tcrms, if the 20ff double helix were expanded so that its width was that of a l c m double-stranded rope, the corresponding human genome (from a single cell) would stretch for -10000 km, or roughly the distance from San Francisco to London. Consider the difficulty in keeping specific regions of the genome appropriately under- or overwound and keeping the 46 individual ropes free from knots and tangles after packaging them into an expanded nucleus less than 50 meters in diameter! Without the means and the machinery necessary to control thc topological state of DNA, the cell as wc know it could not exist. Summary Although the genetic code is defined by a linear array of nucleotides, it is the three-dimensional structure of the double helix that regulates most of its cellular functions. Over the past two decades, it has hecome increasingly clear that aspects of this three-dimensionality which reflect topological relationships within the double helix (i.e., superhelical twisting, knotting, or tangling) influence virtually every facet of nucleic acid physiology. In vivo, DNA topology is modulated by ubiquitous enzymes known as topoisomerases. The type I1 enzyme is essential to the eukaryotic cell and is required for unlinking daughter chromosomes and maintaining chromosome structure. Moreover, topoisomerase I1 also has been identified as the primary cellular target for several widely used antineoplastic drugs. Before the physiological functions of topoisomerase 11 can be effectively dissected or its drug interactions fully exploited, it is imperative to understand the mechanism by which this important enzyme carries out its catalytic cycle. Introduction The genetic code exists in the cell as a one-dimensional array of nucleotides. However, it is the threedimensional properties of DNA that determine how the genetic information is expressed and transmitted from one generation to the next(’,’). Some of the most important three-dimensional properties of DNA rcvolve around to ological relationships within the double helix(’-’? For example, topological alterations such as superhelical twisting (either underwinding or overwinding) govern rates of initiation and elongation for both DNA replication and transcription. Furthermore, intra- and intermolecular knots must be untangled in order to resolve recomnbination products and unlink daughter chromosomes during cell division. The magnitude for potential topological problems within the cell becomes evident once the physical size of the eukaryotic genome is realized. If the 46 chromosomes from a single human nucleus (5 1 0 microns in diameter) were stripped of all proteins and arranged end-to-end, the DNA (at actual size) would be nearly 2 meters in length. To place the problem in more familiar
DNA Topoisomerase \I I n vivo, DNA topology is regulated by two classes o f highly conserved enzymes known as type I and typc 11 topoisotnerases(’,2). The typc IT topoisomerase is a homodimeric protein with a molecular mass of -170 kDa/subunit(’-’). It is essential for the sunival of eukaryotic cells(’-‘) and is rc uircd for the segregation of mitotic(“’) and meiotic‘q chromosomes and the maintenance of chromosome Evidence suggests that it may also be involved in DNA i-eplication, transcription, and r e ~ o m b i n a t i o n ( ~ ~ ’ ~. ~In~ ~ ~ - ” ) addition to its physiological functions, topoisomerase TI is the primary cellular target for a wide variety of clinically relevant antineoplastic drugs(Ls-”O’. The chemotherapeutic potentials of these agents correlate with their abilities to stabilize covalent topoisomerase TI-cleaved DNA complexes, which are reaction intcrmediates in the enzyme’s catalytic c y ~ l e ( ’ ~ - ~ ~ ) . Clearly. before the cellular roles of topoisomerase I1 can be fully defined and its potential as a drug target maxirnally exploited. the reaction niechanism of the enzyme must be understood. To this end, the present review is intended to familiarize the reader with the catalytic cycle of the eukaryotic type I1 enzyme. The basic enzymatic pro erties of topoisomerase I1 were reviewed in 1989(#. Therefore, this article will concentrate on the more recent developments in the field. Finally. an isozyme of topoisomerase 11 with a molecular 7mass of -180 kDa/subunit has been reported(2J,-’). Although the catalytic mechanisms of thc -170 and -180kDa isozymes arc probably identical, preliminary experiments indicate that they can be distinguished on the basis of their biochemical and pharmacological properties(”””). The reader should note that remarks presented below are confined to the -170 kDa form of the type I1 enzyme.
