Genes VII

Topological manipulation of DNA is a central aspect of all its functional activities Vrecombination, replication, and (perhaps) transcription Vas well as of the organization of higher-order structure. All synthetic activities involving double-stranded DNA require the strands to separate. However, the strands do not simply lie side by side; they are intertwined. Their separation therefore requires the strands to rotate about each other in space. Some possibilities for the unwinding reaction are illustrated in Figure 14.15.

We might envisage the structure of DNA in terms of a free end that would allow the strands to rotate about the axis of the double helix for unwinding. Given the length of the double helix, however, this would involve the separating strands in a considerable amount of flailing about, which seems unlikely in the confines of the cell.

A similar result is achieved by placing an apparatus to control the rotation at the free end. However, the effect must be transmitted over a considerable distance, again involving the rotation of an unreasonable length of material.

DNA actually behaves as a closed structure lacking free ends, which excludes these models as a matter of principle and brings home the severity of the topological problem. In a closed structure, a change in the three dimensional organization of DNA in space can affect the winding of the original strands about one another, and vice versa. Supercoiling of the DNA double helix results when it is coiled about itself in space (like twisting a rubber band). The supercoiling creates a tension in the double helix that changes its structure. Positive supercoiling, when the DNA is twisted in space in the same sense as the strands are wound around one another, causes the double helix to be more tightly wound. Negative supercoiling, when the DNA is twisted in space in the opposite sense from the internal winding of the strands, causes the double helix to be les tightly wound. Negative supercoiling can be thought of as creating tension in the DNA that is relieved by unwinding the double helix.

Consider the effects of separating the two strands in a molecule whose ends are not free to rotate. When two intertwined strands are pulled apart from one end, the result is to increase their winding about each other farther along the molecule. So movement of a replication fork would generate increasing positive supercoiling ahead of it, rapidly generating insuperable resistance to further movement. (Similar consequences ensue during transcription, as described in the twin-domain supercoiling model summarized in Figure 9.18.)

The problem can be overcome by introducing a transient nick in one strand. An internal free end allows the nicked strand to rotate about the intact strand, after which the nick can be sealed. Each repetition of the nicking and sealing reaction releases one superhelical turn.

A closed molecule of DNA can be characterized by its linking number, the number of times one strand crosses over the other in space. Closed DNA molecules of identical sequence may have different linking numbers, reflecting different degrees of supercoiling. Molecules of DNA that are the same except for their linking numbers are called topological isomers.

The linking number is made up of two components: the writhing number (W) and the twisting number (T).

The twisting number, T, is a property of the double helical structure itself, representing the rotation of one strand about the other. It represents the total number of turns of the duplex. It is determined by the number of base pairs per turn. For a relaxed closed circular DNA lying flat in a plane, the twist is the total number of base pairs divided by the number of base pairs per turn.

The writhing number, W, represents the turning of the axis of the duplex in space. It corresponds to the intuitive concept of supercoiling, but does not have exactly the same quantitative definition or measurement. For a relaxed molecule, W = 0, and the linking number equals the twist.

We are often concerned with the change in linking number, δ L, given by the equation

δ L = δ W +δ T

The equation states that any change in the total number of revolutions of one DNA strand about the other can be expressed as the sum of the changes of the coiling of the duplex axis in space (δ W) and changes in the screwing of the double helix itself (δ T). In a free DNA molecule, W and T are freely adjustable, and any δ L (change in linking number) is likely to be expressed by a change in W, that is, by a change in supercoiling.

A decrease in linking number, that is, a change of V δ L, corresponds to the introduction of some combination of negative supercoiling and/or underwinding. An increase in linking number, measured as a change of +δ L, corresponds to a decrease in negative supercoiling/underwinding.

