DNA Topoisomerases: Type I

James J. Champoux
University of Washington, Seattle, Washington, USA

The large size of DNA molecules and the double-helical nature of DNA create unique topological problems during replication, transcription, recombination, and chromatin remodeling that are solved by a family of enzymes called DNA topoisomerases. Members of the type II subfamily of DNA topoisomerases alter the supercoiling of DNA and disentangle chromosomes by introducing temporary double-strand breaks into the DNA. Type I DNA topoisomerases, the subject of this review, manage DNA topology in the cell by transiently cleaving only one of the two DNA strands.

Reactions Catalyzed by Type I DNA Topoisomerases

To introduce a temporary single-strand break into duplex DNA, type I DNA topoisomerases must catalyze the cleavage and subsequent religation of a DNA strand. Since these two reactions occur without an external energy source such as ATP, cleavage cannot result from simple hydrolysis of a phosphodiester bond in the DNA. Instead, a covalent enzyme DNA intermediate is generated that makes the religation step energetically feasible. The formation of the covalent intermediate involves nucleophilic attack by the O-4 atom of the active site tyrosine in the enzyme on a phosphodiester bond in the DNA to produce a phosphodiester bond between the tyrosine and the DNA and leave a free DNA hydroxyl end. DNA religation and release of the enzyme is the reverse reaction with the oxygen of the free DNA hydroxyl acting as the nucleophile. The type I enzymes display a loose preference for certain nucleotides in the vicinity of a cleavage site, and therefore a cleavage site typically occurs every 5 20 base pairs along the DNA. Type I topoisomerases act on closed circular DNAs to change the number of times one strand winds around the other, a parameter referred to as the linking number of the DNA. Changes in the linking number are reected in a reduction or an increase in the supercoiling of a plasmid DNA, a property that is most often measured by gel electrophoresis. In addition to altering the supercoiling of a plasmid DNA, many type I topoisomerases are capable of catalyzing a number of other transactions

involving both single- and double-stranded DNAs. Most of these enzymes can catenate (interlock), decatenate, knot, and unknot single-stranded DNA circles. The same series of reactions can be carried out with duplex circular DNAs providing at least one of the circular molecules possesses a nick or gap. In some cases, the enzymes can facilitate the interwinding required for the renaturation of two complementary single-stranded circular DNAs, a reaction that could be important during homologous recombination. Interestingly, topoisomerase V, which has only been described in the hyperthermophilic archaeon Methanopyrus kandleri, possesses, in addition to the usual topoisomerase activity, an apurinic/apyrimidinic site-processing activity that would appear to implicate the enzyme in DNA repair. Finally, with certain unusual DNA substrates, a block to religation leads to permanent suicide cleavage and the enzyme remains covalently linked to the DNA.

Classication, Nomenclature and General Properties

Type I topoisomerases are classied into two structurally and mechanistically distinct subfamilies based on which DNA end becomes covalently attached to the enzyme during the cleavage reaction: type IA enzymes attach via a tyrosine phosphodiester linkage to the 50 end of the DNA, whereas type IB enzymes attach to the 30 end of the DNA. Table I lists the known type I DNA topoisomerases in the two subfamilies with their common names and origins. The common names have generally been assigned in the order of discovery using odd Roman numerals (even Roman numerals are similarly used for type II DNA topoisomerases). Type I enzymes with unusual properties or origins have been given unique names (reverse gyrase, poxviral topoisomerase, and mitochondrial topoisomerase). The recently described IB enzymes in some eubacteria are currently referred to as bacterial topoisomerases IB. The three categories of type IA enzymes listed in Table I can be distinguished on the basis of the types of

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TABLE I Type I DNA Topoisomerases Subfamily IA IA IA IA IB IB IB IB IB IB Common name Topoisomerase I Topoisomerase III Reverse gyrase Reverse gyrase Topoisomerase I Mitochondrial topoisomerase Poxviral topoisomerase Topoisomerase V Topoisomerase IB Topoisomerase I Source All eubacteria and some archaebacteria Some eubacteria and most eukaryotes All hyperthermophilic eubacteria and archaea Archaeon Methanopyrus kandleri Nucleus of all eukaryotes Mitochondria of higher eukaryotes All members of poxviridae family Archaeon Methanopyrus kandleri Some eubacteria (see Table II) Trypanosomatids Trypanosoma brucei and Leishmania donovani Structure Monomer Monomer Monomer Heterodimer Monomer Monomer Monomer Monomer Monomer Heterodimer

reactions they catalyze. Topoisomerases I relax negative but not positive supercoils in plasmid DNAs; but since relaxation does not go to completion, some residual negative supercoils remain in the product. Topoisomerases III require hypernegatively supercoiled plasmid DNA as a substrate and again relaxation is incomplete. Interestingly, topoisomerases III are much more procient than the topoisomerases I in DNA catenation and decatenation (see below). Reverse gyrases, which are only found in hyperthermophilic eubacteria and archaebacteria, introduce positive supercoils into plasmid DNAs at the expense of ATP hydrolysis. All of the type IA enzymes require Mg2 and are monomeric with the exception of the reverse gyrase from the archaeon Methanopyrus kandleri which is a heterodimer. The type IB DNA topoisomerases are capable of relaxing both positive and negative supercoils in a reaction that does not require ATP or divalent cations.

The reactions go to completion to produce a completely relaxed set of plasmid DNA topoisomers. With the exception of the recently discovered heterodimeric topoisomerases I from trypanosomatids, all of the type IB enzymes are monomeric.

Type IA DNA Topoisomerases

PROTEIN DOMAINS
All type IA topoisomerases (Table I) share a highly conserved cleavage/strand passage domain that contains the active site tyrosine. This domain is also responsible for promoting the structural change in the DNA during the interval between the cleavage and religation reactions that results in a linking number change (see below) (Figure 1, red boxes). As indicated in Figure 1, all type IA enzymes contain a poorly conserved

FIGURE 1 Domain structure and sequence relationships between type IA DNA topoisomerases. The domain structure of representative type IA topoisomerases is denoted by colored boxes. The names of the domains for the prototypic E. coli DNA topoisomerase I are given along the top. The cleavage/strand passage domains shared by all the IA enzymes are shown in red. Green is used to denote the basic C-terminal domains, but the different types of ll for these boxes indicate that these domains are poorly conserved. Some type IA enzymes contain a Zn(II) binding domain shown in blue. The helicase-like domain of reverse gyrase is shown in yellow.

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basic C-terminal domain (solid or hatched green boxes) and some contain a Zn(II) binding domain as well (blue boxes). These latter two features appear to be important for the interaction of the enzyme with DNA. Finally, reverse gyrases contain an N-terminal domain, which resembles the ATPase domains of helicases (yellow box), and is connected to two domains that are structurally very similar to the cleavage/strand passage and basic domains of the typical type IA topoisomerases.

CRYSTAL STRUCTURE OF THE CONSERVED CLEAVAGE = STRAND PASSAGE DOMAIN

The crystal structure of the cleavage/strand passage domain of E. coli DNA topoisomerase I shown in Figure 2 provides key insights concerning the substrate preference of the enzyme and the mechanism of DNA relaxation. Notably, the cleavage/strand passage domains of all type IA topoisomerases bear a strong resemblance to the E. coli structure. The hallmark of the crystal structure is a toroidal shape in which the diameter

of the hole in the center of the torus is sufcient to accommodate either single- or double-stranded DNA. The requirement for a negatively supercoiled substrate and the inability to completely relax negative supercoils is best explained by supposing that these enzymes will only bind an otherwise duplex DNA substrate if it contains a single-stranded region resulting from local unwinding of the helix. A plasmid DNA that is highly negatively supercoiled is energetically disposed toward helix unwinding, which explains why such a DNA is a good substrate for the enzyme. However, relaxation ceases when the negative supercoiling falls to a level below which there is insufcient energy to promote the required opening of the helix. As shown in Figure 2, a single strand of DNA (solid black line) binds to a narrow groove on the cleavage/strand passage domain of the enzyme in close proximity to the active site tyrosine (magenta).

