Topoisomerase Functions And Dna Topology Problems

FIGURE 6-17 Topological states of covalently closed, circular (cet) DNA. The figure shows conversion of the relaxed (a) to the negatively supercoiled (b) form of DMA. The stiain in the supetcraled form may be laker t>p by supectwrsting (b) or by local disruption of base pairing (c). [Adapted from a diagram provided by Dt M. Geliert] (Source Modified from Komberg A, and Bake? T.A 1992 DAW replication. 1 1-21, p. 32. © 1992 by W. H Freeman and Company Used with permission.)

a toroid or spiral in which the long axis is wound in a cylindrical manner, as often occurs when DNA wraps around protein (Figure h-18b). The writhing number (Wr) is the total number of interwound and/or spiral writhes in cccDNA. For example, the molecule shown in Figure 6-17b has a writhing number of four.

interwound writhe and spiral writhe are topologically equivalent to each other and are readily interconvertible geometric properties of c.rcDNA. Also, twist and writhe are interconvertible, A molecule of

Twist And Writhe PictureTwist And Writhe Picture

FIGURE 6-18 Two forms of writhe of supercoiled DNA. The figure shows interwound (a) and toroidal (b) writhe of cccDNA of the same length (a) The interwound or ptectonemic writhe is formed by twisting of the double helical DNA molecule over itself as depicted in the example of a blanched molecule (b) Toroidal or spiral writhe is depicted in this example by cylindrical COtls (Source: Modified from Kornberg A. and Baker T.A. 1992 DNA replication, I 1-22, p. 33. <& 1992 by W. H. Freeman and Company. Used with permission. Used by permission ol Dt Nicholas Cozzarefli)

FIGURE 6-18 Two forms of writhe of supercoiled DNA. The figure shows interwound (a) and toroidal (b) writhe of cccDNA of the same length (a) The interwound or ptectonemic writhe is formed by twisting of the double helical DNA molecule over itself as depicted in the example of a blanched molecule (b) Toroidal or spiral writhe is depicted in this example by cylindrical COtls (Source: Modified from Kornberg A. and Baker T.A. 1992 DNA replication, I 1-22, p. 33. <& 1992 by W. H. Freeman and Company. Used with permission. Used by permission ol Dt Nicholas Cozzarefli)

cccDNA can readily undergo distortions that convert some of its twist to writhe or some of its writhe to twist without the breakage of any covalent bonds. The only constraint is that the sum of the twist number (Tiv) and the writhing number (li/-) must remain equat to the linking number {Lk). This constraint is described by the equation;

Lk° Is the Linking Number of Fully Relaxed cccDNA under Physiological Conditions

Consider cccDNA that is free of supercoiling [that is, it is said to be relaxed) and whose twist corresponds to that of the B form of DNA in solution under physiological conditions [about 10.5 base pairs per turn of the helix). The linking number (Lk) of such cccDNA under physiological conditions is assigned the symbol Lka. Lk° for such a molecule is the number of base pairs divided by 10.5. Fnr a cccDNA of 10,500 base pairs, Lk ~ 11,000. [The sign is positive because the twists of DNA are right-handed.) One way to see this is to imagine pulling one strand of the 10,500 base pair cccDNA out into a fiat circle. If we did this, then the other strand would cross the flat circular strand 1,000 times.

How can we remove supercoils From cccDNA if it is not already relaxed? One procedure is to treat the DNA mildly with the enzyme DNase I, so as to break on average one phosphodiester bond (or a small number oF bonds) in each DNA molecule. Once the DNA has been "nicked" in this manner, it is no longer topologically constrained and the strands can rotate freely, allowing writhe to dissipate [Figure 6-19). If the nick is then repaired, the resulting cccDNA molecules will be relaxed and will have on average an Lk that is equal to Lk°. (Due to rotational fluctuation at the time the nick is repaired, some of the resulting cccDNAs will have an Lk that is somewhat greater than Lk° and others will have an Lk that is somewhat tower. Thus, the relaxation procedure will generate a narrow spectrum of cccDNAs whose average Lk is equal to Lk°).

DNA in Cells Is Negatively Supercoiled

The extent of supercoiling is measured by the difference between Lk and Lk°, which is called the linking difference;

Gambar Struktur Dna

FIGURE 6-19 Relaxing DNA With DNAse I.

