topoisomerasas

DNA Topoisomerases: Type IJames J. ChampouxUniversity of Washington, Seattle, Washington, USA

The large size of DNAmolecules 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 IDNA Topoisomerases

To introduce a temporary single-strand break intoduplex DNA, type I DNA topoisomerases must catalyzethe cleavage and subsequent religation of a DNA strand.Since these two reactions occur without an externalenergy source such as ATP, cleavage cannot result fromsimple hydrolysis of a phosphodiester bond in the DNA.Instead, a covalent enzyme–DNA intermediate isgenerated that makes the religation step energeticallyfeasible. The formation of the covalent intermediateinvolves nucleophilic attack by the O-4 atom of theactive site tyrosine in the enzyme on a phosphodiesterbond in the DNA to produce a phosphodiester bondbetween the tyrosine and the DNA and leave a free DNAhydroxyl end. DNA religation and release of the enzymeis the reverse reaction with the oxygen of the free DNAhydroxyl acting as the nucleophile. The type I enzymesdisplay a loose preference for certain nucleotides in thevicinity of a cleavage site, and therefore a cleavage sitetypically occurs every 5–20 base pairs along the DNA.

Type I topoisomerases act on closed circular DNAs tochange the number of times one strand winds around theother, a parameter referred to as the linking number ofthe DNA. Changes in the linking number are reflected ina reduction or an increase in the supercoiling of aplasmid DNA, a property that is most often measured bygel electrophoresis. In addition to altering the super-coiling of a plasmid DNA, many type I topoisomerasesare capable of catalyzing a number of other transactions

involving both single- and double-stranded DNAs. Mostof these enzymes can catenate (interlock), decatenate,knot, and unknot single-stranded DNA circles. Thesame series of reactions can be carried out with duplexcircular DNAs providing at least one of the circularmolecules possesses a nick or gap. In some cases, theenzymes can facilitate the interwinding required for therenaturation of two complementary single-strandedcircular DNAs, a reaction that could be importantduring homologous recombination. Interestingly, topo-isomerase V, which has only been described in thehyperthermophilic archaeon Methanopyrus kandleri,possesses, in addition to the usual topoisomeraseactivity, an apurinic/apyrimidinic site-processingactivity that would appear to implicate the enzyme inDNA repair. Finally, with certain unusual DNA sub-strates, a block to religation leads to permanent suicidecleavage and the enzyme remains covalently linked tothe DNA.

Classification, Nomenclatureand General Properties

Type I topoisomerases are classified into two structurallyand mechanistically distinct subfamilies based on whichDNA end becomes covalently attached to the enzymeduring the cleavage reaction: type IA enzymes attach viaa tyrosine phosphodiester linkage to the 50 end of theDNA, whereas type IB enzymes attach to the 30 end ofthe DNA. Table I lists the known type I DNAtopoisomerases in the two subfamilies with theircommon names and origins. The common names havegenerally been assigned in the order of discovery usingodd Roman numerals (even Roman numerals aresimilarly used for type II DNA topoisomerases). Type Ienzymes with unusual properties or origins have beengiven unique names (reverse gyrase, poxviral topo-isomerase, and mitochondrial topoisomerase). Therecently described IB enzymes in some eubacteria arecurrently referred to as bacterial topoisomerases IB.

The three categories of type IA enzymes listed inTable I can be distinguished on the basis of the types of

Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved. 798

reactions they catalyze. Topoisomerases I relax negativebut not positive supercoils in plasmid DNAs; but sincerelaxation does not go to completion, some residualnegative supercoils remain in the product. Topoisome-rases III require hypernegatively supercoiled plasmidDNA as a substrate and again relaxation is incomplete.Interestingly, topoisomerases III are much more profi-cient than the topoisomerases I in DNA catenation anddecatenation (see below). Reverse gyrases, which areonly found in hyperthermophilic eubacteria and archae-bacteria, introduce positive supercoils into plasmidDNAs at the expense of ATP hydrolysis. All of thetype IA enzymes require Mg2! and are monomeric withthe exception of the reverse gyrase from the archaeonMethanopyrus kandleri which is a heterodimer.

The type IB DNA topoisomerases are capable ofrelaxing both positive and negative supercoils in areaction that does not require ATP or divalent cations.

The reactions go to completion to produce a completelyrelaxed set of plasmid DNA topoisomers. With theexception of the recently discovered heterodimerictopoisomerases I from trypanosomatids, all of the typeIB enzymes are monomeric.

Type IA DNA Topoisomerases

PROTEIN DOMAINS

All type IA topoisomerases (Table I) share a highlyconserved “cleavage/strand passage” domain that con-tains the active site tyrosine. This domain is alsoresponsible for promoting the structural change in theDNA during the interval between the cleavage andreligation reactions that results in a linking numberchange (see below) (Figure 1, red boxes). As indicated inFigure 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 IAtopoisomerases is denoted by colored boxes. The names of the domains for the prototypic E. coliDNA topoisomerase I are given along the top. Thecleavage/strand passage domains shared by all the IA enzymes are shown in red. Green is used to denote the basic C-terminal domains, but thedifferent types of fill for these boxes indicate that these domains are poorly conserved. Some type IA enzymes contain a Zn(II) binding domainshown in blue. The helicase-like domain of reverse gyrase is shown in yellow.

TABLE I

Type I DNA Topoisomerases

Subfamily Common name Source Structure

IA Topoisomerase I All eubacteria and some archaebacteria Monomer

IA Topoisomerase III Some eubacteria and most eukaryotes Monomer

IA Reverse gyrase All hyperthermophilic eubacteria and archaea Monomer

IA Reverse gyrase Archaeon Methanopyrus kandleri Heterodimer

IB Topoisomerase I Nucleus of all eukaryotes Monomer

IB Mitochondrial topoisomerase Mitochondria of higher eukaryotes Monomer

IB Poxviral topoisomerase All members of poxviridae family Monomer

IB Topoisomerase V Archaeon Methanopyrus kandleri Monomer

IB Topoisomerase IB Some eubacteria (see Table II) Monomer

IB Topoisomerase I Trypanosomatids Trypanosoma bruceiand Leishmania donovani

Heterodimer

DNA TOPOISOMERASES: TYPE I 799

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

CRYSTAL STRUCTURE OF THE

CONSERVED CLEAVAGE=STRAND

PASSAGE DOMAIN

The crystal structure of the cleavage/strand passagedomain of E. coli DNA topoisomerase I shown inFigure 2 provides key insights concerning the substratepreference of the enzyme and the mechanism of DNArelaxation. Notably, the cleavage/strand passagedomains of all type IA topoisomerases bear a strongresemblance to the E. coli structure. The hallmark of thecrystal structure is a toroidal shape in which the diameter

of the hole in the center of the torus is sufficient toaccommodate either single- or double-stranded DNA.

The requirement for a negatively supercoiled substrateand the inability to completely relax negative supercoilsis best explained by supposing that these enzymes willonly bind an otherwise duplex DNA substrate if itcontains a single-stranded region resulting from localunwinding of the helix. A plasmid DNA that is highlynegatively supercoiled is energetically disposed towardhelix unwinding, which explains why such a DNA is agood substrate for the enzyme. However, relaxationceaseswhen thenegative supercoiling falls toa levelbelowwhich there is insufficient energy to promote the requiredopening of the helix. As shown in Figure 2, a singlestrandofDNA (solid black line) binds to a narrowgrooveon the cleavage/strand passage domain of the enzymein close proximity to the active site tyrosine (magenta).