FOR

ENZYME- BRIDGING MECHANISM STRAND PASSAGE

FIGURE 2 A ribbon diagram showing the crystal structure of the cleavage/strand passage domain of E. coli DNA topoisomerase I (pdb entry 1ecl) drawn with Swiss-Pdb Viewer software (Glaxo Wellcome Experimental Research). The approximate path of the bound single-stranded substrate DNA is indicated by the solid black line, and the active site tyrosine is shown in magenta. The two regions of the protein that move relative to each other (double-headed arrow) to open and close the torus during the strand passage reaction and to release the DNA are shown in green and orange.

To change the linking number of a closed circular DNA during relaxation, one strand of DNA must pass through a break in the other strand. Knotting, catenation, and decatenation of either single or doublestranded DNAs similarly require such a strand passage event. The strand passage reaction for type IA topoisomerases occurs by what is referred to as an enzyme-bridging mechanism. Once the scissile DNA strand is cleaved, both DNA ends remain tightly associated with the enzyme; the 50 end is bound covalently to the active site tyrosine and the 30 end is bound noncovalently to the enzyme. To orchestrate strand passage, the enzyme undergoes a conformational change in which the top half of the protein containing the 50 end of the cleaved strand (Figure 2, shown in green) lifts upward to generate a gate in the DNA through which another strand of DNA is passed. After strand passage, the broken strand is religated and the enzyme opens up a second time to release the strand that had been passed into the hole of the torus. A correlate of this model is that the linking number can only be changed in steps of one and this prediction has been veried biochemically. This same scheme can explain how the enzyme can catenate or decatenate a DNA containing a nick or gap. However, it is unclear whether the DNA that passes through the temporary gate in the cleaved strand is captured in the hole of the torus before strand cleavage and is then passed out of the hole after cleavage or vice versa as described above.

REVERSE GYRASE MECHANISM

Despite the presence of a helicase-like ATPase domain in the N-terminal region of reverse gyrases, these enzymes

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lack helicase activity when assayed under conditions that would require processive translocation along the DNA. Instead, the binding of the N-terminal domain to DNA is believed to simply unwind a region of the helix. Subsequently, in a reaction dependent on ATP, one of the two strands is cleaved and the other strand is passed through the resultant gate by the cleavage/strand passage domain present in the C-terminal half of the molecule. The key to positive supercoiling is that the strand passage event that occurs in the presence of ATP is directional such that the linking number of the DNA is increased and therefore the DNA ends up positively supercoiled. The structural basis for the unidirectional nature of the strand passage event remains unknown.

important core domain as can be seen from the color scheme in Figure 3 (blue boxes). Where present, the sequence of the mitochondrial enzyme is very similar to the nuclear enzyme with the exception of the N-terminal region (green box), which contains the organelle targeting signals. The eubacterial IB enzyme and the vaccinia topoisomerase are very similar to each other, but they lack most of the core domain as well as the conserved C-terminal domain that is characteristic of the other IB enzymes; instead, they share unique N-terminal and C-terminal regions (white and magenta boxes). The topoisomerase I found in the trypanosomatids is a heterodimer with one subunit containing the catalytic core (blue box) and the other subunit containing a region homologous to the C-terminal domain of the prototypic eukaryotic sequence (yellow box).

Type IB DNA Topoisomerases

DOMAIN STRUCTURE AND SEQUENCE CONSERVATION
Type IB DNA topoisomerases are present in the nucleus of all eukaryotic cells and the mitochondria of higher eukaryotes, as well as in at least one archaeon and some eubacteria (Table I). The typical eukaryotic type IB enzyme possesses the four domains shown in Figure 3. A highly charged and poorly conserved N-terminal domain (red box) is followed by the core domain (blue box), which binds DNA and contains most of the catalytic residues. The active site tyrosine is found in the C-terminal domain (yellow box) that is connected to the remainder of the protein by a poorly conserved linker region (orange box). All of the other type IB enzymes share at least partial sequence and structural homology with the catalytically

CRYSTAL STRUCTURE OF HUMAN TOPOISOMERASE I

Two views of the crystal structure of human topoisomerase I (missing the N-terminal domain) with a bound 22 base pair duplex oligonucleotide are shown in Figure 4. The protein is a bi-lobed structure that clamps completely around the DNA with the active site tyrosine (shown in black in Figure 4A) juxtaposed to the scissile phosphate. The linker region comprises the coiled-coil structure that protrudes conspicuously from the bottom portion of the enzyme and has an unknown function (Figure 4A). To release the DNA, the top half of the protein (shown in blue) must shift upward relative to the bottom half as indicated in Figure 4B by the doubleheaded arrow. Likewise, DNA binding requires that the protein clamp be in an open conformation. The region of

FIGURE 3 Domain structure and sequence relationships between type IB topoisomerases. The domain structure of the type IB topoisomerases from the indicated sources are denoted by colored boxes with similar domains aligned vertically. The names of the domains for the eukaryotic type IB topoisomerases are shown along the top. Regions that are similar in amino acid sequence share the same color; distinct sequences are assigned different colors. The two subunits of the heterodimeric topoisomerase I from trypanosomatids are shown with these same color conventions.

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FIGURE 4 The ribbon diagrams show two views of the crystal structure of human topoisomerase I clamped around a 22 base pair duplex DNA, shown in gray (pdb entry 1a36). The top lobe of the enzyme is shown in blue and the bottom lobe is shown in red and yellow. The poxviral topoisomerase and the eubacterial topoisomerases IB are structurally very similar to the region of the bottom lobe, shown in yellow. The coiled-coil linker region in bottom lobe (in red) is most easily seen in the side view shown in (A). The active site tyrosine (in black) is shown in (A). Panel (B) shows a view of the structure looking down the axis of the DNA. The double-headed arrow in (B) indicates the nature of the conformational change that is required to open and close the clamp during binding and release of the DNA.

the human topoisomerase I structure shared by the poxviral and eubacterial IB enzymes corresponds to the portion of the core domain depicted in yellow in Figure 4. Tyrosine recombinases such as the bacteriophage l and HP1 integrases, and cre recombinase are also structurally very similar to approximately this same region.

CATALYSIS
The co-crystal structure of human topoisomerase I with bound DNA reveals which amino acid residues in the protein are directly involved in catalysis. It is worth noting that the nucleophilic tyrosine O-4 does not appear to be activated for cleavage by general base catalysis, although a lysine residue acts as a general acid to protonate the leaving 50 oxygen. The pentavalent transition state is stabilized by hydrogen-bonding interactions between three basic amino acid side chains and the scissile phosphate oxygens. An interaction between a lysine residue and the base of the nucleotide where cleavage occurs is also important for catalysis. Religation is likely to proceed by a pathway that is essentially the reverse of cleavage.

DNA during DNA relaxation occurs by a rotational mechanism rather than by the enzyme-bridging mechanism described above for the type IA enzymes. However, there appears to be insufcient space within the connes of the enzyme to accommodate unrestricted rotation of the DNA. This feature of the structure suggests that the enzyme probably undergoes a conformational change after cleavage to open up the space downstream of the cleavage site to allow rotation. Unlike the enzyme-bridging model, a rotational mechanism places no a priori limit on the number of rotational events that can occur for each cycle of cleavage and religation. Indeed, in the case of vaccinia topoisomerase, ve rotations of the DNA occur on the average between each cleavage and religation reaction.

Cellular Roles
Although much is yet to be learned about how the various topoisomerases collaborate to manage DNA topology in the cell, a partial picture has emerged based on work in bacteria and simple eukaryotes. Although the type II topoisomerases are not the subject of this review, the activities of these enzymes are briey considered in the sections to follow for the sake of completeness. The type II enzymes are important for any cellular process that requires the passage of a region of duplex DNA through a double-strand break in the same or a different DNA molecule. The allocation of functions to

FOR

ROTATIONAL MECHANISM STRAND PASSAGE

Examination of the crystal structure of human topoisomerase I (Figure 4A) suggests that the strand passage reaction required to change the linking number of the

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the known topoisomerases in setting the global levels of supercoiling, in transcription and in DNA replication, are discussed below.