Labeled Dna Model

^-pivot

If the ALk of a cccDNA is significantly different from zero, then the DMA is torsioually strained and hence it is supercoiled. If Lk < Lk° and ALk < 0, then the DNA is said to be "negatively supercoiled." Conversely, if Lk>Lk° and ¿¿£>0, then the DNA is "positively supercoiled." For example, the molecule shown in Figure 6-17b is negatively supercoiled and has a linking difference of 4 because its Lk (32) is four less than that [36) for the relaxed form of the molecule shown in Figure 6-17a,

Because A Lk and Lk° are dependent upon the length of the DNA molecule, it is more convenient to refer to a normalized measure of supercoiling. This is the superhelica! density, which is assigned the symbol e and is defined as:

Circular DNA molecules purified both from bacteria and eukaryotes are usually negatively supercoiled. having values of a of about —0.06. The electron micrograph shown in Figure 6-20 compares the structures of bacteriophage DNA in its relaxed form with its supercoiled form.

What does superhelical density mean biologically? Negative super-coils can be thought of as a store of free energy that aids in processes that require strand separation, such as DNA replication and transcription. Because Lk = TW + W'r, negative supercoils can be converted into untwisting of the double helix (compare Figure 6-17a with G-17b). Regions of negatively supercoiled DNA, therefore, have a tendency to partially unwind. Thus, strand Separation can be accomplished more easily in negatively supercoiled DNA than in relaxed DNA,

The only organisms that have been found to have positively supercoiled DNA are certain thermophiles, microorganisms that live under conditions of extreme high temperatures, such as in hot springs. In this case, the positive supercoils can be thought of as a store of free energy that helps keep the DNA from denaturing at the elevated temperatures. In so far as positive supercoils can be converted into more twist (positively supercoiled DNA can be thought of as being overwound), strand separation requires more energy in thermophiles than in organisms whose DNA is negatively supercoiled.

Nucleosomes Introduce Negative Supercoiling in Eukaryotes

As we shall see in the next chapter, DNA in the nucleus of eukaryotic cells is packaged in small particles known as nucleosomes in which the double helix is wrapped almost two times around the outside circumference of a protein core. You will be able to recognize this wrapping as the toroid or spiral form of writhe. Importantly, it occurs in a left-handed manner. (Convince yourself of this by applying the handedness rule in your mind's eye to DNA wrapped around the nueleosome in Chapter 7, Figure 7-18). It turns out that writhe in the form of left-handed spirals is equivalent to negative supercoils. Thus, the packaging of DNA into nucleosomes introduces negative superhelical density.

Topoisomerases Can Relax Supercoiled DNA

As we have seen, the linking number is an invariant property of DNA that is topologically constrained. It can only be changed by introducing interruptions into the sugar-phosphate backbone. A remarkable class of enzymes known as topoisomerases are able to do just this by introducing transient single-stranded or double-stranded breaks into the DNA.

FIGURE 6-20 Electron micrograph of supercoiled DNA. The upper electron micro graph ¡5 a relaxed (nonsupercoiied) DNA rnole-cule of bacterinphage PM2. The fowei electron micrograph shows the phage jn its Supertwsted form. (Source: Electron micrographs courtesy of Wang J.C- 1982. Scientific AmencGn 247 97)

Duplex Micrographs

t^wVi-j topoisomerase II. Topoisomerase II binds to DNA, creates a doubte-stranded break, passes uncut DNA through the gap, then reseats the break.

FIGURE 6-21 Schematic for changing the linking number in DNA with

Dna Topology

cut top duplex pass back duplex through break reseat break cut top duplex pass back duplex through break reseat break

Topoisomerases are of two general types. Type II topoisomerases change the linking number in steps of two. They make transient double-stranded breaks in the DNA through which they pass a segment of uncut duplex DNA before resealing the break. This type of reaction is shown schematically in Figure 6-21. Type II topoisomerases require the energy of ATP hydrolysis for their action. Type I topoisomerases, in contrast, change the linking number of DNA in steps of one. They make transient single-stranded breaks in the DNA, allowing the uncut strand to pass through the break before resealing the nick (Figure 6-22). In contrast to the type U topoisomerases, type I topoisomerases do not require ATP. How topoisomerases relax DNA and promote other related reactions in a controlled and concerted manner is explained below.

Prokaryotes Have a Special Topoisomerase that Introduces Supercoils into DNA

Both prokaryotes and eukarytoes have type 1 and type II topoisomerases that are capable of removing supercoils from DNA. In addition, however, prokaryotes have a special type II topoisomerase known as DNA gyrase that introduces, rather than removes, negative supercoils. DNA gyrase is responsible for the negative supercoiling of chromosomes in prokaryotes. This negative supercoiling facilitates the unwinding of the DNA duplex, which stimulates many reactions of DNA including initiation of both transcription and DNA replication.

cuts a srngle strand of the PNA duplex, passes the uncut strand through the break, then reseals the break The process increases the linking number by +1.

