NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

  • By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.
Cover of Molecular Cell Biology

Molecular Cell Biology. 4th edition.

Show details

Section 12.3The Role of Topoisomerases in DNA Replication

DNA molecules can coil and bend in space, leading to changes in topology, including formation of negative or positive supercoils. For example, as discussed in Chapter 4, local unwinding of a DNA duplex whose ends are fixed causes stress that is relieved by supercoiling. The enzymes that control the topology of DNA function at several different steps in replication in both prokaryotic and eukaryotic cells. In this section we describe the two different classes of topoisomerases and their role in DNA replication.

Type I Topoisomerases Relax DNA by Nicking and Then Closing One Strand of Duplex DNA

The first topoisomerase to be discovered, E. coli topoisom-erase I, can remove negative supercoils without leaving nicks in the DNA molecule (Figure 12-14). After the enzyme binds to a DNA molecule, it cuts one strand, simultaneously generating a covalent phosphoester bond between the released 5′ phosphate on the DNA and a tyrosine residue in the enzyme. Formation of this phosphotyrosine bond does not require ATP or another source of energy. The free 3′-hydroxyl end of the DNA is held noncovalently by the enzyme. The DNA strand that has not been cleaved is then passed through the single-stranded break. The cleaved strand is then resealed, forming a structure with the same chemical bonds as the starting DNA, but with one less negative supercoil. By this mechanism, the enzyme removes one negative supercoil at a time.

Figure 12-14. Action of E. coli type I topoisomerase (Topo I).

Figure 12-14

Action of E. coli type I topoisomerase (Topo I). The DNA-enzyme intermediate contains a covalent bond between the 5′-phosphoryl end of the nicked DNA and a tyrosine residue in the protein (inset). After the free 3′-hydroxyl end of the (more...)

Any enzyme that cleaves only one strand of a DNA duplex and then reseals it is classified as a type I topoisomerase (Topo I). The Topo I from E. coli acts on negative, but not positive, supercoils. In contrast, Topo I enzymes from eukaryotic cells can remove both positive and negative supercoils. Because the relaxation (removal) of DNA supercoils by Topo I is energetically favorable, the reaction proceeds without an energy requirement. The sequential action of E. coli Topo I can remove essentially all the supercoils in a DNA molecule (Figure 12-15).

Figure 12-15. Separation of SV40 DNA topoisomers containing different numbers of supercoils by gel electrophoresis.

Figure 12-15

Separation of SV40 DNA topoisomers containing different numbers of supercoils by gel electrophoresis. DNA was extracted from SV40 virions under conditions that ensure the maximal number of supercoils (lane 1). Aliquots were treated with E. coli type I (more...)

Studies with gene-targeted knockout strains of E. coli have established that Topo I is essential for viability. The enzyme is thought to help maintain the proper superhelical density of the E. coli chromosome by removing negative supercoils formed by action of type II topoisomerase (DNA gyrase), which is discussed below. Since E. coli Topo I cannot remove positive supercoils, it is unlikely to play a role in growing fork progression, which generates positive supercoils. In yeast, either Topo I or Topo II, both of which can relax positive and negative supercoils, functions in the movement of growing forks. Mutations in the gene encoding Topo I affect the growth rate of yeast cells but are not lethal; thus, this enzyme is not essential for viability. However, yeast Topo II, which separates the two DNA duplexes following replication, is essential for viability. In both yeast and E. coli, Topo I enzymes may be important in regulating aspects of transcription, as well as in repairing damaged DNA.

Type II Topoisomerases Change DNA Topology by Breaking and Rejoining Double-Stranded DNA

The first type II topoisomerase (Topo II) to be described was isolated from E. coli. and named DNA gyrase. Topo II enzymes have the ability to cut both strands of a double-stranded DNA molecule, pass another portion of the duplex through the cut, and reseal the cut in a process that utilizes ATP (Figure 12-16a). Depending on the DNA substrate, these maneuvers will have the effect of changing a positive supercoil into a negative supercoil or of increasing the number of negative supercoils by 2. The Topo II enzymes from mammalian cells cannot, like E. coli DNA gyrase, increase the superhelical density at the expense of ATP; presumably no such activity is required in eukaryotes, since binding of histones increases the potential superhelicity. All type II topoisomerases catalyze catenation and decatenation, that is, the linking and unlinking, of two different DNA duplexes (Figure 12-16b).

Figure 12-16. Action of E. coli DNA gyrase, a type II topoisomerase.

Figure 12-16

Action of E. coli DNA gyrase, a type II topoisomerase. (a) Introduction of negative supercoils. The initial folding introduces no stable change, but the subsequent activity of gyrase produces a stable structure with two negative supercoils. Eukaryotic Topo (more...)