TABLE II Occurrence of DNA Topoisomerase IB in Eubacteria Known species possessing type IB topoisomerase Examples of species lacking type IB topoisomerase

TYPES OF SUPERCOILING IN EUKARYOTIC VERSUS PROKARYOTIC CELLS

Two different situations lead to the supercoiling of DNA in vivo. First, DNA will assume a supercoiled conguration through an interaction with certain proteins or other cellular components. Alternatively, a closed domain of DNA (e.g., a closed circular DNA) will spontaneously supercoil if the linking number is not the same as the helical winding (referred to as twist) of the DNA helix. This latter type of supercoil is often referred to as torsionally strained supercoils. The chromosomal DNA of eukaryotes is wrapped into a proteinconstrained solenoidal superhelix in nucleosomes and, except for the transient occurrence of torsionally strained supercoils associated with replication and transcription, is maintained in a relaxed state by DNA topoisomerases. However, in prokaryotes it appears that although some supercoils are constrained by virtue of an interaction with proteins as in eukaryotes, there exists, in addition, a xed steady-state level of torsionally strained supercoiling generated by gyrases (see below).

Mycobacterium avium Mycobacterium smegmatis Cytophaga hutchinsonii Agrobacterium tumefaciens Bradyrhizobium japonicum Mesorhizobium loti Sinorhizobium meliloti Rhodobacter sphaeroides Novosphingobium aromaticivorans Bordetella parapertussis Burkholderia fungorum Xanthomonas axonopodis Xanthomonas campestris Pseudomonas aeruginosa Pseudomonas uorescens Pseudomonas putida Pseudomonas syringae Deinococcus radiodurans Streptomyces coelicolor Chlamydia trachomatis Bacillus anthracis Bacillus subtilis Clostridium tetani Mycoplasma pneumoniae Listeria monocytogenes Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Caulobacter crescentus Rickettsia conorii Neisseria meningitidis Helicobacter pylori Escherichia coli Yersinia pestis Vibrio cholerae Xylella fastidiosa Haemophilus inuenzae Salmonella typhimurium Borrelia burgdorferi Treponema pallidum

GENERATION OF SUPERCOILING STRESS IN PROKARYOTES

Mesophilic Bacteria The DNA in all mesophilic eubacteria and archaebacteria contains torsionally strained negative supercoils that are introduced by the type II enzyme called DNA gyrase. It appears that this steady-state level of negative superhelicity is required to facilitate helix opening during the initiation of DNA replication and transcription. To prevent the introduction of excess negative supercoils by the gyrase, these bacteria also contain one or two type IA DNA topoisomerases (topoisomerases I or III or both) to counteract the effects of DNA gyrase. The inability of the type IA enzymes to remove negative supercoils below a critical threshold level prevents these enzymes from negating the effects of DNA gyrase and is crucial for ne-tuning the negative supercoiling levels in these organisms. Some eubacteria also contain a type IB enzyme (Table II), which could also balance the effects of DNA gyrase; but the apparent ability of these enzymes to completely relax the DNA suggests that their activity would have to be regulated in some way. The exact role played by the bacterial topoisomerase IB and why this enzyme is only present in a subset of the mesophilic eubacteria remains unknown.

Hyperthermophilic Bacteria All hyperthermophilic eubacteria and archaebacteria possess a reverse gyrase that actively maintains positive supercoils in the chromosomal DNA. It appears this positive supercoiling is necessary to stabilize the DNA helix against denaturation at the high growth temperatures of these organisms. The mechanism for preventing excess positive supercoiling is not known, but it is likely that a type II enzyme that can relax positive supercoils (DNA gyrase or the archaeal topoisomerase VI) counteracts the effects of reverse gyrase to set the nal steadystate level of positive supercoiling.

TRANSCRIPTION
During transcription, the movement of RNA polymerase along a DNA that is rotationally xed transiently generates positive supercoils in front of the translocating polymerase and negative supercoils behind the polymerase (Figure 5A). The type IA enzymes present in all organisms relax the negative supercoils that accompany transcription, but the mechanism for the removal of the positive supercoils depends on the organism.

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FIGURE 5 Topological transformations of DNA during transcription and replication. (A) The generation of positive supercoils ( ) in front of and negative supercoils (2) behind a translocating RNA polymerase during transcription are depicted. (B) Replication fork movement in a closed domain results in an overwinding of the DNA ahead of the replication fork and the interwinding of the two daughter helices to form precatenanes behind the replication fork, as shown in (B). Any precatenanes remaining at the end of replication for a circular replicon will result in the catenation of the two daughter circular molecules. A simple catenane with a single interlink is also shown.

In prokaryotes, positive supercoils are relaxed by a type II enzyme such as DNA gyrase or in the archaea by topoisomerase VI, and in the case of select eubacteria (see Table II) probably by a combination of a type II enzyme and the type IB topoisomerase. In eukaryotes, it is likely that the type IB DNA topoisomerase I relaxes the supercoils of both signs associated with transcription. Based on the known complete genome sequences, most eukaryotes, but not fungi or Caenorhabditis elegans, contain two distinct genes for type IB enzymes, one for the nuclear enzyme and a second for an enzyme that is imported into the mitochondrion to presumably function in transcription. In those eukaryotic organisms with only a single type IB enzyme, topoisomerase I likely plays a dual role, acting in both the nucleus and the mitochondrion.

relaxing positive supercoils in front of the fork, or by decatenating (or unlinking) the precatenanes behind the moving fork, or both. As in transcription, positive supercoils can be removed in prokaryotes by a type II topoisomerase such as the DNA gyrase, the archaeal topoisomerase VI, or, for those eubacteria that have it, topoisomerase IB. Precatenanes can be resolved by a type II topoisomerase, most notably topoisomerase IV in eubacteria or topoisomerase VI in archaea. As long as gaps exist during discontinuous DNA synthesis, precatenanes can also be unlinked in some eubacteria either by topoisomerase IB (Table II) or by the potent type IA decatenating enzyme, topoisomerase III. Segregation of true catenanes lacking nicks or gaps in either of the two strands can only be accomplished by a type II enzyme. In eukaryotes, the type IB topoisomerase relaxes the positive supercoils ahead of the replication fork and during synthesis can probably decatenate the precatenanes by acting at gaps in the DNA. However, most of the precatenanes and terminally interlinked or catenated structures are likely resolved by a type II topoisomerase. In mitochondria, replication-associated positive supercoils are relaxed by a type IB topoisomerase as described above for transcription. A mitochondrial type II topoisomerase and a type IA enzyme (topoisomerase III) are likely involved in unlinking precatenanes and catenated circular DNAs that occur during mitochondrial DNA replication.

SEE ALSO

THE

FOLLOWING ARTICLES

DNA Supercoiling DNA Topoisomerases: Type II

GLOSSARY
catenane Two interlocked circular DNA molecules in which the two duplexes are wound around each other one or more times. catenate The process whereby two circular DNAs are interlocked to form a catenane. The unlocking of catenated DNAs is referred to as decatenation. closed circular DNA Circular DNA in which both strands are intact. DNA gyrase DNA topoisomerase that couples the hydrolysis of ATP to the introduction of negative supercoils into a closed circular DNA. DNA supercoiling The coiling of the axis of a DNA molecule in three-dimensional space. Supercoiling may result from an interaction of the DNA with protein or from an inequality between the number of helical turns dictated by the structure of the DNA helix under a particular set of conditions (the twist of the DNA) and the linking number of the DNA. linking number Topological property of a closed circular DNA that is a measure of the xed interwinding of the two DNA strands. precatenanes The interwinding of the two daughter duplexes behind a replication fork. reverse gyrase DNA topoisomerase that couples the hydrolysis of ATP to the introduction of positive supercoils into a closed circular DNA.

DNA REPLICATION
As the two DNA strands are separated during DNA replication, the DNA helix in front of the replication fork becomes at least transiently overwound or positively supercoiled (Figure 5B). This overwinding of the helix has been shown to be at least partially transmitted to the region behind the replication fork to cause an interwinding of the two daughter helices. These interwindings are referred to as precatenanes (Figure 5B), since in a circular replicon, if any remain at the end of replication, the two daughter circular molecules will be catenated. Resolution of the overwound structure of a replicating chromosome can be accomplished by

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scissile strand The strand of DNA that is cleaved by a type I topoisomerase. topoisomerase Enzyme that changes the linking number of a closed circular DNA by temporarily breaking one (type I) or both (type II) of the strands of the DNA. topoisomers Variants of a closed circular DNA that have different linking numbers. torsionally strained supercoils Supercoils that result from an inequality between the number of helical turns dictated by the structure of the DNA helix under a particular set of conditions and the linking number of the DNA.