ENZYME-BRIDGING MECHANISM

FOR STRAND PASSAGE

To change the linking number of a closed circular DNAduring relaxation, one strand of DNA must passthrough a break in the other strand. Knotting, catena-tion, and decatenation of either single or double-stranded DNAs similarly require such a strandpassage event. The strand passage reaction for type IAtopoisomerases occurs by what is referred to as anenzyme-bridging mechanism. Once the scissile DNAstrand is cleaved, both DNA ends remain tightlyassociated with the enzyme; the 50 end is boundcovalently to the active site tyrosine and the 30 end isbound noncovalently to the enzyme. To orchestratestrand passage, the enzyme undergoes a conformationalchange in which the top half of the protein containing the50 end of the cleaved strand (Figure 2, shown in green)lifts upward to generate a gate in the DNA throughwhich another strand of DNA is passed. After strandpassage, the broken strand is religated and the enzymeopens up a second time to release the strand that had beenpassed into the hole of the torus. A correlate of thismodelis that the linking number can only be changed in steps ofone and this prediction has been verified biochemically.

This same scheme can explain how the enzyme cancatenate or decatenate a DNA containing a nick or gap.However, it is unclear whether the DNA that passesthrough the temporary gate in the cleaved strand iscaptured in the hole of the torus before strand cleavageand is then passed out of the hole after cleavage or viceversa as described above.

REVERSE GYRASE MECHANISM

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

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

800 DNA TOPOISOMERASES: TYPE I

lack helicase activity when assayed under conditionsthat would require processive translocation along theDNA. Instead, the binding of the N-terminal domain toDNA is believed to simply unwind a region of the helix.Subsequently, in a reaction dependent on ATP, one of thetwo strands is cleaved and the other strand is passedthrough the resultant gate by the cleavage/strandpassage domain present in the C-terminal half of themolecule. The key to positive supercoiling is that thestrand passage event that occurs in the presence of ATPis directional such that the linking number of the DNA isincreased and therefore the DNA ends up positivelysupercoiled. The structural basis for the unidirectionalnature of the strand passage event remains unknown.

Type IB DNA Topoisomerases

DOMAIN STRUCTURE AND

SEQUENCE CONSERVATION

Type IB DNA topoisomerases are present in the nucleusof all eukaryotic cells and the mitochondria of highereukaryotes, as well as in at least one archaeon and someeubacteria (Table I). The typical eukaryotic type IBenzyme possesses the four domains shown in Figure 3.A highly charged and poorly conserved N-terminaldomain (red box) is followed by the core domain (bluebox), which binds DNA and contains most of thecatalytic residues. The active site tyrosine is found inthe C-terminal domain (yellow box) that is connected tothe remainder of the protein by a poorly conservedlinker region (orange box).

All of the other type IB enzymes share at least partialsequence and structural homology with the catalytically

important core domain as can be seen from the colorscheme in Figure 3 (blue boxes). Where present, thesequence of the mitochondrial enzyme is very similar tothe nuclear enzyme with the exception of the N-terminalregion (green box), which contains the organelletargeting signals. The eubacterial IB enzyme and thevaccinia topoisomerase are very similar to each other,but they lack most of the core domain as well as theconserved C-terminal domain that is characteristic of theother IB enzymes; instead, they share unique N-terminaland C-terminal regions (white and magenta boxes). Thetopoisomerase I found in the trypanosomatids is aheterodimer with one subunit containing the catalyticcore (blue box) and the other subunit containing aregion homologous to the C-terminal domain of theprototypic eukaryotic sequence (yellow box).

CRYSTAL STRUCTURE OF

HUMAN TOPOISOMERASE I

Two views of the crystal structure of human topoiso-merase I (missing the N-terminal domain) with a bound22 base pair duplex oligonucleotide are shown inFigure 4. The protein is a bi-lobed structure that clampscompletely around the DNAwith the active site tyrosine(shown in black in Figure 4A) juxtaposed to the scissilephosphate. The linker region comprises the coiled-coilstructure that protrudes conspicuously from the bottomportion of the enzyme and has an unknown function(Figure 4A). To release the DNA, the top half of theprotein (shown in blue) must shift upward relative to thebottom half as indicated in Figure 4B by the double-headed arrow. Likewise, DNA binding requires that theprotein 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 topoisomerasesfrom the indicated sources are denoted by colored boxes with similar domains aligned vertically. The names of the domains for the eukaryotic typeIB topoisomerases are shown along the top. Regions that are similar in amino acid sequence share the same color; distinct sequences are assigneddifferent colors. The two subunits of the heterodimeric topoisomerase I from trypanosomatids are shown with these same color conventions.


the human topoisomerase I structure shared by thepoxviral and eubacterial IB enzymes corresponds to theportion of the core domain depicted in yellow in Figure 4.Tyrosine recombinases such as the bacteriophage l andHP1 integrases, and cre recombinase are also structu-rally very similar to approximately this same region.

CATALYSIS

The co-crystal structure of human topoisomerase I withbound DNA reveals which amino acid residues in theprotein are directly involved in catalysis. It is worthnoting that the nucleophilic tyrosine O-4 does notappear to be activated for cleavage by general basecatalysis, although a lysine residue acts as a general acidto protonate the leaving 50 oxygen. The pentavalenttransition state is stabilized by hydrogen-bondinginteractions between three basic amino acid side chainsand the scissile phosphate oxygens. An interactionbetween a lysine residue and the base of the nucleotidewhere cleavage occurs is also important for catalysis.Religation is likely to proceed by a pathway that isessentially the reverse of cleavage.

ROTATIONAL MECHANISM

FOR STRAND PASSAGE

Examination of the crystal structure of human topo-isomerase I (Figure 4A) suggests that the strand passagereaction required to change the linking number of the

DNA during DNA relaxation occurs by a rotationalmechanism rather than by the enzyme-bridging mech-anism described above for the type IA enzymes.However, there appears to be insufficient space withinthe confines of the enzyme to accommodate unrestrictedrotation of the DNA. This feature of the structuresuggests that the enzyme probably undergoes a confor-mational change after cleavage to open up the spacedownstream of the cleavage site to allow rotation.Unlike the enzyme-bridging model, a rotational mech-anism places no a priori limit on the number ofrotational events that can occur for each cycle ofcleavage and religation. Indeed, in the case of vacciniatopoisomerase, five rotations of the DNA occur on theaverage between each cleavage and religation reaction.

Cellular Roles

Although much is yet to be learned about how thevarious topoisomerases collaborate to manage DNAtopology in the cell, a partial picture has emerged basedon work in bacteria and simple eukaryotes. Although thetype II topoisomerases are not the subject of this review,the activities of these enzymes are briefly consideredin the sections to follow for the sake of completeness.The type II enzymes are important for any cellularprocess that requires the passage of a region of duplexDNA through a double-strand break in the same or adifferent DNA molecule. The allocation of functions to

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 poxviraltopoisomerase and the eubacterial topoisomerases IB are structurally very similar to the region of the bottom lobe, shown in yellow. The coiled-coillinker 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 changethat is required to open and close the clamp during binding and release of the DNA.


