Dna Topoisomerases Prevent dna Tangling During Replication
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As a replication fork moves along double-stranded DNA, it creates what has been called the “winding problem.” Every 10 base pairs replicated at the fork corresponds to one complete turn about the axis of the parental double helix. Therefore, for a replication fork to move, the entire chromosome ahead of the fork would normally have to rotate rapidly (Figure 5-24). This would require large amounts of energy for long chromosomes, and an alternative strategy is used instead: a swivel is formed in the DNA helix by proteins known as DNA topoisomerases.
Figure 5-24
The “winding problem” that arises during DNA replication. For a bacterial replication fork moving at 500 nucleotides per second, the parental DNA helix ahead of the fork must rotate at 50 revolutions per second.
A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.
One type of topoisomerase, called topoisomerase I, produces a transient single-strand break (or nick); this break in the phosphodiester backbone allows the two sections of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point (Figure 5-25). Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. As a result, DNA replication can occur with the rotation of only a short length of helix—the part just ahead of the fork. The analogous winding problem that arises during DNA transcription (discussed in Chapter 6) is solved in a similar way. Because the covalent linkage that joins the DNA topoisomerase protein to a DNA phosphate retains the energy of the cleaved phosphodiester bond, resealing is rapid and does not require additional energy input. In this respect, the rejoining mechanism is different from that catalyzed by the enzyme DNA ligase, discussed previously (see Figure 5-14).
Figure 5-25
The reversible nicking reaction catalyzed by a eucaryotic DNA topoisomerase I enzyme. As indicated, these enzymes transiently form a single covalent bond with DNA; this allows free rotation of the DNA around the covalent backbone bonds linked to the (more...)
A second type of DNA topoisomerase, topoisomerase II, forms a covalent linkage to both strands of the DNA helix at the same time, making a transient double-strand break in the helix. These enzymes are activated by sites on chromosomes where two double helices cross over each other. Once a topoisomerase II molecule binds to such a crossing site, the protein uses ATP hydrolysis to perform the following set of reactions efficiently: (1) it breaks one double helix reversibly to create a DNA “gate;” (2) it causes the second, nearby double helix to pass through this break; and (3) it then reseals the break and dissociates from the DNA (Figure 5-26). In this way, type II DNA topoisomerases can efficiently separate two interlocked DNA circles (Figure 5-27).
Figure 5-26
A model for topoisomerase II action. As indicated, ATP binding to the two ATPase domains causes them to dimerize and drives the reactions shown. Because a single cycle of this reaction can occur in the presence of a non-hydrolyzable ATP analog, ATP hydrolysis (more...)
Figure 5-27
The DNA-helix-passing reaction catalyzed by DNA topoisomerase II. Identical reactions are used to untangle DNA inside the cell. Unlike type I topoisomerases, type II enzymes use ATP hydrolysis and some of the bacterial versions can introduce superhelical (more...)
The same reaction also prevents the severe DNA tangling problems that would otherwise arise during DNA replication. This role is nicely illustrated by mutant yeast cells that produce, in place of the normal topoisomerase II, a version that is inactive at 37°C. When the mutant cells are warmed to this temperature, their daughter chromosomes remain intertwined after DNA replication and are unable to separate. The enormous usefulness of topoisomerase II for untangling chromosomes can readily be appreciated by anyone who has struggled to remove a tangle from a fishing line without the aid of scissors.