backbone of both strands of the DNA, allowing passage of an unbroken stretch of double helix through the gap. The mechanism of action of topo II is outlined in Figure 14. Topo II inhibitors exert their effect in one of two ways: so-called catalytic inhibitors stabilise the topological complex (step 3, Figure 14), thereby preventing initial DNA cleavage; while cleavage agents inhibit topo II activity by preventing the already cleaved complex (step 5, Figure 14) from re-ligation.

Figure 14
Figure 14 The mechanism of topoisomerase II action. Step 1: Topo II binds non-covalently to the gate (G) duplex (so called because it opens like a gate). Step 2: The G duplex–topo II complex binds at the crossover region with the transported (T) duplex (so called because this duplex will pass through the G duplex). Step 3: ATP binds and promotes formation of the topological complex. Step 4: Mg2+-dependent cleavage of the G dupex. Step 5: The T duplex passes through the gap formed in the G duplex. Step 6: The G duplex is re-ligated and the bound ATP is hydrolysed. The enzyme is now free to enter another catalytic cycle.
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