DNA gyrase is composed of two identical subunits. Hydrolysis of ATP by gyrase’s inherent ATPase activity powers the conformational changes that are critical to the enzyme’s operation (Figure 12-17). The enzyme functions to introduce negative supercoils at or near the oriC site in the DNA template; as noted earlier, DnaA can initiate replication only on a negatively supercoiled template. Measurements of the degree of DNA supercoiling in E. coli suggest that there is one negative supercoil for each 15 – 20 turns of the DNA helix. A second crucial function of gyrase is to remove the positive supercoils that form ahead of the growing fork during elongation of the growing strands (Figure 12-18).

Figure 12-17. Molecular model for the catalytic activity of E. coli topoisomerase II (DNA gyrase).

Figure 12-17

Molecular model for the catalytic activity of E. coli topoisomerase II (DNA gyrase). The enzyme is a dimer of two identical subunits. Initially, the enzyme binds one part of a DNA strand, the G segment (dark blue), inducing a conformational change in (more...)

Figure 12-18. Movement of the growing fork during DNA replication induces formation of positive supercoils in the duplex DNA ahead of the fork.

Figure 12-18

Movement of the growing fork during DNA replication induces formation of positive supercoils in the duplex DNA ahead of the fork. In order for extensive DNA synthesis to proceed, the positive supercoils must be removed (relaxed). This can be accomplished (more...)

Replicated Circular DNA Molecules Are Separated by Type II Topoisomerases

During DNA replication the parental strands remain intact and retain their superhelicity. This poses steric and topological constraints to the completion of replication of a circular DNA molecule as the two growing forks approach each other. (A similar situation arises wherever two growing forks in a linear eukaryotic chromosome approach each other.) Figure 12-19 illustrates how the last few helical turns in the parental DNA could be removed by changing the topology of the already replicated regions, leaving the two nearly complete daughter helices linked together as catenanes, covalently linked but not yet completely finished circles. Replication then could be completed before or after decatenation to yield two separated complete daughter helices.

Figure 12-19. Completion of replication of circular DNA molecules.

Figure 12-19

Completion of replication of circular DNA molecules. Denaturation of the unreplicated terminus followed by supercoiling overcomes the steric and topological constraints of copying the terminus. At least with SV40 DNA, the final two steps (synthesis and decatenation) (more...)

In E. coli, decatenation is catalyzed by DNA gyrase and a second type II enzyme, called topoisomerase IV, which genetic studies suggest is responsible for separating newly replicated molecules in vivo. Furthermore, temperature-sensitive Topo IV mutants carrying plasmids accumulate interlocked plasmids — that is, catenanes of plasmid DNA, which appear similar to the SV40 catenane shown in Figure 12-19 (inset). To separate DNA catenanes, Topo IV presumably binds to the interlocked duplexes and makes a double-stranded break in one molecule; while remaining attached to the substrate, it then passes the other molecule through the break and finally reseals the break in the cut molecule (see Figure 12-17). Interestingly, although DNA gyrase can carry out decatenation in vitro, it cannot fully substitute for Topo IV in vivo, as demonstrated by the lethal effects of Topo IV mutations.

Linear Daughter Chromatids Also Are Separated by Type II Topoisomerases

The use of temperature-sensitive mutations in Topo II has shown the importance of this protein in yeast-cell viability; furthermore, light-microscopic studies have revealed the participation of Topo II in separating the linear chromosomes of yeast cells. Although individual yeast chromosomes are too small to be visualized by light microscopy, a structure called the nuclear body, which contains all the chromosomes clumped together, can be seen. When temperature-sensitive Topo II mutants are shifted to a nonpermissive temperature, the nuclear body, which usually divides at the junction of the mother and daughter cell, appears to get stuck in the passageway between the two cells.

Fluorescent antibody staining of metaphase chromosomes reveals that topoisomerase II is a principal component of the nonhistone protein scaffolding to which long DNA loops are attached. This finding and the genetic studies in yeast strongly suggest that eukaryotic Topo II resolves tangles that exist in newly replicated linear chromosomes. And finally, the similarity between the phenotypes of Topo II mutants in yeast and Topo IV mutants in bacteria argues that control of topological domains may be analogous in eukaryotic chromosomes and in small circular DNA molecules.

SUMMARY

  •  Among the proteins involved in DNA replication are several that change the topology of DNA: helicases, which can unwind the DNA duplex, thereby inducing formation of supercoils, and topoisomerases, which catalyze addition or removal of supercoils.
  •  Type I topoisomerases relax DNA (i.e., remove supercoils) by nicking and closing one strand of duplex DNA (see Figure 12-14).
  •  Type II topoisomerases change DNA topology by breaking and rejoining double-stranded DNA. These enzymes can introduce or remove supercoils and can separate two DNA duplexes that are intertwined (see Figure 12-16).
  •  Topoisomerases are important both in growing fork movement and in resolving (untangling) finished chromosomes after DNA duplication. Both replicated circular and linear DNA chromosomes are separated by type II topoisomerases.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21703

Views

  • Cite this Page
  • Disable Glossary Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...