Champoux, J. J. (2001). DNA topoisomerases: Structure, function, and mechanism. Annu. Rev. Biochem. 70, 369 413. Wang, J. C. (1996). DNA topoisomerases. Annu. Rev. Biochem. 65, 635692. Wang, J. C. (2002). Cellular roles of DNA topoisomerases: A molecular perspective. Nat. Rev. Mol. Cell. Biol. 3, 430 440.

BIOGRAPHY
James J. Champoux is a Professor in the Department of Microbiology in the School of Medicine at the University of Washington. His research focuses on topoisomerases and reverse transcription. He holds a Ph.D. from Stanford University and carried out his postdoctoral work at the Salk Institute in San Diego, California. He discovered the eukaryotic type IB topoisomerase and was the rst to show that the reaction proceeds through an enzyme DNA covalent intermediate. He has been instrumental in elucidating the roles of the RNase H activity of reverse transcriptase in retroviral replication.

FURTHER READING
Alexandrov, A. I., Cozzarelli, N. R., Holmes, V. F., Khodursky, A. B., Peter, B. J., Postow, L., Rybenkov, V., and Vologodskii, A. V. (1999). Mechanisms of separation of the complementary strands of DNA during replication. Genetica 106, 131140.

DNA Topoisomerases: Type II

Renier Velez-Cruz and Neil Osheroff
Vanderbilt University School of Medicine, Nashville, Tennessee, USA

Although the genetic information of an organism is encoded by the linear array of DNA bases that make up its genome, the three-dimensional properties of the double helix dramatically affect how this information is expressed and passed from generation to generation. Some of the most important threedimensional relationships in the genetic material are topological in nature, including DNA under- and overwinding, knotting, and tangling. The enzymes that modulate the topological properties of DNA are termed DNA topoisomerases. There are two classes of topoisomerases, type I and type II, which are dened by their reaction mechanisms. Type I topoisomerases alter DNA topology by creating a transient single-stranded break in the genetic material and facilitating controlled rotation of the double helix about (or strand passage through) the nick. Type II topoisomerases act by passing an intact double helix through a transient doublestranded break that they generate in a separate DNA segment. As a consequence of their reaction mechanisms, both classes of enzymes can regulate DNA under- and overwinding. However, because type II topoisomerases cut both strands of the double helix, they also are able to resolve knots and tangles in the genetic material. Type II topoisomerases are essential to all species. Beyond their critical physiological functions, these enzymes are the targets for some of the most important anticancer and antibacterial drugs in clinical use.

about itself to form superhelical twists. Negative supercoiling puts energy into the genetic material and makes it easier to separate the two strands of the double helix for replication and transcription. Thus, DNA underwinding dramatically increases the rates of these two fundamental processes. In contrast, the movement of DNA tracking systems (such as replication forks and transcription complexes) through the double helix locally overwinds the DNA ahead of their actions. Because overwinding makes it much harder to pull apart the double helix, it blocks many essential cellular processes. The second issue is related to the extreme length of genomic DNA. Nucleic acid knots (intramolecular) and tangles (intermolecular) are formed routinely during a variety of ongoing cellular processes including DNA recombination and replication. Both knots and tangles must be resolved in order for daughter chromosomes to segregate properly during meiosis and mitosis.

DNA Topoisomerases
Cells contain ubiquitous enzymes known as DNA topoisomerases that maintain the appropriate level of DNA supercoiling and remove knots and tangles from the genetic material. These enzymes modulate the topological structure of the genetic material by creating transient breaks in the backbone of DNA. There are two classes of topoisomerases that can be distinguished by the number of DNA strands that they cleave during their catalytic cycles. Type I enzymes create transient singlestranded DNA breaks, whereas type II enzymes create transient double-stranded breaks. To maintain genomic integrity during their DNA cleavage events, topoisomerases form covalent linkages between active-site tyrosyl residues and the newly generated DNA termini. These covalent protein-cleaved DNA complexes, known as cleavage complexes, are the hallmarks of all topoisomerases irrespective of enzyme classication. Because type I topoisomerases create single-stranded breaks in the genetic material, they can regulate DNA supercoiling. However, because type II topoisomerases generate double-stranded breaks in the DNA backbone, they can

DNA Topology
The topological properties of DNA are dened as those that cannot be altered without breaking one or both strands of the double helix. Because DNA comprises two interwound nucleic acid strands and the genomes of all known organisms are very long or circular (or both), two distinct topological issues arise as a result of the genetic material. Proliferating cells must be able to cope with both of these in order to survive. The rst issue is related to the torsional stress on the double helix. The DNA from all species of eukaryotes and eubacteria is globally underwound , 5 10%. DNA under torsional stress is termed supercoiled (underwound molecules are negatively supercoiled and overwound molecules are positively supercoiled) because underwound or overwound DNA writhes

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resolve knots and tangles in addition to removing torsional stress from the genetic material. Type II topoisomerases are essential to all eukaryotic and prokaryotic organisms. They are highly conserved among species, and the eukaryotic enzymes appear to be direct descendents of ancestral bacterial proteins.

Eukaryotic Type II Topoisomerases

The eukaryotic type II enzyme is called topoisomerase II. It was discovered in 1980 and is a member of the type IIA homology subfamily. Topoisomerase II can remove positive and negative superhelical twists from the double helix and can resolve DNA knots and tangles.

ENZYME MECHANISM
Topoisomerase II interconverts different topological forms of DNA by the double-stranded DNA passage reaction depicted in Figure 1, which shows the products of each individual step. Briey, it is proposed that topoisomerase II (1) binds two DNA segments, (2) creates

a double-stranded break in one of the segments, (3) translocates the other DNA segment through the cleaved double helix, (4) rejoins (i.e., ligates) the cleaved DNA, (5) releases the translocated segment through a gate in the protein, and (6) closes the protein gate and regains the ability to start a new round of catalysis. The scissile bonds on the two strands of the double helix that are cut by topoisomerase II are staggered. Thus, the enzyme generates cleaved DNA molecules that contain four-base single-stranded ends at their 50 -termini. During its cleavage event, topoisomerase II covalently attaches to these newly generated 50 -termini. Topoisomerase II requires two cofactors in order to carry out its catalytic double-stranded DNA passage reaction. First, it needs a divalent cation for all steps beyond enzyme-DNA binding (Figure 1, complex 1). Magnesium(II) appears to be the divalent cation that the enzyme uses in vivo. Second, topoisomerase II uses the energy of adenosine triphosphate (ATP) to drive the overall DNA strand passage reaction. Although ATP is not required for either DNA cleavage or ligation, the binding of this nucleoside triphosphate triggers DNA translocation (which converts complex 2 to complex 3)

FIGURE 1 Catalytic cycle of type IIA topoisomerases. The complete double-stranded DNA passage reaction is shown as a series of discrete steps (the products of each step are shown). (1) EnzymeDNA binding, (2) DNA cleavage (formation of cleavage complex), (3) doublestranded DNA passage, (4) DNA ligation, (5) gate opening and release of the translocated DNA helix, and (6) enzyme recycling. The protein (shown in blue) is based on the crystallographic structure of the catalytic core of yeast topoisomerase II. Modeled DNA helices are shown in green (horizontal) and orange (coming out of the plane of the paper). Structures are courtesy of Dr. James M. Berger, University of California, Berkeley.

808

DNA TOPOISOMERASES: TYPE II

and its hydrolysis to adenosine diphosphate (ADP) and inorganic phosphate (Pi) is necessary for enzyme recycling (which converts complex 5 to complex 6). Normally, topoisomerase II binds two molecules of ATP. Although hydrolysis of the cofactor is not a prerequisite for the strand passage event, it appears that this step proceeds more rapidly if it is preceded by hydrolysis of one of the bound ATP molecules.