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

TYPES OF SUPERCOILING

IN EUKARYOTIC VERSUS

PROKARYOTIC CELLS

Two different situations lead to the supercoiling of DNAin vivo. First, DNA will assume a supercoiled configur-ation through an interaction with certain proteins orother cellular components. Alternatively, a closeddomain of DNA (e.g., a closed circular DNA) willspontaneously supercoil if the linking number is not thesame as the helical winding (referred to as twist) of theDNA helix. This latter type of supercoil is often referredto as torsionally strained supercoils. The chromo-somal DNA of eukaryotes is wrapped into a protein-constrained solenoidal superhelix in nucleosomes and,except for the transient occurrence of torsionallystrained supercoils associated with replication andtranscription, is maintained in a relaxed state by DNAtopoisomerases. However, in prokaryotes it appears thatalthough some supercoils are constrained by virtue of aninteraction with proteins as in eukaryotes, there exists,in addition, a fixed steady-state level of torsionallystrained supercoiling generated by gyrases (see below).

GENERATION OF SUPERCOILING

STRESS IN PROKARYOTES

Mesophilic Bacteria

The DNA in all mesophilic eubacteria and archaebac-teria contains torsionally strained negative supercoilsthat are introduced by the type II enzyme called DNAgyrase. It appears that this steady-state level of negativesuperhelicity is required to facilitate helix openingduring the initiation of DNA replication and transcrip-tion. To prevent the introduction of excess negativesupercoils by the gyrase, these bacteria also contain oneor two type IA DNA topoisomerases (topoisomerases Ior III or both) to counteract the effects of DNA gyrase.The inability of the type IA enzymes to remove negativesupercoils below a critical threshold level prevents theseenzymes from negating the effects of DNA gyrase and iscrucial for fine-tuning the negative supercoiling levels inthese organisms. Some eubacteria also contain a type IBenzyme (Table II), which could also balance the effects ofDNA gyrase; but the apparent ability of these enzymesto completely relax the DNA suggests that their activitywould have to be regulated in some way. The exact roleplayed by the bacterial topoisomerase IB and why thisenzyme is only present in a subset of the mesophiliceubacteria remains unknown.

Hyperthermophilic Bacteria

All hyperthermophilic eubacteria and archaebacteriapossess a reverse gyrase that actively maintains positivesupercoils in the chromosomal DNA. It appears thispositive supercoiling is necessary to stabilize the DNAhelix against denaturation at the high growth tempera-tures of these organisms. The mechanism for preventingexcess positive supercoiling is not known, but it is likelythat a type II enzyme that can relax positive supercoils(DNA gyrase or the archaeal topoisomerase VI) counter-acts the effects of reverse gyrase to set the final steady-state level of positive supercoiling.

TRANSCRIPTION

During transcription, the movement of RNA polymerasealong a DNA that is rotationally fixed transientlygenerates positive supercoils in front of the translocatingpolymerase and negative supercoils behind the poly-merase (Figure 5A). The type IA enzymes present inall organisms relax the negative supercoils that accom-pany transcription, but the mechanism for the removalof the positive supercoils depends on the organism.

TABLE II

Occurrence of DNA Topoisomerase IB in Eubacteria

Known species possessingtype IB topoisomerase

Examples of species lackingtype IB topoisomerase

Mycobacterium avium

Mycobacterium smegmatis Streptomyces coelicolor

Cytophaga hutchinsonii Chlamydia trachomatis

Agrobacterium tumefaciens Bacillus anthracis

Bradyrhizobium japonicum Bacillus subtilis

Mesorhizobium loti Clostridium tetani

Sinorhizobium meliloti Mycoplasma pneumoniae

Rhodobacter sphaeroides Listeria monocytogenes

Novosphingobium aromaticivorans Staphylococcus aureus

Bordetella parapertussis Streptococcus pneumoniae

Burkholderia fungorum Streptococcus pyogenes

Xanthomonas axonopodis Caulobacter crescentus

Xanthomonas campestris Rickettsia conorii

Pseudomonas aeruginosa Neisseria meningitidis

Pseudomonas fluorescens Helicobacter pylori

Pseudomonas putida Escherichia coli

Pseudomonas syringae Yersinia pestis

Deinococcus radiodurans Vibrio cholerae

Xylella fastidiosa

Haemophilus influenzae

Salmonella typhimurium

Borrelia burgdorferi

Treponema pallidum


In prokaryotes, positive supercoils are relaxed by a typeII enzyme such as DNA gyrase or in the archaea bytopoisomerase VI, and in the case of select eubacteria(see Table II) probably by a combination of a type IIenzyme and the type IB topoisomerase.

In eukaryotes, it is likely that the type IB DNAtopoisomerase I relaxes the supercoils of both signsassociated with transcription. Based on the knowncomplete genome sequences, most eukaryotes, but notfungi or Caenorhabditis elegans, contain two distinctgenes for type IB enzymes, one for the nuclear enzymeand a second for an enzyme that is imported into themitochondrion to presumably function in transcription.In those eukaryotic organisms with only a single typeIB enzyme, topoisomerase I likely plays a dual role,acting in both the nucleus and the mitochondrion.

DNA REPLICATION

As the two DNA strands are separated during DNAreplication, the DNA helix in front of the replicationfork becomes at least transiently overwound or posi-tively supercoiled (Figure 5B). This overwinding of thehelix has been shown to be at least partially transmittedto the region behind the replication fork to cause aninterwinding of the two daughter helices. These inter-windings are referred to as precatenanes (Figure 5B),since in a circular replicon, if any remain at the end ofreplication, the two daughter circular molecules will becatenated. Resolution of the overwound structure of areplicating chromosome can be accomplished by

relaxing positive supercoils in front of the fork, or bydecatenating (or unlinking) the precatenanes behind themoving fork, or both.

As in transcription, positive supercoils can beremoved in prokaryotes by a type II topoisomerasesuch 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 topo-isomerase VI in archaea. As long as gaps exist duringdiscontinuous DNA synthesis, precatenanes can also beunlinked in some eubacteria either by topoisomerase IB(Table II) or by the potent type IA decatenating enzyme,topoisomerase III. Segregation of true catenanes lackingnicks or gaps in either of the two strands can only beaccomplished by a type II enzyme.

In eukaryotes, the type IB topoisomerase relaxes thepositive supercoils ahead of the replication fork andduring synthesis can probably decatenate the precate-nanes by acting at gaps in the DNA. However, most ofthe precatenanes and terminally interlinked or catenatedstructures are likely resolved by a type II topoisomerase.In mitochondria, replication-associated positive super-coils are relaxed by a type IB topoisomerase as describedabove for transcription. A mitochondrial type IItopoisomerase and a type IA enzyme (topoisomeraseIII) are likely involved in unlinking precatenanes andcatenated circular DNAs that occur during mitochon-drial DNA replication.

SEE ALSO THE FOLLOWING ARTICLES

DNA Supercoiling † DNA Topoisomerases: Type II

GLOSSARY

catenane Two interlocked circular DNA molecules in which the twoduplexes are wound around each other one or more times.

catenate The process whereby two circular DNAs are interlockedto form a catenane. The unlocking of catenated DNAs is referredto 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 closedcircular DNA.

DNA supercoiling The coiling of the axis of a DNA molecule inthree-dimensional space. Supercoiling may result from an inter-action of the DNAwith protein or from an inequality between thenumber of helical turns dictated by the structure of the DNA helixunder a particular set of conditions (the twist of the DNA) and thelinking number of the DNA.

linking number Topological property of a closed circular DNA that isa measure of the fixed interwinding of the two DNA strands.

precatenanes The interwinding of the two daughter duplexes behinda replication fork.

reverse gyrase DNA topoisomerase that couples the hydrolysis ofATP to the introduction of positive supercoils into a closed circularDNA.