FIGURE 6-22 Schematic mechanism of action for topoisomerase I. The enzyme

FIGURE 6-22 Schematic mechanism of action for topoisomerase I. The enzyme

Topoisomerase Mechanism

Topoisomerases also Unknot and Disentangle DNA Molecules

In addition to relaxing supercoiled DNA, topoisomerases promote several other reactions important to maintaining the proper DNA structure within cells. The enzymes use the same transient DNA break and strand passage reaction that they use to relax DNA to carry out these reactions.

Topoisomerases can both catenate and dec a ten ate circular DNA molecules. Circular DNA molecules are said to be catenated if they are linked together like two rings of a chain (Figure 6-23a), Of these two activities, the ability of topoisomerases to decatenate DNA is of clear biological imparlance. As we will see in Chapter 8, catenated DNA molecules are commonly produced as a round of DNA replication is finished (see Figure 8-33). Topoisomerases play the essential role of unlinking these DNA molecules to allow them to separate into the two daughter cells tor cell division. Decatenation of two covalently closed circular DNA molecules requires passage of the two DNA strands of one molecule through a double-stranded break in the second DNA molecule. This reaction therefore depends on a type El topoisomerase. The requirement for decatenation explains why type 1! topoisomerases are essential cellular proteins. However, if at least one of the two catenated DNA molecules carries a nick or a gap, then a type I enzyme may also unlink the two molecules (Figure 6-23b).

Although we often focus on circular DNA molecules when considering topological issues, the long linear chromosomes of eukaryotic organisms also experience topological problems, For example, during a round a type II topoisomerase b type 1 topoisomerase catenation ^ v decatenation catenation ^ s decatenation

What Decatenating

FIGURE 6-23 Topoisomerases decatenate, disentangle, and unknot DNA.

(a) Type II topoisomerases can catenate and decatenate covalently closed, circular DNA molecules by introducing a dcufcJe-strended break in one DMA and passing the other DMA molecule through the break (b) Type I topoisomerases can only catenate and decatenate molecules if one OIMA strand has a rack or a gap. Ibis is because these en/ymes cleave only one DNA strand at a time (c) Entangled long linear DNA molecules, generated, tor example, during the replication of eukaryctic chromosomes, can be disentangled by a topoisomerase. (d) DMA knots can also be unknotted by lopotsomerase action.

a type II topoisomerase catenation ^ v decatenation b type 1 topoisomerase catenation ^ s decatenation d type II topoisomerase

Catenation

d type II topoisomerase of DNA replication, the two double-stranded daughter DNA molecules wil! often become entangled {Figure 6-23c), These sites of entanglement, just like the links between catenated DNA molecules, block the separation of the daughter chromosomes during mitosis. Therefore, DNA disentanglement, generally catalyzed by a type 11 topoisornerase, is also required for a successful round of DNA replication and cell division in eukaryotes.

On occasion, a DNA molecule becomes knotted (Figure 6-23d). For example, some site-specific recombination reactions, which we shall discuss in detail in Chapter 11, give rise to knotted DNA products. Once again, a type II topoisomerase can "untie" a knot in duplex DNA. If the DNA molecule is nicked or gapped, then a type 1 enzyme can also do this job.

Topoisomerases Use a Covalent Protein-DNA Linkage to Cleave and Rejoin DNA Strands

To perform their functions, topoisomerases must cleave a DNA strand (or two strands) and then rejoin the cleaved strand (or strands). Topoisomerases are able to promote both DNA cleavage and rejoining without the assistance of other proteins or high-energy co-factors (for example, ATP; also see below) because they use a cuvalent-intermediate mechanism. DNA cleavage occurs when a tyrosine residue in the active site of the topoisomerase attacks a phosphodiester bond in the backbone of the target DNA [Figure 6-24), This attack generates a break in the DNA, whereby the topoisomerase is covalently joined to one of the broken ends via a phospho-tyrosme linkage. The other end of the DNA terminates with a free OH group. This end is also held lightly by the enzyme, as we will see below.