ENZYME DOMAIN STRUCTURES

AND ISOFORMS

Eukaryotic type II topoisomerases are homodimeric enzymes with protomer molecular masses ranging from , 160 to 180 kDa (depending on the species). On the basis of amino-acid-sequence comparisons with the bacterial type II enzyme, DNA gyrase, each enzyme monomer can be divided into three distinct domains (Figure 2). The N-terminal domain of the enzyme is homologous to the B-subunit of DNA gyrase (GyrB) and

contains consensus sequences for ATP binding. The central domain is homologous to the A-subunit of DNA gyrase (GyrA) and contains the active-site tyrosyl residue that forms the covalent bond with DNA during scission. The C-terminal domain is not highly conserved and appears to have no corresponding region of homology with DNA gyrase. This variable region of the eukaryotic enzyme contains nuclear localization sequences as well as amino acid residues that are phosphorylated in vivo. Although some eukaryotic species such as yeast and Drosophila appear to have only a single type II topoisomerase (i.e., topoisomerase II), vertebrates contain two closely related isoforms, topoisomerase IIa and b. These two isoforms share extensive amino acid sequence identity (, 70%), but are encoded by separate genes (located at chromosomal bands 17q21 22 and 3p24 in humans, respectively) and can be distinguished by their protomer molecular masses (, 170 and , 180 kDa, respectively).

Human topoisomerase IIa NH2 ATP binding motifs ATPase DNA cleavage/ligation Tyr805 NLS and PO4 -COOH 1531

E. coli DNA gyrase

NH2 GyrB -COOH NH2 804 Tyr122 GyrA -COOH 875

E. coli topoisomerase IV
NH2 ParE A -COOH NH2 637 Tyr120 ParC -COOH 766

S. shibatae topoisomerase VI
NH2 -COOH NH2 530 Top6B B
FIGURE 2 Domain structures of type II topoisomerases. (A) The domain structures of three type IIA topoisomerases: human topoisomerase IIa and bacterial (Escherichia coli) DNA gyrase and topoisomerase IV. Regions of homology among the enzymes are indicated by colors. The N-terminal (i.e., GyrB) homology domains contain the regions responsible for ATP binding and hydrolysis. The vertical white stripes represent the three conserved motifs of the Bergerat fold that dene the ATP-binding domain. The central (i.e., GyrA) homology domains contain the active site tyrosyl residue (Tyr805 in human topoisomerase IIa) that forms the covalent bond with DNA during scission. For human topoisomerase IIa, the variable C-terminal domain contains nuclear localization sequences (NLS) and phosphorylation sites (PO4). (B) The Top6A and Top6B subunits of the archaeal type IIB topoisomerase, Sulfolobus shibatae topoisomerase VI, shown for comparison.

-COOH Tyr106 Top6A 389

DNA TOPOISOMERASES: TYPE II

809

PHYSIOLOGICAL FUNCTIONS
Topoisomerase II plays a number of essential roles in eukaryotic cells and participates in virtually every major process that involves the genetic material. It unlinks daughter chromosomes that are tangled following replication and resolves DNA knots that are formed during recombination. It also helps to remove the positive DNA supercoils that are generated ahead of replication forks and transcription complexes. Topoisomerase II is required for proper chromosome condensation, cohesion, and segregation and appears to play roles in centromere function and chromatin remodeling. Finally, the type II enzyme is important for the maintenance of proper chromosome organization and structure, and it is the major nonhistone protein of the mitotic chromosome scaffold and the interphase nuclear matrix. It is not obvious why vertebrate species possess two distinct topoisomerase II isoforms. Enzymological differences between topoisomerase IIa and b are subtle and the relationships between these isoforms are not well dened. Although either enzyme can complement yeast strains lacking topoisomerase II activity, topoisomerase IIa is essential for proliferating mammalian cells and its loss cannot be compensated by the b isoform. Topoisomerase IIb appears to be dispensable at the cellular level, but is required for proper neural development in mice. The specic cellular functions of topoisomerase IIa and b probably reect their physiological regulation more than their enzymological characteristics. Topoisomerase IIa is regulated over both cell and growth cycles. Enzyme levels increase throughout the S-phase of the cell cycle and peak at the G2-M boundary. Furthermore, this isoform is found almost exclusively in rapidly proliferating tissues. In contrast, the concentration of topoisomerase IIb is independent of the cell cycle and this isoform is found in most cell types regardless of proliferation status. Taken together, these characteristics suggest that topoisomerase IIa is the isoform responsible for events associated with DNA replication and chromosome segregation, whereas topoisomerase IIb is the isoform that probably functions in ongoing nuclear processes.

DNA GYRASE
DNA gyrase was discovered in 1976. It was the rst type II topoisomerase to be described and is the only one to retain its historical name (in the modern nomenclature, type II topoisomerases are denoted by even numbers). In contrast to the eukaryotic type II enzymes, DNA gyrase is comprised of two distinct subunits, GyrA and GyrB (molecular mass < 96 kDa and 88 kDa, respectively) and is arranged as an A2B2 tetramer. GyrA contains the active site tyrosine used in DNA cleavage and ligation, and GyrB contains the binding site for ATP (Figure 2A). In contrast to all other type II topoisomerases, DNA gyrase is the only enzyme that is capable of actively underwinding (i.e., negatively supercoiling) the double helix. It accomplishes this feat by wrapping DNA around itself in a right-handed fashion and carrying out its strand-passage reaction in a unidirectional manner. The negative supercoiling activity of DNA gyrase far exceeds the ability of the enzyme to remove either knots or tangles from the genetic material. Consequently, the major physiological roles of DNA gyrase stem directly from its ability to underwind the double helix. DNA gyrase plays a critical role in opening DNA replication origins and removing positive supercoils that accumulate in front of replication forks and transcription complexes. In addition, this enzyme works in conjunction with the v protein (a type I topoisomerase that removes negative supercoils from the double helix) to maintain the global balance of DNA supercoiling in bacterial cells.

TOPOISOMERASE IV
Topoisomerase IV is an A2B2 tetramer that is comprised of two distinct subunits, ParC (molecular mass < 88 kDa), and ParE (molecular mass < 70 kDa), which are homologous to the A- and B-subunits of DNA gyrase (Figure 2B). (In gram-positive bacterial species, the subunits of topoisomerase IV are designated GrlA and GrlB, respectively.) It was known for several years that the ParC and ParE proteins were necessary for proper chromosome segregation in bacteria. However, it was not discovered until 1990 that these two subunits together constituted a type II topoisomerase. The catalytic properties of topoisomerase IV can be distinguished from those of DNA gyrase in two important ways. First, although topoisomerase IV can remove positive and negative superhelical twists from DNA, it cannot actively underwind the double helix. Second, the ability of topoisomerase IV to resolve DNA knots and tangles is dramatically better than that of DNA gyrase. Because of these differences, the physiological roles of topoisomerase IV are distinct from those of DNA gyrase. The primary cellular functions of topoisomerase IV are to unlink daughter chromosomes following DNA replication and to resolve DNA knots

Prokaryotic Type II Topoisomerases

Eubacteria contain two distinct type II topoisomerases, DNA gyrase and topoisomerase IV. Both are members of the type IIA subfamily. In addition to these two enzymes, many archaeal species contain a third type II enzyme, topoisomerase VI. This last enzyme is a member of the type IIB subfamily.

810

DNA TOPOISOMERASES: TYPE II

that are formed during recombination. Recently, it was found that topoisomerase IV removes positive supercoils from DNA more efciently than it removes negative supercoils. This has led to speculation that the enzyme also may act ahead of DNA tracking systems to alleviate overwinding of the double helix. However, the precise role of topoisomerase IV in this process has yet to be dened.

evidence that Spo11 has topoisomerase (i.e., DNA strand passage) activity.

Type II Topoisomerases as Therapeutic Targets

In addition to their varied and critical physiological functions, the type IIA topoisomerases are targets for some of the most active anticancer and antibacterial drugs in clinical use. In contrast to most enzymetargeted drugs, these agents do not act by robbing cells of an essential enzyme activity. Rather, drugs that target type II topoisomerases kill cells by dramatically increasing the concentration of covalent enzyme-cleaved DNA complexes (i.e., cleavage complexes) that are requisite intermediates formed during the double-stranded DNA passage reaction. Normally, cleavage complexes are present at low steady-state levels and are tolerated by cells. However, conditions that signicantly increase either their concentration or lifetime trigger numerous mutagenic events. The potential lethality of cleavage complexes rises dramatically when DNA tracking enzymes such as polymerases or helicases attempt to traverse the covalently bound topoisomerase roadblock in the genetic material. Such an action disrupts cleavage complexes and converts transient enzyme-mediated DNA breaks to permanent DNA breaks. These permanent breaks in the genome trigger the generation of chromosomal insertions, deletions, translocations, and other aberrations, and, when present in sufcient numbers, they initiate a series of events that culminates in cell death. Because the drugs that target type II topoisomerases convert these essential enzymes to potent cellular toxins that fragment the genome, they are referred to as topoisomerase poisons to distinguish them from drugs that act as catalytic inhibitors.