FIGURE 5 Topological transformations of DNA during transcrip-tion and replication. (A) The generation of positive supercoils (! ) infront of and negative supercoils (2) behind a translocating RNApolymerase during transcription are depicted. (B) Replication forkmovement in a closed domain results in an overwinding of the DNAahead of the replication fork and the interwinding of the twodaughter helices to form precatenanes behind the replication fork, asshown in (B). Any precatenanes remaining at the end of replicationfor a circular replicon will result in the catenation of the twodaughter circular molecules. A simple catenane with a single interlinkis also shown.


scissile strand The strand of DNA that is cleaved by a type Itopoisomerase.

topoisomerase Enzyme that changes the linking number of a closedcircular 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 differentlinking numbers.

torsionally strained supercoils Supercoils that result from an inequal-ity between the number of helical turns dictated by the structure ofthe DNA helix under a particular set of conditions and the linkingnumber of the DNA.

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 ofDNA during replication. Genetica 106, 131–140.

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. 65,635–692.

Wang, J. C. (2002). Cellular roles of DNA topoisomerases: Amolecularperspective. Nat. Rev. Mol. Cell. Biol. 3, 430–440.

BIOGRAPHY

James J. Champoux is a Professor in the Department of Microbiologyin the School ofMedicine at the University ofWashington. His researchfocuses on topoisomerases and reverse transcription. He holds a Ph.D.from Stanford University and carried out his postdoctoral work at theSalk Institute in San Diego, California. He discovered the eukaryotictype IB topoisomerase and was the first to show that the reactionproceeds through an enzyme–DNA covalent intermediate. He hasbeen instrumental in elucidating the roles of the RNase H activity ofreverse transcriptase in retroviral replication.


DNA Topoisomerases: Type IIRenier Velez-Cruz and Neil OsheroffVanderbilt 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 three-

dimensional relationships in the genetic material are topolo-

gical in nature, including DNA under- and overwinding,

knotting, and tangling. The enzymes that modulate the

topological properties of DNA are termed DNA topoisome-

rases. There are two classes of topoisomerases, type I and type

II, which are defined 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 double-

stranded 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.

DNA Topology

The topological properties of DNA are defined as thosethat cannot be altered without breaking one or bothstrands of the double helix. Because DNA comprises twointerwound nucleic acid strands and the genomes of allknown organisms are very long or circular (or both),two distinct topological issues arise as a result of thegenetic material. Proliferating cells must be able to copewith both of these in order to survive.

The first issue is related to the torsional stress on thedouble helix. The DNA from all species of eukaryotesand eubacteria is globally underwound ,5–10%.DNA under torsional stress is termed supercoiled(underwound molecules are negatively supercoiled andoverwound molecules are positively supercoiled)because underwound or overwound DNA writhes

about itself to form superhelical twists. Negative super-coiling puts energy into the genetic material and makes iteasier to separate the two strands of the double helix forreplication and transcription. Thus, DNA underwindingdramatically increases the rates of these two funda-mental processes. In contrast, the movement of DNAtracking systems (such as replication forks and tran-scription complexes) through the double helix locallyoverwinds the DNA ahead of their actions. Becauseoverwinding makes it much harder to pull apart thedouble helix, it blocks many essential cellular processes.

The second issue is related to the extreme length ofgenomic DNA. Nucleic acid knots (intramolecular) andtangles (intermolecular) are formed routinely during avariety of ongoing cellular processes including DNArecombination and replication. Both knots and tanglesmust be resolved in order for daughter chromosomes tosegregate properly during meiosis and mitosis.

DNA Topoisomerases

Cells contain ubiquitous enzymes known as DNAtopoisomerases that maintain the appropriate level ofDNA supercoiling and remove knots and tangles fromthe genetic material. These enzymes modulate thetopological structure of the genetic material by creatingtransient breaks in the backbone of DNA. There are twoclasses of topoisomerases that can be distinguished bythe number of DNA strands that they cleave during theircatalytic cycles. Type I enzymes create transient single-stranded DNA breaks, whereas type II enzymes createtransient double-stranded breaks. To maintain genomicintegrity during their DNA cleavage events, topoisome-rases form covalent linkages between active-site tyrosylresidues and the newly generated DNA termini. Thesecovalent protein-cleaved DNA complexes, known ascleavage complexes, are the hallmarks of all topoisome-rases irrespective of enzyme classification. Because type Itopoisomerases create single-stranded breaks in thegenetic material, they can regulate DNA supercoiling.However, because type II topoisomerases generatedouble-stranded breaks in the DNA backbone, they can


resolve knots and tangles in addition to removingtorsional stress from the genetic material.

Type II topoisomerases are essential to all eukaryoticand prokaryotic organisms. They are highly conservedamong species, and the eukaryotic enzymes appear to bedirect 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 typeIIA homology subfamily. Topoisomerase II canremove positive and negative superhelical twists fromthe double helix and can resolve DNA knots and tangles.

ENZYME MECHANISM

Topoisomerase II interconverts different topologicalforms of DNA by the double-stranded DNA passagereaction depicted in Figure 1, which shows the productsof each individual step. Briefly, it is proposed thattopoisomerase II (1) binds twoDNAsegments, (2) creates

a double-stranded break in one of the segments,(3) translocates the other DNA segment through thecleaved double helix, (4) rejoins (i.e., ligates) the cleavedDNA, (5) releases the translocated segment through agate in the protein, and (6) closes the protein gate andregains the ability to start a new round of catalysis. Thescissile bonds on the two strands of the double helix thatare cut by topoisomerase II are staggered. Thus, theenzyme generates cleaved DNA molecules that containfour-base single-stranded ends at their 50-termini. Duringits cleavage event, topoisomerase II covalently attaches tothese newly generated 50-termini.

Topoisomerase II requires two cofactors in order tocarry out its catalytic double-stranded DNA passagereaction. First, it needs a divalent cation for all stepsbeyond enzyme-DNA binding (Figure 1, complex 1).Magnesium(II) appears to be the divalent cation thatthe enzyme uses in vivo. Second, topoisomerase II usesthe energy of adenosine triphosphate (ATP) to drive theoverall DNA strand passage reaction. Although ATP isnot required for either DNA cleavage or ligation, thebinding of this nucleoside triphosphate triggers DNAtranslocation (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 discretesteps (the products of each step are shown). (1) Enzyme–DNA binding, (2) DNA cleavage (formation of cleavage complex), (3) double-stranded 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 shownin green (horizontal) and orange (coming out of the plane of the paper). Structures are courtesy of Dr. James M. Berger, University ofCalifornia, Berkeley.

DNA TOPOISOMERASES: TYPE II 807

and its hydrolysis to adenosine diphosphate (ADP) andinorganic phosphate (Pi) is necessary for enzymerecycling (which converts complex 5 to complex 6).Normally, topoisomerase II binds two molecules of ATP.Although hydrolysis of the cofactor is not a prerequisitefor the strand passage event, it appears that this stepproceeds more rapidly if it is preceded by hydrolysis ofone of the bound ATP molecules.

ENZYME DOMAIN STRUCTURES

AND ISOFORMS

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

contains consensus sequences for ATP binding. Thecentral domain is homologous to the A-subunit of DNAgyrase (GyrA) and contains the active-site tyrosylresidue that forms the covalent bond with DNA duringscission. The C-terminal domain is not highly conservedand appears to have no corresponding region ofhomology with DNA gyrase. This variable region ofthe eukaryotic enzyme contains nuclear localizationsequences as well as amino acid residues that arephosphorylated in vivo.