The phospho-tyrosine linkage conserves the energy of the phosphodiester bond that was cleaved. Therefore, the DNA can be re-sealed simply by reversing the original reaction: the OH group from one broken DNA end attacks the phospho-tyrosme bond reforming the DNA phosphodiester bond. This reaction rejoins the DNA strand and releases the topoisomerase, which can then go on to catalyze another reaction cycle. Although as noted above, type II topoisomerases require ATP-hydrolysis for activity, the. energy released by this hydrolysis is used to promote conformational changes in the topoisomerase~DNA complex rather than to cleave nr rejoin DNA.

Topoisomerases Form an Enzyme Bridge and Pass DNA Segments through Each Other

Between the steps of DNA cleavage and DNA rejoining, the topoisomerase promotes passage of a second segment of DNA through the break. Topoisomerase function thus requires that DNA cleavage, strand passage, and DNA rejoining all occur in a highly coordinated manner. Structures of several different topoisomerases have provided insight into how the reaction cycle occurs. Here we will explain a model for how a type I topoisomerase relaxes DNA.

To initiate a relaxation cycle, the topoisomerase binds to a segment of duplex DNA in which the two strands are melted (Figure 6-25a). Melting of the DNA strands is favored in highly negatively superceded DNA (see above}, making this DNA an excellent substrate for relaxation. One of the DNA strands binds in a clefl in the enzyme that places it near the

Topological Problems The DnaTopoisomerase

FIGURE 6-24 lopoisomerases cleave PNA using a covalent tyrosine-DNA intermediate.

(a) Schematic of the cfeavage and rejoining reaction, For sirnplioty, only a single strand of DNA is shown. See figure 6-25 for a more realistic picture. The same mechanism ts used by type II topoisomerases. although wo enzyme subunits are required, one to cleave each of the two DMA strands Topoisomerases sometimes cut to the 5' side end sometimes to the 3' side, (b) Close-up view of the phospho-tytosine cwalcnt intermediate.

FIGURE 6-24 lopoisomerases cleave PNA using a covalent tyrosine-DNA intermediate.

(a) Schematic of the cfeavage and rejoining reaction, For sirnplioty, only a single strand of DNA is shown. See figure 6-25 for a more realistic picture. The same mechanism ts used by type II topoisomerases. although wo enzyme subunits are required, one to cleave each of the two DMA strands Topoisomerases sometimes cut to the 5' side end sometimes to the 3' side, (b) Close-up view of the phospho-tytosine cwalcnt intermediate.

tyrosine intermediate (Figure 6-25b). The success of the reaction requires that the other end of the newly cleaved DNA is also tightly bound by the enzyme. After cleavage, the topoisomerase undergoes a large conformational change to open up a gap in the cleaved strand, with the enzyme bridging the gap. The second (uncleaved) DNA strand then passes though the gap, and binds to a DNA-binding site in an internal "donut-shapud " hole in the protein (Figure 6-25c). After strand passage occurs, a second conformational change in the topoisomerase-DNA complex brings the cleaved DNA ends back together (Figure t5-25d); rejoining of the DNA strand occurs by attack of the OH end on the phosopho-tyrosine bond (see above). After rejoining, the enzyme must open up one final time to release the DNA (Figure G-25e). This product DNA is identical to the starting DNA molecule, except that the linking number has been increased by one.

This general mechanism, in which the enzyme provides a "protein bridge" during the strand passage reaction can also be applied to the type II topafeomerases. The type II enzymes, however, are dimeric (or in some cases tetrameric). Two lopoisomerase subunits, with their active site tyrosine residues, are required to cleave the two DNA strands and make the double-stranded DNA hreak that is an essential feature of the type II topoisomerase mechanism.

Topoisomerase Mechanism

rejoining I of cfeaved 1 strand |

FIGURE 6-25 Model for the reaction cycle catalyzed by a type I topoisomerase. The figure shows a series of proposed steps for the relaxation of one turn of a negatively supercorled plasm td ONA. The two strands of UNA are shown as dark gray (and not drawn to scate) The four domains of the protein are labeled in panel (a). Domain t is shown in red, 11 is blue, lit is green, and IV is orange (Source: Adapted from ChampoLix j. 2001 DMA topotsorneiases Annual Review of Biochemistry 70: 369-413 Copyright © 2001 by Annoat Reviews www.annualreviews.org.) A B C D

rejoining I of cfeaved 1 strand |

FIGURE 6-25 Model for the reaction cycle catalyzed by a type I topoisomerase. The figure shows a series of proposed steps for the relaxation of one turn of a negatively supercorled plasm td ONA. The two strands of UNA are shown as dark gray (and not drawn to scate) The four domains of the protein are labeled in panel (a). Domain t is shown in red, 11 is blue, lit is green, and IV is orange (Source: Adapted from ChampoLix j. 2001 DMA topotsorneiases Annual Review of Biochemistry 70: 369-413 Copyright © 2001 by Annoat Reviews www.annualreviews.org.) A B C D

FIGURE 6-26 Schematic of elertrophoretic separation of DNA topoisomers. ! ane A represents relaxed or nicked circular DM; lane 8, linear DNA; lane C, highly supercoiled cccDNA; and fane D, a ladder of topotsomers.

