Figure 5–22 The DNA-helix-passing reaction catalyzed by DNA topoisomerase II. Unlike type I topoisomerases, type II enzymes hydrolyze ATP (red), which is needed to release and reset the enzyme after each cycle. Type II topoisomerases are largely confined to proliferating cells in eukaryotes; partly for that reason, they have been effective targets for anticancer drugs. Some of these drugs inhibit topoisomerase II at the third step in the figure and thereby produce high levels of double-strand breaks that kill rapidly dividing cells. The small yellow circles represent the phosphates in the DNA backbone that become covalently bonded to the topoisomerase (see Figure 5–21).
double-strand break in the helix. These enzymes are activated by sites on chromosomes where two double helices cross over each other such as those generated by supercoiling in front of a replication fork (see Figure 5–20). 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 opening; and (3) it then reseals the break and dissociates from the DNA. At crossover points generated by supercoiling, passage of the double helix through the gate occurs in the direction that will reduce supercoiling. In this way, type II topoisomerases can relieve the overwinding tension generated in front of a replication fork. Their reaction mechanism also allows type II DNA topoisomerases to efficiently separate two interlocked DNA circles (Figure 5–22).
Topoisomerase II 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 above 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.
Much of what we know about DNA replication was first derived from studies of purified bacterial and bacteriophage multienzyme systems capable of DNA replication in vitro. The development of these systems in the 1970s was greatly facilitated by the prior isolation of mutants in a variety of replication genes; these mutants were exploited to identify and purify the corresponding replication proteins. The first mammalian replication system that accurately replicated DNA in vitro was described in the mid-1980s, and mutations in genes encoding nearly all of the replication components have now been isolated and analyzed in the yeast Saccharomyces cerevisiae. As a result, much is known about the detailed enzymology of DNA replication in eukaryotes, and it is clear that the fundamental features of DNA replication—including replication-fork geometry and the use of a multi-protein replication machine—have been conserved during the long evolutionary process that separated bacteria from eukaryotes.