Although some eukaryotic species such as yeast andDrosophila appear to have only a single type IItopoisomerase (i.e., topoisomerase II), vertebrates con-tain two closely related isoforms, topoisomerase IIaand b. These two isoforms share extensive amino acidsequence identity (,70%), but are encoded by separategenes (located at chromosomal bands 17q21–22 and3p24 in humans, respectively) and can be distingui-shed by their protomer molecular masses (,170 and,180 kDa, respectively).

NLS and PO4

Human topoisomerase IIa

NH2- -COOH

1531Tyr805

E. coli DNA gyrase

NH2- -COOH

875Tyr122GyrB GyrA

E. coli topoisomerase IV

NH2- -COOH

766Tyr120ParE ParC

NH2--COOH

804

637A

B

NH2-

Top6B

530

NH2--COOH

Top6A

-COOHNH2--COOH

389

S. shibatae topoisomerase VI

Tyr106

ATPase DNA cleavage/ ligation

ATP binding motifs

FIGURE 2 Domain structures of type II topoisomerases. (A) The domain structures of three type IIA topoisomerases: human topoisomeraseIIa 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 stripesrepresent the three conserved motifs of the Bergerat fold that define the ATP-binding domain. The central (i.e., GyrA) homology domainscontain 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 forcomparison.

808 DNA TOPOISOMERASES: TYPE II

PHYSIOLOGICAL FUNCTIONS

Topoisomerase II plays a number of essential roles ineukaryotic cells and participates in virtually every majorprocess that involves the genetic material. It unlinksdaughter chromosomes that are tangled followingreplication and resolves DNA knots that are formedduring recombination. It also helps to remove thepositive DNA supercoils that are generated ahead ofreplication forks and transcription complexes. Topo-isomerase II is required for proper chromosome conden-sation, cohesion, and segregation and appears to playroles in centromere function and chromatin remodeling.Finally, the type II enzyme is important for themaintenance of proper chromosome organizationand structure, and it is the major nonhistone protein ofthe mitotic chromosome scaffold and the interphasenuclear matrix.

It is not obvious why vertebrate species possess twodistinct topoisomerase II isoforms. Enzymologicaldifferences between topoisomerase IIa and b are subtleand the relationships between these isoforms are notwell defined. Although either enzyme can complementyeast strains lacking topoisomerase II activity, topo-isomerase IIa is essential for proliferating mammaliancells and its loss cannot be compensated by the bisoform. Topoisomerase IIb appears to be dispensable atthe cellular level, but is required for proper neuraldevelopment in mice.

The specific cellular functions of topoisomerase IIaand b probably reflect their physiological regulationmore than their enzymological characteristics. Topoi-somerase IIa is regulated over both cell and growthcycles. Enzyme levels increase throughout the S-phase ofthe cell cycle and peak at the G2-M boundary.Furthermore, this isoform is found almost exclusivelyin rapidly proliferating tissues. In contrast, the concen-tration of topoisomerase IIb is independent of the cellcycle and this isoform is found in most cell typesregardless of proliferation status. Taken together, thesecharacteristics suggest that topoisomerase IIa is theisoform responsible for events associated with DNAreplication and chromosome segregation, whereastopoisomerase IIb is the isoform that probably functionsin ongoing nuclear processes.

Prokaryotic Type IITopoisomerases

Eubacteria contain two distinct type II topoisomerases,DNA gyrase and topoisomerase IV. Both are members ofthe 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 thetype IIB subfamily.

DNA GYRASE

DNA gyrase was discovered in 1976. It was the first typeII topoisomerase to be described and is the only one toretain its historical name (in the modern nomenclature,type II topoisomerases are denoted by even numbers). Incontrast to the eukaryotic type II enzymes, DNA gyraseis 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 theactive 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 activelyunderwinding (i.e., negatively supercoiling) the doublehelix. It accomplishes this feat bywrapping DNA arounditself in a right-handed fashion and carrying out itsstrand-passage reaction in a unidirectional manner.

The negative supercoiling activity of DNA gyrase farexceeds the ability of the enzyme to remove either knotsor tangles from the genetic material. Consequently, themajor physiological roles of DNA gyrase stem directlyfrom its ability to underwind the double helix. DNAgyrase plays a critical role in opening DNA replicationorigins and removing positive supercoils that accumulatein front of replication forks and transcription complexes.In addition, this enzyme works in conjunction with the vprotein (a type I topoisomerase that removes negativesupercoils from the double helix) to maintain the globalbalance of DNA supercoiling in bacterial cells.

TOPOISOMERASE IV

Topoisomerase IV is an A2B2 tetramer that is comprisedof two distinct subunits, ParC (molecular mass<88 kDa), and ParE (molecular mass <70 kDa),which are homologous to the A- and B-subunits ofDNA gyrase (Figure 2B). (In gram-positive bacterialspecies, the subunits of topoisomerase IV are designatedGrlA and GrlB, respectively.) It was known for severalyears that the ParC and ParE proteins were necessary forproper chromosome segregation in bacteria. However, itwas not discovered until 1990 that these two subunitstogether constituted a type II topoisomerase.

The catalytic properties of topoisomerase IV can bedistinguished from those of DNA gyrase in twoimportant ways. First, although topoisomerase IV canremove positive and negative superhelical twists fromDNA, it cannot actively underwind the double helix.Second, the ability of topoisomerase IV to resolve DNAknots and tangles is dramatically better than that ofDNA gyrase. Because of these differences, the physio-logical roles of topoisomerase IV are distinct from thoseof DNA gyrase. The primary cellular functions oftopoisomerase IV are to unlink daughter chromosomesfollowing DNA replication and to resolve DNA knots


that are formed during recombination. Recently, it wasfound that topoisomerase IV removes positive supercoilsfrom DNA more efficiently than it removes negativesupercoils. This has led to speculation that the enzymealso may act ahead of DNA tracking systems to alleviateoverwinding of the double helix. However, the preciserole of topoisomerase IV in this process has yet tobe defined.

ARCHAEAL TOPOISOMERASE VI

In 1997, a novel type II topoisomerase, topoisomeraseVI, was discovered in hyperthermophilic archaealspecies. This enzyme was designated as the first memberof the topoisomerase IIB subfamily due to its lack ofhomology to previously identified type II enzymes.

Topoisomerase VI has two subunits, Top6A andTop6B (molecular masses <47 and 60 kDa, respect-ively), and is arranged as an A2B2 tetramer. Bothsubunits are considerably smaller than those of bacterialDNA gyrase or topoisomerase IV (Figure 2B). Althoughshort regions of Top6B surrounding the ATP-bindingdomain are homologous to portions of GyrB, andTop6A contains an active-site tyrosine that is requiredfor DNA cleavage, the primary structure of topoisome-rase VI displays little similarity to the type IIA enzymes.

Archaeal topoisomerase VI appears to alter DNAtopology by using a double-stranded DNA passagereaction like that described for other type II topoisome-rases. During this reaction, it generates DNAbreaks with50 overhangs that are covalently attached to its active-sitetyrosyl residues. Topoisomerase VI relaxes positively andnegatively supercoiled DNA, but cannot actively under-wind the double helix. In addition, it can unlink (i.e.,untangle) interwound double-stranded DNA circles.

The catalytic properties of topoisomerase VI differfrom those of the type IIA enzymes in two significantaspects. First, topoisomerase VI requires ATP binding inorder to cleave its DNA substrate. Second, in markedcontrast to the type IIA enzymes (which produce four-base staggered ends during scission), topoisomerase VI-mediated DNA cleavage generates DNA termini thatcontain only two-base overhangs. Although the physio-logical functions of topoisomerase VI have yet to bedetermined, the enzyme is believed to play a role inunlinking daughter chromosomes following replicationin archaeal cells.