ARCHAEAL TOPOISOMERASE VI
In 1997, a novel type II topoisomerase, topoisomerase VI, was discovered in hyperthermophilic archaeal species. This enzyme was designated as the rst member of the topoisomerase IIB subfamily due to its lack of homology to previously identied type II enzymes. Topoisomerase VI has two subunits, Top6A and Top6B (molecular masses < 47 and 60 kDa, respectively), and is arranged as an A2B2 tetramer. Both subunits are considerably smaller than those of bacterial DNA gyrase or topoisomerase IV (Figure 2B). Although short regions of Top6B surrounding the ATP-binding domain are homologous to portions of GyrB, and Top6A contains an active-site tyrosine that is required for DNA cleavage, the primary structure of topoisomerase VI displays little similarity to the type IIA enzymes. Archaeal topoisomerase VI appears to alter DNA topology by using a double-stranded DNA passage reaction like that described for other type II topoisomerases. During this reaction, it generates DNA breaks with 50 overhangs that are covalently attached to its active-site tyrosyl residues. Topoisomerase VI relaxes positively and negatively supercoiled DNA, but cannot actively underwind the double helix. In addition, it can unlink (i.e., untangle) interwound double-stranded DNA circles. The catalytic properties of topoisomerase VI differ from those of the type IIA enzymes in two signicant aspects. First, topoisomerase VI requires ATP binding in order to cleave its DNA substrate. Second, in marked contrast to the type IIA enzymes (which produce fourbase staggered ends during scission), topoisomerase VImediated DNA cleavage generates DNA termini that contain only two-base overhangs. Although the physiological functions of topoisomerase VI have yet to be determined, the enzyme is believed to play a role in unlinking daughter chromosomes following replication in archaeal cells. With the exception of plants, no Top6B homologue has been identied in eukaryotic species. However, a Top6A homologue, Spo11, has been found in eukaryotes ranging from yeast to humans. Spo11 generates the double-stranded DNA breaks that initiate meiotic recombination. Like its topoisomerase relatives, Spo11 forms a covalent bond between an active-site tyrosyl residue and the 50 -DNA termini generated by its scission reaction. At the present time there is no

ANTICANCER DRUGS
At the present time, six topoisomerase II-targeted anticancer agents (Figure 3A) are approved for use in the United States. Drugs such as etoposide and doxorubicin are front-line therapy for breast and lung cancers, as well as for a variety of leukemias, lymphomas, and germ-line malignancies. Approximately onehalf of all cancer chemotherapy regimens contain drugs targeted to topoisomerase II. Moreover, every form of cancer that can be cured by systemic chemotherapy is treated with these agents. Due to the high concentration of topoisomerase IIa in rapidly proliferating cells, this isoform probably is the major important target of anticancer therapy. However, circumstantial evidence suggests that the b-isoform also contributes to drug efcacy.

DNA TOPOISOMERASES: TYPE II

811

R O O O HO OH O O O H O O H R2

OH

O C R1 OH

OH O NH3
+

H 3C OH

R1 CH2OH CH3 CH3

R2 OCH3 OCH3 H

H3CO OH R CH3

OCH3

Doxorubicin: Danorubicin: Idarubicin:

O

Etoposide: Teniposide:

OH S

HN(CH2)2NH(CH2)2OH

OH

HN(CH2)2NH(CH2)2OH

A
O F N HN N

Mitoxantrone
O COOH F N H 3C N O CH3 N COOH

cell cycle The process by which a cell grows, replicates its genome, and divides. The cell cycle is divided in four distinct phases: G1, a growth phase; S, the phase in which the cell duplicates (i.e., synthesizes) its genetic material; G2, a second growth phase in which the cell prepares to divide; and M, the phase in which the cell divides (i.e., mitosis). DNA recombination The process by which the cell reorganizes its genetic material in order to repair certain forms of DNA damage (including double-stranded DNA breaks) or promote genetic diversity. DNA replication The process by which the cell duplicates (i.e., synthesizes) its genetic material. DNA supercoiling The underwinding (i.e., negative supercoiling) or overwinding (i.e., positive supercoiling) of the genetic material. topoisomerase poison A drug that increases levels of topoisomerasecleaved DNA complexes. topology A eld of mathematics that deals with relationships that are not altered by elastic deformation. transcription The process by which the cell expresses its genetic material; the generation of messenger RNAs from a DNA template.

FURTHER READING
Anderson, V. E., and Osheroff, N. (2001). Type II topoisomerases as targets for quinolone antibacterials: Turning Dr. Jekyll into Mr. Hyde. Curr. Pharm. Des. 7, 337 353. Champoux, J. J. (2001). DNA topoisomerases: Structure, function, and mechanism. Annu. Rev. Biochem. 70, 369 413. Fortune, J. M., and Osheroff, N. (2000). Topoisomerase II as a target for anticancer drugs: When enzymes stop being nice. Prog. Nucl. Acid Res. Mol. Biol. 64, 221253. Gadelle, D., Filee, J., Buhler, C., and Forterre, P. (2003). Phylogenomics of type II DNA topoisomerases. BioEssays 25, 232242. Heddle, J. G., Barnard, F. M., Wentzell, L. M., and Maxwell, A. (2000). The interaction of drugs with DNA gyrase: A model for the molecular basis of quinolone action. Nucleosides Nucleotides Nucleic Acids 19, 12491264. Li, T. K., and Liu, L. F. (2001). Tumor cell death induced by topoisomerase-targeting drugs. Annu. Rev. Pharmacol. Toxicol. 41, 5377. Osheroff, N (ed.) (1998). DNA topoisomerases. Biochim. Biophys. Acta 1400. Wang, J. C. (2002). Cellular roles of DNA topoisomerases: A molecular perspective. Nat. Rev. Mol. Cell. Biol. 3, 430 440.

Ciprofloxacin

Levofloxacin

FIGURE 3 Structures of selected (A) anticancer drugs targeted to topoisomerase II and (B) antibacterial drugs targeted to DNA gyrase and topoisomerase IV.

ANTIBACTERIAL DRUGS
DNA gyrase and topoisomerase IV are the targets for quinolone-based antibacterial agents (Figure 3B). Quinolones are the most active and broad-spectrum antibacterial drugs currently available. Drugs such as ciprooxacin are prescribed routinely for a wide variety of gram-negative bacterial infections, including gastrointestinal tract, respiratory tract, and bone and joint infections. Ciprooxacin also is used to treat a number of sexually transmitted diseases as well as infection with anthrax. Newly developed quinolones, such as levooxacin, display signicant efcacy against gram-positive bacterial infections. DNA gyrase is the primary cytotoxic target of quinolones in gram-negative bacteria. However, topoisomerase IV appears to be the more important target for many of these drugs in gram-positive species.

BIOGRAPHY
Renier Velez-Cruz is completing his doctoral studies in the Department of Biochemistry, Vanderbilt University School of Medicine. Neil Osheroff is a Professor in the Departments of Biochemistry and Medicine at the Vanderbilt University School of Medicine and holds the John G. Coniglio Chair in Biochemistry. His principal research interests are the elds of DNA topoisomerases, topoisomerase-targeted drugs, and DNA repair. He holds a Ph.D. in Biochemistry and Molecular Biology from Northwestern University and received his postdoctoral training in the Department of Biochemistry at the Stanford University School of Medicine. He has authored more than 170 articles and has contributed signicantly to our understanding of the mechanism of action of type II topoisomerases and topoisomerase II poisons.

SEE ALSO

THE

FOLLOWING ARTICLES

DNA Supercoiling DNA Topoisomerases: Type I

GLOSSARY
adenosine triphosphate (ATP) A cofactor that supplies energy for many enzymatic processes.

