Chromosomal Elements:-Topoisomerases Catalyze Changes in the Linking Number of DNA
DNA supercoiling is a precisely regulated process that influences many aspects of DNA metabolism. Every cell has enzymes with the sole function of underwinding and/or relaxing DNA. The enzymes that increase or de crease the extent of DNA underwinding are topoisomerases; the property of DNA that they change is the linking number. These enzymes play an especially important role in processes such as replication and DNA packaging. There are two classes of topoisomerases. Type I topoisomerases act by transiently breaking one of the two DNA strands, passing the unbroken strand through the break, and rejoining the broken ends; they change Lk in increments of 1. Type II topoisomerases break both DNA strands and change Lk in increments of 2.
The effects of these enzymes can be demonstrated using agarose gel electrophoresis (Fig. 2). A population of identical plasmid DNAs with the same linking number migrates as a discrete band during electrophoresis. Topoisomers with Lk values differing by as little as 1 can be separated by this method, so changes in linking number induced by topoisomerases are readily detected.
FIGURE 1 Promotion of cruciform structures by DNA under winding. In principle, cruciforms can form at palindromic sequences , but they seldom occur in relaxed DNA because the linear DNA accommodates more paired bases than does the cruciform structure. Underwinding of the DNA facilitates the partial strand separation needed to promote cruciform formation at appropriate sequences.
FIGURE 2 Visualization of topoisomers. In this experiment, all DNA molecules have the same number of base pairs but exhibit some range in the degree of supercoiling. Because supercoiled DNA molecules are more compact than relaxed molecules, they migrate more rapidly during gel electrophoresis. The gels shown here separate topoisomers (moving from top to bottom) over a limited range of superhelical density. In lane 1, highly supercoiled DNA migrates in a single band, even though different topoisomers are probably present. Lanes 2 and 3 illustrate the effect of treating the supercoiled DNA with a type I topoisomerase; the DNA in lane 3 was treated for a longer time than that in lane 2. As the superhelical density of the DNA is reduced to the point where it corresponds to the range in which the gel can resolve individual topoisomers, distinct bands appear. Individual bands in the region indicated by the bracket next to lane 3 each contain DNA circles with the same linking number; the linking number changes by 1 from one band to the next.
E. coli has at least four different individual topo isomerases (I through IV). Those of type I (topoisomerases I and III) generally relax DNA by removing negative supercoils (increasing Lk). The way in which bacterial type I topoisomerases change linking number is illustrated in Figure 1. A bacterial type II enzyme, called either topoisomerase II or DNA gyrase, can introduce negative supercoils (decrease Lk). It uses the energy of ATP to accomplish this. To alter DNA linking number, type II topoisomerases cleave both strands of a DNA molecule and pass another duplex through the break. The degree of supercoiling of bacterial DNA is maintained by regulation of the net activity of topoisomerases I and II.
Eukaryotic cells also have type I and type II topo isomerases. The type I enzymes are topoisomerases I and III; the type II enzymes are topoisomerases II and II. The eukaryotic type II topoisomerases cannot under wind DNA (introduce negative supercoils), but they can relax both positive and negative supercoils. We consider one probable origin of negative supercoils in eukaryotic cells in our discussion of chromatin in Section 24.3. The process catalyzed by eukaryotic type II topoisomerases is illustrated in Figure 3.
FIGURE 3 Proposed mechanism for the alteration of linking number by eukaryotic type IIA topoisomerases. 1 The multisubunit enzyme binds one DNA molecule (blue). Gated cavities above and below the bound DNA are called the N-gate and the C-gate. 2 A second segment of the same DNA molecule (red) is bound at the N gate and 3 trapped. Both strands of the first DNA are now cleaved (the chemistry is similar to that in Fig. 3b), and 4 the second DNA segment is passed through the break. 5 The broken DNA is re ligated, and the second DNA segment is released through the C-gate. Two ATPs are bound and hydrolyzed during this cycle; it is likely that one is hydrolyzed in the step leading to the complex in step 4. Additional details of the ATP hydrolysis component of the reaction remain to be worked out.
MECHANISM FIGURE 4 Bacterial type I topoisomerases alter linking number. A proposed reaction sequence for the bacterial topoi somerase I is illustrated. The enzyme has closed and open conformations. (a) A DNA molecule binds to the closed conformation and one DNA strand is cleaved. (b) The enzyme changes to its open conformation, and the other DNA strand moves through the break in the first strand. (c) In the closed conformation, the DNA strand is religated.