With the exception of plants, no Top6B homologuehas been identified in eukaryotic species. However, aTop6A homologue, Spo11, has been found in eukar-yotes ranging from yeast to humans. Spo11 generatesthe double-stranded DNA breaks that initiate meioticrecombination. Like its topoisomerase relatives, Spo11forms a covalent bond between an active-site tyrosylresidue and the 50-DNA termini generated by itsscission reaction. At the present time there is no

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

Type II Topoisomerasesas Therapeutic Targets

In addition to their varied and critical physiologicalfunctions, the type IIA topoisomerases are targets forsome of the most active anticancer and antibacterialdrugs in clinical use. In contrast to most enzyme-targeted drugs, these agents do not act by robbing cellsof an essential enzyme activity. Rather, drugs that targettype II topoisomerases kill cells by dramatically increas-ing the concentration of covalent enzyme-cleaved DNAcomplexes (i.e., cleavage complexes) that are requisiteintermediates formed during the double-stranded DNApassage reaction. Normally, cleavage complexes arepresent at low steady-state levels and are tolerated bycells. However, conditions that significantly increaseeither their concentration or lifetime trigger numerousmutagenic events.

The potential lethality of cleavage complexes risesdramatically when DNA tracking enzymes such aspolymerases or helicases attempt to traverse thecovalently bound topoisomerase roadblock in thegenetic material. Such an action disrupts cleavagecomplexes and converts transient enzyme-mediatedDNA breaks to permanent DNA breaks. These perma-nent breaks in the genome trigger the generation ofchromosomal insertions, deletions, translocations, andother aberrations, and, when present in sufficientnumbers, they initiate a series of events that culminatesin cell death. Because the drugs that target type IItopoisomerases convert these essential enzymes topotent cellular toxins that fragment the genome, theyare referred to as topoisomerase poisons to distinguishthem from drugs that act as catalytic inhibitors.

ANTICANCER DRUGS

At the present time, six topoisomerase II-targetedanticancer agents (Figure 3A) are approved for use inthe United States. Drugs such as etoposide anddoxorubicin are front-line therapy for breast and lungcancers, as well as for a variety of leukemias, lympho-mas, and germ-line malignancies. Approximately one-half of all cancer chemotherapy regimens contain drugstargeted to topoisomerase II. Moreover, every form ofcancer that can be cured by systemic chemotherapy istreated with these agents.

Due to the high concentration of topoisomerase IIa inrapidly proliferating cells, this isoform probably is themajor important target of anticancer therapy. However,circumstantial evidence suggests that the b-isoform alsocontributes to drug efficacy.

810 DNA TOPOISOMERASES: TYPE II

ANTIBACTERIAL DRUGS

DNA gyrase and topoisomerase IV are the targetsfor quinolone-based antibacterial agents (Figure 3B).Quinolones are the most active and broad-spectrumantibacterial drugs currently available. Drugs such asciprofloxacin are prescribed routinely for a wide varietyof gram-negative bacterial infections, including gastro-intestinal tract, respiratory tract, and bone and jointinfections. Ciprofloxacin also is used to treat a numberof sexually transmitted diseases as well as infection withanthrax. Newly developed quinolones, such as levoflox-acin, display significant efficacy against gram-positivebacterial infections.

DNA gyrase is the primary cytotoxic target ofquinolones in gram-negative bacteria. However, topo-isomerase IVappears to be the more important target formany of these drugs in gram-positive species.


DNA Supercoiling † DNA Topoisomerases: Type I

GLOSSARY

adenosine triphosphate (ATP) A cofactor that supplies energy formany enzymatic processes.

cell cycle The process by which a cell grows, replicates its genome,and divides. The cell cycle is divided in four distinct phases: G1, agrowth phase; S, the phase in which the cell duplicates (i.e.,synthesizes) its genetic material; G2, a second growth phase inwhich the cell prepares to divide; andM, the phase in which the celldivides (i.e., mitosis).

DNA recombination The process by which the cell reorganizes itsgenetic material in order to repair certain forms of DNA damage(including double-stranded DNA breaks) or promote geneticdiversity.

DNA replication The process by which the cell duplicates (i.e.,synthesizes) its genetic material.

DNA supercoiling The underwinding (i.e., negative supercoiling) oroverwinding (i.e., positive supercoiling) of the genetic material.

topoisomerase poison A drug that increases levels of topoisomerase-cleaved DNA complexes.

topology A field of mathematics that deals with relationships that arenot altered by elastic deformation.

transcription The process by which the cell expresses its geneticmaterial; the generation of messenger RNAs from a DNA template.

FURTHER READING

Anderson, V. E., and Osheroff, N. (2001). Type II topoisomerases astargets for quinolone antibacterials: Turning Dr. Jekyll intoMr. Hyde. Curr. Pharm. Des. 7, 337–353.


Fortune, J. M., and Osheroff, N. (2000). Topoisomerase II as a targetfor anticancer drugs: When enzymes stop being nice. Prog. Nucl.Acid Res. Mol. Biol. 64, 221–253.

Gadelle, D., Filee, J., Buhler, C., and Forterre, P. (2003). Phyloge-nomics of type II DNA topoisomerases. BioEssays 25, 232–242.

Heddle, J. G., Barnard, F. M., Wentzell, L. M., and Maxwell, A.(2000). The interaction of drugs with DNA gyrase: A model for themolecular basis of quinolone action. Nucleosides NucleotidesNucleic Acids 19, 1249–1264.

Li, T. K., and Liu, L. F. (2001). Tumor cell death induced bytopoisomerase-targeting drugs. Annu. Rev. Pharmacol. Toxicol.41, 53–77.

Osheroff, N (ed.) (1998). DNA topoisomerases. Biochim. Biophys.Acta 1400.

Wang, J. C. (2002). Cellular roles of DNA topoisomerases: Amolecularperspective. Nat. Rev. Mol. Cell. Biol. 3, 430–440.

BIOGRAPHY

Renier Velez-Cruz is completing his doctoral studies in the Departmentof Biochemistry, Vanderbilt University School of Medicine.

Neil Osheroff is a Professor in the Departments of Biochemistry andMedicine at the Vanderbilt University School of Medicine and holdsthe John G. Coniglio Chair in Biochemistry. His principal researchinterests are the fields of DNA topoisomerases, topoisomerase-targeteddrugs, and DNA repair. He holds a Ph.D. in Biochemistry andMolecular Biology from Northwestern University and received hispostdoctoral training in the Department of Biochemistry at theStanford University School of Medicine. He has authored more than170 articles and has contributed significantly to our understanding ofthe mechanism of action of type II topoisomerases and topoisomeraseII poisons.

S

REtoposide:

Teniposide:

CH3

R1 R2

Doxorubicin:Danorubicin:

Idarubicin:

CH2OHCH3CH3

OCH3OCH3H

OC

OH

OH

O

O

OH

O

R1

R2

OH3C

OHNH3

O

O

H

H3CO OCH3OH

HO

O

OO

O

R

HOOH O

OH

OH

HN(CH2)2NH(CH2)2OH

HN(CH2)2NH(CH2)2OH

O

O

Mitoxantrone

N

O

O

COOH

H3CN

N

F

CH3

Levofloxacin

N

COOHO

F

NHN

Ciprofloxacin

A

B

+

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


DNA Topoisomerases:Type III–RecQ Helicase SystemsRodney Rothstein and Erika ShorColumbia 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 catalyz-

ing 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 identified

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 character-

ized 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 topo-

isomerases has been observed throughout the evolutionary

tree, suggesting that these two proteins act in concert to

promote genomic stability.