Catalytic Cycle of Topoisomerase II Topoisomerase I1 alters the topological statc of nucleic acids by passing an intact helix of DNA through a transient doublc-stranded break which it generates in a separate DNA This double-stranded D N A
passage reaction takes place at the expense of ATP hydrolysis and rcq uires the presence of magnesium. Although the enzyme's catalytic mechanism appears to be concerted and quite complex in nature: it can be broken down into a series of discrete and straightforward events. These reaction steps are outlined in Fig. 1. Four points should be noted before the individual events arc discussed. First, in the absence of additional factors(27),eukaryotic to oisomerase I1 can only work down an energy gradientfi.2).Hence, the catalytic cycle shown in Fig. 1 depicts the removal of negative superhelical twists from DNA. (The enzyme is equally capable of removing positive superhelical twisd2)). This process is known as DNA relaxation. In contrast to its prokaryotic counterpart, DNA gyrase, the purified eukaryotic enzyme has no intrinsic ability to introduce negative or positive supercoils (i.e. underwinds or overwinds) into relaxed molecules(',2,2S), Second, the reaction cycle for topoisomerase 11-catalyzed catenation/decatenation or knotting/unknotting of DNA is fundamentally the same as that shown for its relaxation activity. Third, it is not known when the cofactors for topoisomerase 11, ATP and magnesium, first interact with the enzyme and/or DNA. Therefore, in Fig. 1 they have been introduced into the reaction cycle at the first step in which they are required for enzyme function. Fourth, while the enzyme's catalytic cycle alters the topological state of DNA, both the nucleic acid sequence and the chemical structure of the relaxed
D N A product are identical to those of the original supercoiled substrate. Step 7, Substrate recognition and binding Topoisomerase I1 initiates its catalytic cycle by binding to its nucleic acid substrate. The enzyme's interaction with DNA is determined by two properties of the double helix; nucleotide sequence and topological structure. The nucleotide sequence of DNA dictates the sites at which topoisomerase TI binds the double helix(29330). It also defines sites of enzyme-mediated DNA cleavand catalytic activity(36).Although the type 11 enzyme interacts with preferred sequences: its specificity is far less stringent than, for example, that of a restriction endon~clease('~). This lack of stringency is probably important to the physiological functions of topoisomerase II? as it allows the enzyme to act at numerous sites within a given genome. Several topoisomerase TI recognition/cleavage sites have been but no one consensus fits all of the known sequences (Table 1). Thus, the precise relationship between primary structure and enzyme recognition remains a mystery. DNA recognition specificity does, however, appear to be an intrinsic property of topoisomerase 11, since enzymes from divergent organisms interact at the same nucleic acid sites (Table 1). Finally, DNA sequences recognized by topoisomerase TI are asymmetric in nature (Table 1). This is in contrast to other homodimeric enzymes (such as the type I1 restriction endonucleases) which generally interact with palindromic sites (i.e., sequences which contain a central axis of symmetry)(37). The topological state of D N A modulates the level of topoisomerase I1 binding. The enzyme can discern topological structures and interacts preferentially (3- to 10-fold) with supercoiled nucleic acids over relaxed molecules(3x8s0).This recognition is independent of the enzyme's recognition of DNA primary structure(4"). Table 1. Consensus D N A recognition/cleavuge sequences reported for eukaryotic topoisomeruse II"
Topoisomerase I1 species
A k GTNTAYATTNATNNGN CANATRTABNTANNCN
NCNNGYNGTTNYNYNN NGNNCRNCAANRNRNN C
Fig. 1. Catalytic cycle of topoisomerase 11. The homodimeric enzyme is represented by the paired circles. The double-stranded DNA passage reaction o f topoisomerase I1 is inadc up of six steps: 1) substrate recognition and binding; 2) pre-strand passage DNA cleavage/rel~gation;3) double-stranded DNA passage; 4) post-strand passage DNA cleavage/religation; 5 ) ATP hydrolysis: and 6 ) enzyme turnover. Transient DNA cleakage species are shown in brackets.
Chicken and human("!
GC J. A ACAATGCGYTYATCRT TGTTACGCRARTAGYA CG T1'
Drosophila, calf. and tctrahymena('1r34)
aThe arrows represent points of topoisomerase 11-mediated DNA cleavage; Y represents pyrimidines, R represents purines, and N represents any complementary base.
Fi 2, Recognition of DNA crosovers by Diowphilu topoiwrnerdw ITa5' Elcctron micrograph\ of the enzyme complexed with negatively supercoiled (/eft panel) and lincar (nght panel) DNA are shown.