DNA Topoisomers Can Be Separated by Electrophoresis

Covalently closed, circular DNA molecules of the same length but of different linking numbers are called DNA topoisomers. Even though topoisomers have the same molecular weight, they can be separated from each other by electrophoresis through a gel of agarose (see tliapter 20 for an explanation of gel electrophoresis). The basis for this separation is that the greater the writhe, the more compact the shape of a cccDNA. Once again, think of how supercoiling a telephone cord causes it to become more compact. The more compact the DNA, the more easily (up to a point) it is able to migrate through the gel matrix (Figure 6-26). Thus, a fully relaxed cccDNA migrates more slowly than a highly super-coiled tupoisoiner of the same circular DNA. Figure 6-27 shows a ladder of DNA topoisomers resolved by gel electrophoresis. Molecules in adjacent rungs of the ladder differ from each other by a linking number difference of just one. Obviously, electrophoretic mobility is highly sensitive to the topological state of DNA (see Box 6-2, Proving that DNA Has a Helical Periodicity of about lO.fi Base Pairs per Turn from the Topological Properties of DNA Rings)!'

Ethidium Ions Cause DNA to Unwind

Ethidiurn is a large, flat, multi-ringed cation. Its planar shape enables ethidium to slip, or intercalate, between the stacked base pairs of DNA

Box 6-2 Proving that DNA Has a Helical Periodicity of about 10.5 Base Pairs per Tum from the Topological Properties of ONA Rings

The observation that DNA topoisomers can be separated from each other eiec-trophoreticaity is the basis for a simple expenment that proves that DNA has a helical penodicity of about 10.5 base pairs per turn in solution. Constder three cccDWfe of sizes 3,990, 3,995, and 4,01! base pairs that were relaxed to completion by treatment with type I topoisomerase, When subjected to electrophoresis through agarose, the 3,990- and 4,01 1-base-pair DMAs exhibit essentially identical mobilities. Due to thermal fluctuation, topoisomerase treatment actually generates a narrow spectrum of topoisomers, but for simplicity let us consider the mobility of only the most abundant topoisomer (that corresponding tn the cccDNA rn its most relaxed state). The mobilities of the most abundant topoisomers for the 3,990-and 4,011-base-pair DNAs are indistinguishable because the 21-base-pair difference between them is negligible compared to the sizes of the rings. The most abundant topoisorner for the 3,995-base-patr ring however, is found to migrate slightly more rapidly than the other two rings even though it is only 5 base pairs larger than the 3,990-base-pair ring. Hew are we to explain this anomaly? The 3,990- and 4,011 base-pair rings in their most relaxed states are expected to have linking numbers equal to Lk°, that fs, 380 tn the case of the 3,990'base-pair ring {dividing the size by 10.5 base pairs) and 362 in the case of the 4,011-base-pair nng. Because Lk is equal to Lk°, the linking difference (ALk — Lk - UP) in both cases ts zero and there is no writhe. But because the linking number must be an integer, the most relaxed state for the 3.995-base-pair ring would be either of two topoisomers having linking numbers of 380 or 381 However, Lk° for the 3,995-base-pair nng is 380.5. Thus, even in its most relaxed state, a covalently dosed orcle of 3,995 base pairs would necessarily have about half a unit ot writhe (its linking difference would be 0.5), and hence tt would migrate more rapidly than the 3,990- and 4,011-base-pair circles. In other words, to explain how rings that differ in length by 21 base pairs (two turns of the helix) have the same mobility, whereas a ring lhat differs in length by only 5 base pairs (about half a helical turn) exhibits a different mobility, we must con-dude that DNA in solution has a helical periodicity of about 10,5 base pairs per turn.

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Responses

  • olle
    How topoismomerase enzyme works with picture?
    3 years ago
  • omar
    What are topoisomers with diagram?
    3 years ago
  • lily
    What is the function of topoisomerase?
    1 year ago
  • toivo kasslin
    What does a topoisomerase enzyme do to the structure (base pairs) of DNA To the shape To the size?
    10 months ago

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