Structure and MolecularMechanisms

TOPOISOMERASE III

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

formed. After passage of other DNA strand(s) throughthe break, the reverse transesterification reaction leadsto the rejoining of the DNA backbone. TopoisomeraseIII activity can result in relaxation/removal of negativesupercoiling from DNA, (de)catenation and knotting ofss circular DNA molecules, and (de)catenation of dsDNA molecules that contain ss regions. TopoisomeraseIII does not require energy in the form of nucleosidetriphosphates, such as ATP. Hence, the directionality ofthe topoisomerase III-driven reactions is toward theDNA conformation with the lowest free energy.

RECQ HELICASES

The RecQ family of DNA helicases is defined byhomology to the bacterial RecQ protein. Like otherhelicases, these proteins contain seven signature helicasemotifs, including sequences that contain Walker A(required for ATP binding and hydrolysis) and Bboxes. In addition to the core helicase motifs, all RecQhelicases share additional regions of homology notshared by other families of helicases.

The functional unit of a helicase is generally composedof a dimer or a hexamer that forms a ring around itssubstrate DNA. However, examples of monomerichelicases are also known. Whereas several early studieshave indicated that RecQ-like helicases form hexamers,two recent studies suggest that DNA helicase activitiesin vitro of both E. coli RecQ and human BLM proteinsare associated with a monomeric form of the protein.Thus, the composition of a functional unit of RecQ-likehelicases is still being explored.

All RecQ-type helicases examined to date display30 ! 50 directionality on DNAwith respect to the strandto which the protein is bound (Figure 2A). In vitrosubstrate preference studies have indicated that thesehelicases can act on a variety of DNA structures,possibly reflecting the diversity of their in vivo activities.Among the structures that RecQ helicases can unwindare branched molecules, four-way dsDNA junctions,G-quartets, and D-loops (Figure 2B). ATP and Mg2!!

are necessary cofactors for RecQ-driven reactions.


The RecQHelicase–TopoisomeraseIII Connection

PHYSICAL ASSOCIATION

Two widely used model organisms, budding andfission yeast, each has one RecQ-like helicase andone topo III. In both organisms, the two proteinshave been shown to physically interact with eachother and/or to associate in cellular extracts. In humancells, the RecQ homolog BLM physically interactswith an isoform of topoisomerase III, Top3a, via theN-terminus of BLM. Mutation or deletion of BLM in

human cells has several detrimental consequences ongenome stability, including a marked increase inrecombination between sister chromatids. Interestingly,cells expressing BLM protein that lacks its N-terminusand fails to interact with Top3a (but retains DNAunwinding activity) show increased levels of sisterchromatid exchange, similar to cells that lack theentire BLM protein. This demonstrates that, in orderto perform its role in suppressing recombinationbetween sister chromatids, BLM needs to interactwith Top3a in vivo, which supports other evidencethat the two act as a complex with key roles inmaintenance of genome stability.

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

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 theknown in vitro substrates of RecQ helicases before and after incubation with the protein.

DNA TOPOISOMERASES: TYPE III–RecQ HELICASE SYSTEMS 813

GENETIC INTERACTIONS IN YEAST

As mentioned above, the genomes of both budding andfission yeast encode a single topoisomerase III gene. Inboth organisms, loss or mutation of the topo III proteinis detrimental, resulting in slow growth or lethality.Concomitant loss of RecQ helicase function in mutantslacking topo III largely restores normal growth andviability. These genetic observations have led to thefollowing model of RecQ–Topoisomerase III interactionin vivo. It is proposed that in wild-type cells, the activityof the RecQ helicase creates a DNA structure that isnormally acted on and resolved by topo III. When topoIII is inactivated, this structure is not processed properlyand causes slow growth or lethality. In mutants lackingthe RecQ helicase, this structure is not created,eliminating the need for a functional topo III. Whilethis model is supported by genetic evidence, experi-mental data confirming the existence of such a DNAstructure are lacking to date.

COMBINED MODE OF ACTION

The genetic and physical interactions between RecQ-likehelicases and type III topoisomerases in several organ-isms have prompted investigation and speculationregarding their combined mode of action. One possi-bility first put forth upon the discovery of the buddingyeast RecQ homolog Sgs1 and its genetic and physicalassociation with the budding yeast topo III was that thetwo proteins form a complex that functions like aeukaryotic reverse gyrase. A reverse gyrase is an enzymefound in several species of Archaea. A hallmark featureof the reverse gyrase protein is the presence of a type Itopoisomerase-like domain and a helicase domain aspart of the same polypeptide. In vitro, the enzymeintroduces positive supercoiling into dsDNA molecules.This is thought to happen in the following manner. Thehelicase domain of reverse gyrase unwinds a region ofdsDNA, creating local negative supercoiling on one sideof the enzyme and local positive superoiling on the other.The topoisomerase domain relaxes the negative super-coiling, resulting in a net overall increase in positivesupercoiling in the dsDNA molecule. However,despite the attractiveness of the hypothesis that theRecQ–topoIII complex resembles reverse gyrase, thefew in vitro studies done on RecQ and topo III have notreported increased positive supercoiling as a result of thecombined action of the two enzymes.

Another hypothesis regarding the role of the RecQ–topoIII complex suggests a role in catenation ordecatenation of ds DNA molecules. This idea issupported by a study that examined the consequencesof concerted action of bacterial RecQ and topo III in anin vitro system. It was shown that both bacterial andyeast topo III proteins are specifically stimulated by the

bacterial RecQ helicase to fully catenate or decatenate(depending on the conditions of the reaction) covalentlyclosed circular dsDNA molecules. In vivo, a decatena-tion activity may be used to separate such interlinkeddsDNA substrates as sister chromatids following DNAreplication or homologous chromosomes followingmitotic or meiotic recombination. Their completedecatenation is essential for subsequent faithful chromo-somal segregation, which in turn ensures accuratetransfer of genetic material into daughter cells. On theother hand, a catenating activity may be used in vivo tosuppress recombination, as DNA sequences that arehighly catenated undergo fewer recombination events.

Cellular Roles of theRecQ–TopoIII Complexin DNA Metabolism

THE RECOMBINATION CONNECTION

Inactivation of a RecQ-type helicase or topoisomeraseIII generally causes a variety of detrimental con-sequences on the genomic integrity of an organism.One consequence that is shared by all organismsexamined to date is an increase in genetic recombina-tion. As mentioned above, hyper-recombinationbetween sister chromatids is a hallmark of human cellsexpressing mutant versions of BLM protein. In buddingyeast, inactivation of the RecQ homolog Sgs1 or of thetopo III homolog Top3 leads to an increase in severalkinds of recombination, such as recombination betweentandemly repeated DNA sequences. In E. coli, inacti-vation of the recQ gene leads to an increase inillegitimate recombination (i.e., recombination betweennonhomologous DNA sequences). These observationshave suggested that RecQ helicases generally control orsuppress recombination. This idea is supported by aseries of genetic experiments with budding yeast andmirrored by observations in fission yeast. In theseorganisms, concomitant mutation of the RecQ familymember and of another helicase, Srs2, causes extremeslow growth and frequent lethality. However, thesedefects are fully rescued by mutation of any of severalproteins that perform homologous recombination. Thisindicates that in the absence of a functional homologousrecombination pathway, deletion of the two helicases isno longer detrimental to the cell. This can be explainedby proposing that in the absence of the two helicases,incorrectly regulated or uncontrolled recombination isresponsible for the poor growth and increased lethality.Physical interactions between RecQ helicases andproteins that function in recombination add furthersupport to the connection between the RecQ family andrecombination. In both yeast and humans, a RecQ

814 DNA TOPOISOMERASES: TYPE III–RecQ HELICASE SYSTEMS

family member was shown to physically interact withRad51, a protein that performs key steps in homologousrecombination, such as the invasion of a DNA duplex bya homologous single strand and subsequent branchmigration. Also, biochemical activities of several RecQhelicases are consistent with a direct role in suppressingrecombination. Human BLM and yeast Sgs1 proteinsefficiently unwind or branch-migrate four way DNAjunctions in vitro (Figure 2B). Interestingly, four-wayjunctions closely resemble the structure of an intermedi-ate in genetic recombination, a Holliday junction.Another human RecQ homolog, WRN, unwindsD-loops, DNA intermediates formed by the first stepof homologous recombination—the strand invasion of aduplex by a ssDNA molecule (Figure 2B). Thus, RecQhelicases may control recombination in vivo by disrupt-ing recombination intermediates.