We can describe the change in state of any DNA by the specific linking difference, σ = δL/L₀, where L₀ is the linking number when the DNA is relaxed. If all of the change in linking number is due to change in W (that is, δT = 0), the specific linking difference equals the supercoiling density. In effect, σ as defined in terms of δL/L₀ can be assumed to correspond to superhelix density so long as the structure of the double helix itself remains constant.

The critical feature about the use of the linking number is that this parameter is an invariant property of any individual closed DNA molecule. The linking number cannot be changed by any deformation short of one that involves the breaking and rejoining of strands. A circular molecule with a particular linking number can express it in terms of different combinations of T and W, but cannot change their sum so long as the strands are unbroken. (In fact, the partition of L between T and W prevents the assignment of fixed values for the latter parameters for a DNA molecule in solution.)

The linking number is related to the actual enzymatic events by which changes are made in the topology of DNA. The linking number of a particular closed molecule can be changed only by breaking a strand or strands, using the free end to rotate one strand about the other, and rejoining the broken ends. When an enzyme performs such an action, it must change the linking number by an integer; this value can be determined as a characteristic of the reaction. Then we can consider the effects of this change in terms of δW and δT.

DNA topoisomerases catalyze conversions of this type. Some topoisomerases can relax (remove) only negative supercoils from DNA; others can relax both negative and positive supercoils. Some can introduce negative supercoils.

Topoisomerases are divided into two classes, according to the nature of the mechanisms they employ. Type I enzymes act by making a transient break in one strand of DNA. Type II enzymes act by introducing a transient double-strand break. As well as those enzymes that function as general topoisomerases with DNA irrespective of sequence, enzymes involved in site-specific recombination reactions fit the definition of topoisomerases (see later).

The best characterized type I topoisomerase is the product of the topA gene of E. coli, which relaxes highly negatively supercoiled DNA. The enzyme does not act on positively supercoiled DNA. Mutations in it cause an increase in the level of supercoiling in the nucleoid (and may affect transcription, as described in 9 Transcription).

In addition to the relaxation of negative supercoils in duplex DNA, the enzyme interacts with single-stranded DNA. It may like negative supercoils because they tend to stabilize single-stranded regions, which could provide the substrate bound by the enzyme.

When E. coli topoisomerase I binds to DNA, it forms a stable complex in which one strand of the DNA has been nicked and its 5′ Vphosphate end is covalently linked to a tyrosine residue in the enzyme. This suggests a mechanism for the action of the enzyme; it transfers a phosphodiester bond in DNA to the protein, manipulates the structure of the two DNA strands, and then rejoins the bond in the original strand.

Eukaryotic type I topoisomerases have no sequence or structural similarity with the prokaryotic enzymes. They form a covalent intermediate with the 3′ end of the broken strand, and can relax positive as well as negative supercoils.

A model for the action of topoisomerase I is illustrated in Figure 14.16. The enzyme binds to a region in which duplex DNA becomes separated into its single strands; then it breaks one strand, pulls the other strand through the gap, and finally seals the gap. The transfer of bonds from nucleic acid to protein explains how the enzyme can function without requiring any input of energy. There has been no irreversible hydrolysis of bonds; their energy has been conserved through the transfer reactions.

The reaction changes the linking number in steps of 1. Each time one strand is passed through the break in the other, there is a δL of +1. The figure illustrates the enzyme activity in terms of moving the individual strands. In a free supercoiled molecule, the interchangeability of W and T should let the change in linking number be taken up by a change of δW = +1, that is, by one less turn of negative supercoiling.

The reaction is equivalent to the rotation illustrated in bottom part of Figure 14.15, with the restriction that the enzyme limits the reaction to a single-strand passage per event. (By contrast, the introduction of a nick in a supercoiled molecule allows free strand rotation to relieve all the tension by multiple rotations.)

The type I topoisomerase also can pass one segment of a single-stranded DNA through another. This single-strand passage reaction can introduce knots in DNA and can catenate two circular molecules so that they are connected like links on a chain. We do not understand the uses (if any) to which these reactions are put in vivo.