DNA Topoisomerases: Type IIIRecQ Helicase Systems

Rodney Rothstein and Erika Shor
Columbia University College of Physicians and Surgeons, New York, USA

DNA helicases and topoisomerases belong to the category of proteins that physically manipulate and alter the structure of DNA molecules. DNA helicases are enzymes that separate the strands of double-stranded (ds) DNA molecules, thus catalyzing DNA unwinding. Topoisomerases transiently create breaks in a DNA strand(s), pass other strands through the broken strand(s), and reseal the breaks. Topoisomerase activity can change levels of DNA supercoiling or result in catenation (interlinking) or decatenation (unlinking) of two DNA molecules. Both DNA helicases and topoisomerases are key players in various DNA transactions, such as replication, transcription, and recombination. Different classes and families of helicases and topoisomerases have been identied based on their protein sequence conservation, substrate preference, directionality on DNA, and other properties. DNA helicases of the RecQ family have garnered much interest lately because of the involvement of three human RecQ helicase family members in genetic disorders characterized by genomic instability and cancer predisposition. RecQ helicases are evolutionarily conserved proteins found in organisms ranging from bacteria to humans. Interestingly, an association between RecQ-type helicases and type III topoisomerases has been observed throughout the evolutionary tree, suggesting that these two proteins act in concert to promote genomic stability.

formed. After passage of other DNA strand(s) through the break, the reverse transesterication reaction leads to the rejoining of the DNA backbone. Topoisomerase III activity can result in relaxation/removal of negative supercoiling from DNA, (de)catenation and knotting of ss circular DNA molecules, and (de)catenation of ds DNA molecules that contain ss regions. Topoisomerase III does not require energy in the form of nucleoside triphosphates, such as ATP. Hence, the directionality of the topoisomerase III-driven reactions is toward the DNA conformation with the lowest free energy.

REC Q HELICASES
The RecQ family of DNA helicases is dened by homology to the bacterial RecQ protein. Like other helicases, these proteins contain seven signature helicase motifs, including sequences that contain Walker A (required for ATP binding and hydrolysis) and B boxes. In addition to the core helicase motifs, all RecQ helicases share additional regions of homology not shared by other families of helicases. The functional unit of a helicase is generally composed of a dimer or a hexamer that forms a ring around its substrate DNA. However, examples of monomeric helicases are also known. Whereas several early studies have indicated that RecQ-like helicases form hexamers, two recent studies suggest that DNA helicase activities in vitro of both E. coli RecQ and human BLM proteins are associated with a monomeric form of the protein. Thus, the composition of a functional unit of RecQ-like helicases is still being explored. All RecQ-type helicases examined to date display 30 ! 50 directionality on DNA with respect to the strand to which the protein is bound (Figure 2A). In vitro substrate preference studies have indicated that these helicases can act on a variety of DNA structures, possibly reecting the diversity of their in vivo activities. Among the structures that RecQ helicases can unwind are branched molecules, four-way dsDNA junctions, G-quartets, and D-loops (Figure 2B). ATP and Mg2 are necessary cofactors for RecQ-driven reactions.

Structure and Molecular Mechanisms

TOPOISOMERASE III
Topoisomerase III belongs to the type IA topoisomerases (also known as type I-50 ). This subfamily of topoisomerases acts on DNA that is negatively supercoiled (underwound) and/or contains single-stranded (ss) regions. The topoisomerase, which functions as a monomer, makes a break in a ssDNA region via a transesterication reaction between an active site tyrosine of the enzyme and a DNA phosphate group (Figure 1). A transient covalent linkage between the tyrosine and the 50 -phosphoryl group of the DNA is thus

Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved.

812

DNA TOPOISOMERASES: TYPE III RecQ HELICASE SYSTEMS

813

FIGURE 1 The mechanism of strand passage by a type IA topoisomerase. The topoisomerase makes a break in the blue strand by catalyzing a trans-esterication reaction between the tyrosine at the topoisomerase active site and a 50 phosphate group of the DNA. Only the reacting phosphate is depicted as a P for clarity. The intact red strand is passed through the break, and a reverse trans-esterication reaction reseals the blue strand.

The RecQ Helicase Topoisomerase III Connection

PHYSICAL ASSOCIATION
Two widely used model organisms, budding and ssion yeast, each has one RecQ-like helicase and one topo III. In both organisms, the two proteins have been shown to physically interact with each other and/or to associate in cellular extracts. In human cells, the RecQ homolog BLM physically interacts with an isoform of topoisomerase III, Top3a, via the N-terminus of BLM. Mutation or deletion of BLM in

human cells has several detrimental consequences on genome stability, including a marked increase in recombination between sister chromatids. Interestingly, cells expressing BLM protein that lacks its N-terminus and fails to interact with Top3a (but retains DNA unwinding activity) show increased levels of sister chromatid exchange, similar to cells that lack the entire BLM protein. This demonstrates that, in order to perform its role in suppressing recombination between sister chromatids, BLM needs to interact with Top3a in vivo, which supports other evidence that the two act as a complex with key roles in maintenance of genome stability.

FIGURE 2 (A) RecQ helicases unwind DNA in the 30 to 50 direction with respect to the strand to which the helicase is bound. (B) Several of the known in vitro substrates of RecQ helicases before and after incubation with the protein.

814

DNA TOPOISOMERASES: TYPE III RecQ HELICASE SYSTEMS

GENETIC INTERACTIONS

IN

YEAST

As mentioned above, the genomes of both budding and ssion yeast encode a single topoisomerase III gene. In both organisms, loss or mutation of the topo III protein is detrimental, resulting in slow growth or lethality. Concomitant loss of RecQ helicase function in mutants lacking topo III largely restores normal growth and viability. These genetic observations have led to the following model of RecQ Topoisomerase III interaction in vivo. It is proposed that in wild-type cells, the activity of the RecQ helicase creates a DNA structure that is normally acted on and resolved by topo III. When topo III is inactivated, this structure is not processed properly and causes slow growth or lethality. In mutants lacking the RecQ helicase, this structure is not created, eliminating the need for a functional topo III. While this model is supported by genetic evidence, experimental data conrming the existence of such a DNA structure are lacking to date.

bacterial RecQ helicase to fully catenate or decatenate (depending on the conditions of the reaction) covalently closed circular dsDNA molecules. In vivo, a decatenation activity may be used to separate such interlinked dsDNA substrates as sister chromatids following DNA replication or homologous chromosomes following mitotic or meiotic recombination. Their complete decatenation is essential for subsequent faithful chromosomal segregation, which in turn ensures accurate transfer of genetic material into daughter cells. On the other hand, a catenating activity may be used in vivo to suppress recombination, as DNA sequences that are highly catenated undergo fewer recombination events.

Cellular Roles of the RecQ TopoIII Complex in DNA Metabolism

THE RECOMBINATION CONNECTION
Inactivation of a RecQ-type helicase or topoisomerase III generally causes a variety of detrimental consequences on the genomic integrity of an organism. One consequence that is shared by all organisms examined to date is an increase in genetic recombination. As mentioned above, hyper-recombination between sister chromatids is a hallmark of human cells expressing mutant versions of BLM protein. In budding yeast, inactivation of the RecQ homolog Sgs1 or of the topo III homolog Top3 leads to an increase in several kinds of recombination, such as recombination between tandemly repeated DNA sequences. In E. coli, inactivation of the recQ gene leads to an increase in illegitimate recombination (i.e., recombination between nonhomologous DNA sequences). These observations have suggested that RecQ helicases generally control or suppress recombination. This idea is supported by a series of genetic experiments with budding yeast and mirrored by observations in ssion yeast. In these organisms, concomitant mutation of the RecQ family member and of another helicase, Srs2, causes extreme slow growth and frequent lethality. However, these defects are fully rescued by mutation of any of several proteins that perform homologous recombination. This indicates that in the absence of a functional homologous recombination pathway, deletion of the two helicases is no longer detrimental to the cell. This can be explained by proposing that in the absence of the two helicases, incorrectly regulated or uncontrolled recombination is responsible for the poor growth and increased lethality. Physical interactions between RecQ helicases and proteins that function in recombination add further support to the connection between the RecQ family and recombination. In both yeast and humans, a RecQ