The ability to recognize DNA topology is important to the biological function of topoisomcrase I1 since i t allows the enzyme to distinguish between the substrate and products of its catalytic reaction. A recent study demonstrated that topoisomerase TI recognizes iiucleic acid topology because it binds to DNA at regions of helix-helix juxtaposition (i.e., crossovers or nodes)("')). As illustrated in Fig. 1, such D N A crossovers are more prevalent in supercoiled than relaxed molecules. When visualized by electron microscopy, the cn;.yme is observed at crossovers -90 % of the time when bound to circular DNA substrates which contained as few as three or four nodes (Fig. 2). Topoisomerase I1 also showc an affinity for crossovers formed in linear DNA s ~ b s t r a t e s ( ~ ' 3 ~as~ ) well as intermolecular crossovers formed by two independent DNA molecules(") (Fig. 2). Moreover. the enzyme binds D N A nodes even in the absence of divalent cations (magnesium is required for the enzyme's catalytic function(1,2)). Thus, recognition of DNA crossovers by topoisomerase TI is not dependent upon enzymatic activity. In addition to its impact on the recognition of DNA topology. the eniyme's preference for D N A crossovers provides a link between its essential catalytic and structural roles in the cell. The ability to bind intermolecular D N A nodes allows topoisomerase IT to untangle topologically intertwined daughter chromosomes at the time of cell The ability to bind two DNA helices prior to catalytic activation allows the enzyme to anchor chromosomal loops to the nuclear
Step 2. Pre-strand passage DNA cleavage/religation Following recognition of its nucleic acid substrate, topoisomerase 11 rapidly establishes a double-stranded DNA cleavage/religation equilibrium(2). This equilibrium highly favors the religation event (c1eavage:religation, -1:99). Without an efficient religatioii activity. the type I1 enzyme would not be able to maintain the topological integrity of DNA. DNA cleavage and religation require the presence of a divalent While magnesium is employed by topoisomerase I1 in vivo. calcium (and to a lesser extent, manganese or cobalt) can substitute in the cleavage/religation step(422-M).These latter divalent
cations cannot efficiently support the enzyme's overall catalytic cycle because of the requirement for a Mg .ATP cofactor in the strand passage ~ t e p ( ~ . ~ * ) . Upon hydrolysis of the D N A backbone, topoisomerase I1 becomes covalentlv attached to both newly generated 5'-termini via 0';phosphotyrosine bonds(4s). Breakage of each strand of the double helix is mediated by a scparate subunit of the homodimeric enzyme(46). As shown in Table 1, topoisomerase I1 makes a staggered break in DNA which results in 4-base 5 ' ~verhangd~~~"). Recent evidence indicates that the type 11 enzyme performs double-stranded DNA cleavage by generating two coordinated sin le stranded breaks in the nucleic acid backbone (33,34.-a,46). Furthermore, kinetic studies demonstrate that nicked DNA is an obligatory intermediate in the enz me's pathway for religating double-stranded breaksg6). Taken together. these findings lead to the conclusion that the coordinated single-stranded breaks generated by topoisomerase I1 are made in a sequential rather than a concerted fashion. The asymmetric nature of its recognition sites requires topoisoinerase I1 to cleave DNA between different nucleotides on the two complementary strands of the double helix (Table 1). Therefore, it is not surprisiiig that the enzyme can discriininate between the two strands and in many cases cleaves one preferentially over the other(33.34.44).In a sequence that aligns with the bottom consensus shown in Table 1, topoisomerase TI cleaves the top strand 3- to 10-fold more efficiently than it does the bottom(34). The physiological ramifications of this strand preference are not known at the present time. Besides being a critical step in the catalytic cycle of topoisomerase 11, the enzyme's pre-strand passage DNA cleavage/religation equilibrium is an important target for several classes of antineoplastic The chemotherapeutic potentials of these agents relate to their abilities to stabilize the covalent topoisomerase 11-DNA cleavage complex(18-20). Despite the clinical importance of many of these drugs. their interactions with the enzyme-DNA complex have yet to be detailed. However, both etoposide (a representative nonintercalative dru ) and amsacrine (a representative intercalative drug6"'")) have been shown to enhaiice DNA breakage primarily by decreasing the enzyme's apparent first order rate constant for religation(21.'2). Finally, topoisomerase 11-targeted drugs alter the enzyme's utilization of DNA cleavage sites(1"~2").Unfortunately, relationships between site alteration. inhibition of religation. and clinical efficacy have not been defined. The nature of the cleavage/religation reaction of topoisoinerase I1 has led to speculation that the enzyme mediates some forms of illegitimate DNA recombination in the Support for this hypothesis stems from in vivo as wcll as in vitro investigations. Topoisomerase 11-targeted antineoplastic drugs that stabilize the enzyme's D N A cleavage complex increase
-CIRC - LIN
Step 3. Double-stranded DNA passage Once topoisomerase I1 has created a double-stranded break in the nucleic acid backbone, it passes a separate double-stranded segment of DNA through the break. The strand passage event is completely dependent on the binding of Mg.ATP(2,3X).Hydrolysis of the hlgh encrgy cofactor is not required for DNA translocation. This is evidenced by the ability of non-hydrolyzable ATP analo s to support a single round of strand passage('.2'%
-0LIGO 1 2 3 4 5 6 7 Fig. 3. Intermolecular ligation catalyzed by Drasvphzla topoisornera.je II("). A time course for the enzvme-mediated ligation of cleaved @X174(+) strand DNA to a 56 bp (i~P]phospliale-labeled doublestrandcd oligonucleotide is shown. Reaction products were scparated by eleclruphoresis uii an a g a r u x gel and visualized by autoradiography. The electrophorcric mobilities of circular (CIRC, unclcaiul) $Xl74(+) strand DNA: Iinear (LIN, cleaved) @X174(T) strand DKA. and the oligonucleoticie (OLIGO) are indicated. Note that the oligoiiucleotidc is ligated to the cleavcd linear. but not thc uiicleaved circular single-strarided bacteriophage DNA.
the frequency of chi ornosoinal translocations, inutations, arid sister chromatid exchange in mammals and mammalian cell culturesi16 18-20) . In a dd.Ition, topoiwmerase I1 can mediate low levels (