Type II topoisomerases generally relax both negative and positive supercoils. The reaction requires ATP; probably one ATP is hydrolyzed for each catalytic event. As illustrated in Figure 14.17, the reaction is mediated by making a double-stranded break in one DNA duplex, and passing another duplex region through it.

A formal consequence of two-strand transfer is that the linking number is always changed in multiples of two. The topoisomerase II activity can be used also to introduce or resolve catenated duplex circles and knotted molecules.

The reaction probably represents a nonspecific recognition of duplex DNA in which the enzyme binds any two double-stranded segments that cross each other. The hydrolysis of ATP may be used to drive the enzyme through conformational changes that provide the force needed to push one DNA duplex through the break made in the other. Because of the topology of supercoiled DNA, the relationship of the crossing segments allows supercoils to be removed from either positively or negatively supercoiled circles.

Bacterial DNA gyrase is a topoisomerase of type II that is able to introduce negative supercoils into a relaxed closed circular molecule. DNA gyrase binds to a circular DNA duplex and supercoils it processively and catalytically: it continues to introduce supercoils into the same DNA molecule. One molecule of DNA gyrase can introduce ~100 supercoils per minute.

The supercoiled form of DNA has a higher free energy than the relaxed form, and the energy needed to accomplish the conversion is supplied by the hydrolysis of ATP. In the absence of ATP, the gyrase can relax negative but not positive supercoils, although the rate is more than 10 slower than the rate of introducing supercoils.

The E. coli DNA gyrase is a tetramer consisting of two types of subunit, each of which is a target for antibiotics (the most often used being nalidixic acid which acts on GyrA, and novobiocin which acts on GyrB). The drugs inhibit replication, which suggests that DNA gyrase is necessary for DNA synthesis to proceed. Mutations that confer resistance to the antibiotics identify the loci that code for the subunits.

Gyrase binds its DNA substrate around the outside of the protein tetramer. Gyrase protects ~140 bp of DNA from digestion by micrococcal nuclease. The sign inversion model for gyrase action is illustrated in Figure 14.18. The enzyme binds the DNA in a crossover configuration that is equivalent to a positive supercoil. This induces a compensating negative supercoil in the unbound DNA. Then the enzyme breaks the double strand at the crossover of the positive supercoil, passes the other duplex through, and seals the break.

The reaction directly inverts the sign of the supercoil: it has been converted from a +1 turn to a V1 turn. So the linking number has changed by δL = V2, conforming with the demand that all events involving double-strand passage must change the linking number by a multiple of two.

Gyrase then releases one of the crossing segments of the (now negative) bound supercoil; this allows the negative turns to redistribute along DNA (as change in either T or W or both), and the cycle begins again. The same type of topological manipulation is responsible for catenation and knotting.

On releasing the inverted supercoil, the conformation of gyrase changes. For the enzyme to undertake another cycle of supercoiling, its original conformation must be restored. This process is called enzyme turnover. It is thought to be driven by the hydrolysis of ATP, since the replacement of ATP by an analog that cannot be hydrolyzed allows gyrase to introduce only one inversion ( V2 supercoils) per substrate. So it does not need ATP for the supercoiling reaction, but does need it to undertake a second cycle. Novobiocin interferes with the ATP Vdependent reactions of gyrase, by preventing ATP from binding to the B subunit.

The (ATP-independent) relaxation reaction is inhibited by nalidixic acid. This implicates the A subunit in the breakage and reunion reaction. Treating gyrase with nalidixic acid allows DNA to be recovered in the form of fragments generated by a staggered cleavage across the duplex. The termini all possess a free 3′ VOH group and a 4-base 5′ single-strand extension covalently linked to the A subunit. The covalent linkage retains the energy of the phosphate bond; this can be used to drive the sealing reaction, explaining why gyrase can undertake relaxation without ATP. The sites of cleavage are fairly specific, occurring about once every 100 bp.