COMBINED MODE

OF

ACTION

The genetic and physical interactions between RecQ-like helicases and type III topoisomerases in several organisms have prompted investigation and speculation regarding their combined mode of action. One possibility rst put forth upon the discovery of the budding yeast RecQ homolog Sgs1 and its genetic and physical association with the budding yeast topo III was that the two proteins form a complex that functions like a eukaryotic reverse gyrase. A reverse gyrase is an enzyme found in several species of Archaea. A hallmark feature of the reverse gyrase protein is the presence of a type I topoisomerase-like domain and a helicase domain as part of the same polypeptide. In vitro, the enzyme introduces positive supercoiling into dsDNA molecules. This is thought to happen in the following manner. The helicase domain of reverse gyrase unwinds a region of dsDNA, creating local negative supercoiling on one side of the enzyme and local positive superoiling on the other. The topoisomerase domain relaxes the negative supercoiling, resulting in a net overall increase in positive supercoiling in the dsDNA molecule. However, despite the attractiveness of the hypothesis that the RecQ topoIII complex resembles reverse gyrase, the few in vitro studies done on RecQ and topo III have not reported increased positive supercoiling as a result of the combined action of the two enzymes. Another hypothesis regarding the role of the RecQ topoIII complex suggests a role in catenation or decatenation of ds DNA molecules. This idea is supported by a study that examined the consequences of concerted action of bacterial RecQ and topo III in an in vitro system. It was shown that both bacterial and yeast topo III proteins are specically stimulated by the

DNA TOPOISOMERASES: TYPE III RecQ HELICASE SYSTEMS

815

family member was shown to physically interact with Rad51, a protein that performs key steps in homologous recombination, such as the invasion of a DNA duplex by a homologous single strand and subsequent branch migration. Also, biochemical activities of several RecQ helicases are consistent with a direct role in suppressing recombination. Human BLM and yeast Sgs1 proteins efciently unwind or branch-migrate four way DNA junctions in vitro (Figure 2B). Interestingly, four-way junctions closely resemble the structure of an intermediate in genetic recombination, a Holliday junction. Another human RecQ homolog, WRN, unwinds D-loops, DNA intermediates formed by the rst step of homologous recombinationthe strand invasion of a duplex by a ssDNA molecule (Figure 2B). Thus, RecQ helicases may control recombination in vivo by disrupting recombination intermediates.

THE REPLICATION CONNECTION

Several lines of evidence indicate that RecQ-like helicases and topo III function during DNA replication. Analyses of mRNA and protein levels of the budding yeast RecQ and topo III homologs have shown that expression of these genes uctuates throughout the cell cycle, peaking during and right after the period of active DNA replication. Also, yeast mutants lacking the RecQ helicase or topo III are sensitive to chemicals that arrest DNA replication by depleting cellular pools of deoxynucleotide triphosphates (dNTPs)the building blocks necessary to assemble new DNA molecules. For instance, wild-type ssion yeast cells are able to resume normal replication after transient exposure to one such chemical, hydroxyurea, whereas mutants that lack the RecQ helicase fail to make the recovery. These observations indicate that the function of RecQ family proteins is especially important for cellular survival if DNA replication is stalled. Whereas chemicals such as hydroxyurea can arrest the progress of DNA replication throughout the entire genome, occasional pausing in the progression of individual replication forks is thought to occur spontaneously in a high proportion of cells. Accordingly, proteins that are necessary for proper handling and eventual restart of these paused forks are required throughout DNA replication even in cells unchallenged by exogenous agents. Consistent with a role for RecQ helicases during normal cell cycle, analysis of human cells expressing mutant versions of BLM or WRN proteins has shown that these cells exhibit delayed replication progression and accumulate abnormal replication intermediates. There are several non-mutually exclusive possibilities for the roles of RecQ family members during DNA replication. These proteins could physically manipulate or maintain replication fork structure. The importance

of RecQ helicases for normal progression of DNA replication combined with their previously discussed roles in controlling recombination have led to the idea that these proteins function specically to suppress recombination initiated at stalled replication forks. Also, RecQ helicases may be involved in replicating difcult regions of the genome, such as telomeres. Telomeric DNA is very GC-rich and may form alternative conformations, such as G-quartets (Figure 2B). Several RecQ homologs can unwind G-quartet DNA in vitro, and yeast RecQ homolog Sgs1 has been implicated in telomere maintenance in vivo. Alternatively, these proteins could detect fork stalling and signal to other molecules, resulting in recruitment of factors necessary for resumption of fork movement. Indeed, recent evidence suggests that some RecQ family members may be involved in DNA damage surveillance and signaling mechanisms called checkpoints. In particular, the budding yeast RecQ homolog Sgs1 has been shown to participate in such signaling mechanisms specic to DNA damage occurring during DNA replication.

RecQ Helicases and Human Disease

The human genome encodes ve proteins that belong to the RecQ class of helicases. Mutations in three of these proteins, BLM, WRN, and RECQ4, cause genetic disorders: Bloom, Werner, and (at least a subset of) Rothmund Thomson syndromes, respectively. Among other symptoms, Bloom syndrome patients exhibit short stature, immunodeciency, impaired fertility, and a predisposition to a variety of cancers. At the cellular level, the syndrome is characterized by genomic instability, including hyper-recombination between sister-chromatids and homologous chromosomes. Werner and Rothmund-Thomson syndrome patients exhibit symptoms of premature aging, as well as a predisposition to certain types of cancer. Both syndromes are also characterized by increased genomic instability at the cellular level. Cells cultured from Werner syndrome patients exhibit an increase in illegitimate recombination, resulting in chromosomal deletions and translocations. Less is known about the cellular characteristics of the Rothmund Thomson syndrome, but these cells also display increased chromosomal abnormalities. Whereas no genetic disorder is known to result from mutation of a human topo III homolog, mouse knock-out strains have provided important information about the role of these proteins in higher eukaryotes. Deletion of the TOP3a gene, encoding one of the two isoforms of topo III in mice and humans, results in embryonic lethality, indicating that this protein has essential functions during development. Deletion of the other topo III isoform, TOP3b, does not result in lethality, but

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causes a decrease in lifespan, reduced fertility, and increased incidence of aneuploidy. Thus, topo III-like proteins, similar to RecQ helicases, play important roles in mammalian development, aging, and chromosomal integrity.

FURTHER READING
Champoux, J. J. (2001). DNA topoisomerases: Structure, function, and mechanism. Annu. Rev. Biochem. 70, 369 413. Lohman, T. M., and Bjornson, K. P. (1996). Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169214. Lombard, D. B. (2001). Biochemistry and Genetics of RecQ-Helicases. Kluwer Academic, Boston. Oakley, T. J., and Hickson, I. D. (2002). Defending genome integrity during S-phase: Putative roles for RecQ helicases and topoisomerase III. DNA Repair 1, 175207. van Brabant, A. J., Stan, R., and Ellis, N. A. (2000). DNA helicases, genomic instability, and human genetic disease. Annu. Rev. Genomics Hum. Genet. 1, 409 459. Wang, J. C. (2002). Cellular roles of DNA topoisomerases: A molecular prospective. Nature Rev. 3, 430 440.

SEE ALSO

THE

FOLLOWING ARTICLES

DNA Helicases: Dimeric Enzyme Action DNA Helicases: Hexameric Enzyme Action DNA Replication Fork, Eukaryotic DNA Supercoiling Glutamate Receptors, Ionotropic

GLOSSARY
DNA recombination Exchange or transfer of genetic material between two DNA molecules, such as two chromosomes in the cell. DNA replication The process of faithful copying of genetic information in a cellular genome prior to cell division. This is accomplished by separating the strands of duplex chromosomal DNA and synthesizing new DNA strands that are complementary to the parental strands. replication fork Y-shaped DNA structure formed during DNA synthesis when the parental DNA strands are separated to provide a template for DNA replication. sister chromatids The identical copies of a single chromosome produced after DNA replication. supercoiling The topological state achieved by twisting a duplex DNA molecule around its axis. telomere Region of DNA at the end of a linear chromosome.

BIOGRAPHY
Rodney Rothstein is a Professor of Genetics and Development at Columbia University College of Physicians and Surgeons in New York. His principal research interests are in the mechanisms of DNA recombination and the cellular response to DNA damage. He holds a Ph.D. from The University of Chicago and received postdoctoral training at the University of Rochester and Cornell University. He developed one-step gene disruption in yeast and is one of the authors of the double-strand break repair model. His laboratory discovered the rst eukaryotic topoisomerase III gene family member and the rst eukaryotic RecQ homolog, Sgs1. Erika Shor, a senior graduate student in the Rothstein laboratory, studies the budding yeast RecQ and topoisomerase III family members.