THE REPLICATION CONNECTION

Several lines of evidence indicate that RecQ-likehelicases and topo III function during DNA replication.Analyses of mRNA and protein levels of the buddingyeast RecQ and topo III homologs have shown thatexpression of these genes fluctuates throughout the cellcycle, peaking during and right after the period of activeDNA replication. Also, yeast mutants lacking the RecQhelicase or topo III are sensitive to chemicals that arrestDNA replication by depleting cellular pools of deoxy-nucleotide triphosphates (dNTP’s)—the building blocksnecessary to assemble new DNA molecules. Forinstance, wild-type fission yeast cells are able to resumenormal replication after transient exposure to one suchchemical, hydroxyurea, whereas mutants that lack theRecQ helicase fail to make the recovery. Theseobservations indicate that the function of RecQ familyproteins is especially important for cellular survival ifDNA replication is stalled.

Whereas chemicals such as hydroxyurea can arrestthe progress of DNA replication throughout the entiregenome, occasional pausing in the progression ofindividual replication forks is thought to occur spon-taneously in a high proportion of cells. Accordingly,proteins that are necessary for proper handling andeventual restart of these paused forks are requiredthroughout DNA replication even in cells unchallengedby exogenous agents. Consistent with a role for RecQhelicases during normal cell cycle, analysis of humancells expressing mutant versions of BLM or WRNproteins has shown that these cells exhibit delayedreplication progression and accumulate abnormal repli-cation intermediates.

There are several non-mutually exclusive possibilitiesfor the roles of RecQ family members during DNAreplication. These proteins could physically manipulateor maintain replication fork structure. The importance

of RecQ helicases for normal progression of DNAreplication combined with their previously discussedroles in controlling recombination have led to the ideathat these proteins function specifically to suppressrecombination initiated at stalled replication forks.Also, RecQ helicases may be involved in replicating“difficult” regions of the genome, such as telomeres.Telomeric DNA is very GC-rich and may form alterna-tive conformations, such as G-quartets (Figure 2B).Several RecQ homologs can unwind G-quartet DNAin vitro, and yeast RecQ homolog Sgs1 has beenimplicated in telomere maintenance in vivo. Alterna-tively, these proteins could detect fork stalling and signalto other molecules, resulting in recruitment of factorsnecessary for resumption of fork movement. Indeed,recent evidence suggests that some RecQ family mem-bers may be involved in DNA damage surveillance andsignaling mechanisms called checkpoints. In particular,the budding yeast RecQ homolog Sgs1 has been shownto participate in such signaling mechanisms specific toDNA damage occurring during DNA replication.

RecQ Helicasesand Human Disease

The human genome encodes five proteins that belong tothe RecQ class of helicases. Mutations in three of theseproteins, BLM, WRN, and RECQ4, cause geneticdisorders: Bloom, Werner, and (at least a subset of)Rothmund–Thomson syndromes, respectively. Amongother symptoms, Bloom syndrome patients exhibit shortstature, immunodeficiency, impaired fertility, and apredisposition to a variety of cancers. At the cellularlevel, the syndrome is characterized by genomic instabil-ity, including hyper-recombination between sister-chro-matids and homologous chromosomes. Werner andRothmund-Thomson syndrome patients exhibit symp-toms of premature aging, as well as a predisposition tocertain types of cancer. Both syndromes are alsocharacterized by increased genomic instability at thecellular level. Cells cultured from Werner syndromepatients exhibit an increase in illegitimate recombina-tion, resulting in chromosomal deletions and transloca-tions. Less is known about the cellular characteristics ofthe Rothmund–Thomson syndrome, but these cells alsodisplay increased chromosomal abnormalities.

Whereas no genetic disorder is known to result frommutation of a human topo III homolog,mouse knock-outstrains have provided important information about therole of these proteins in higher eukaryotes. Deletion ofthe TOP3a gene, encoding one of the two isoformsof topo III in mice and humans, results in embryoniclethality, indicating that this protein has essentialfunctions during development. Deletion of the othertopo III isoform, TOP3b, does not result in lethality, but

DNA TOPOISOMERASES: TYPE III–RecQ HELICASE SYSTEMS 815

causes a decrease in lifespan, reduced fertility, andincreased incidence of aneuploidy. Thus, topo III-likeproteins, similar to RecQ helicases, play importantroles in mammalian development, aging, and chromo-somal integrity.


DNA Helicases: Dimeric Enzyme Action † DNAHelicases: Hexameric Enzyme Action † DNA Replica-tion Fork, Eukaryotic † DNA Supercoiling † GlutamateReceptors, Ionotropic

GLOSSARY

DNA recombination Exchange or transfer of genetic materialbetween two DNAmolecules, such as two chromosomes in the cell.

DNA replication The process of faithful copying of genetic infor-mation in a cellular genome prior to cell division. This isaccomplished by separating the strands of duplex chromosomalDNA and synthesizing new DNA strands that are complementaryto the parental strands.

replication fork Y-shaped DNA structure formed during DNAsynthesis when the parental DNA strands are separated to providea template for DNA replication.

sister chromatids The identical copies of a single chromosomeproduced after DNA replication.

supercoiling The topological state achieved by twisting a duplexDNA molecule around its axis.

telomere Region of DNA at the end of a linear chromosome.

FURTHER READING


Lohman, T. M., and Bjornson, K. P. (1996). Mechanisms ofhelicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65,169–214.

Lombard, D. B. (2001). Biochemistry and Genetics of RecQ-Helicases.Kluwer Academic, Boston.

Oakley, T. J., and Hickson, I. D. (2002). Defending genome integrityduring S-phase: Putative roles for RecQ helicases and topoiso-merase III. DNA Repair 1, 175–207.

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: Amolecular prospective. Nature Rev. 3, 430–440.

BIOGRAPHY

Rodney Rothstein is a Professor of Genetics and Development atColumbia University College of Physicians and Surgeons in New York.His principal research interests are in the mechanisms of DNArecombination and the cellular response to DNA damage. He holds aPh.D. from The University of Chicago and received postdoctoraltraining at the University of Rochester and Cornell University. Hedeveloped one-step gene disruption in yeast and is one of the authors ofthe double-strand break repair model. His laboratory discovered thefirst eukaryotic topoisomerase III gene family member and the firsteukaryotic RecQ homolog, Sgs1.

Erika Shor, a senior graduate student in the Rothstein laboratory,studies the budding yeast RecQ and topoisomerase III familymembers.

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