Human topoisomerase IIalpha uses a two-metal-ion mechanism for DNA cleavage

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen.     

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.     

Using 3'-bridging phosphorothiolates to isolate the forward DNA cleavage reaction of human topoisomerase IIalpha

The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3'-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3'-terminal -SH moiety that is a poor nucleophile relative to the normal 3'-terminal -OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIalpha-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIalpha and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one scissile bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional "suicide substrates" and should be valuable for future studies on DNA scission and the topoisomerase II-DNA cleavage complex.     

Metal-ion-dependent catalysis and specificity of CCA-adding enzymes: a comparison of two classes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases] catalyze synthesis of the conserved and essential CCA sequence to the tRNA 3' end. These enzymes are divided into two classes of distinct structures that differ in the overall orientation of the head to tail domains. However, the catalytic core of the two classes is conserved and contains three carboxylates in a geometry commonly found in DNA and RNA polymerases that use the two-metal-ion mechanism for phosphoryl transfer. Two important aspects of the two-metal-ion mechanism are tested here for CCA enzymes: the dependence on metal ions for catalysis and for specificity of nucleotide addition. Using the archaeal Sulfolobus shibabae enzyme as an example of the class I, and the bacterial Escherichia coli enzyme as an example of the class II, we show that both enzymes depend on metal ions for catalysis, and that both use primarily Mg2+ and Mn2+ as the "productive" metal ions, but several other metal ions such as Ca2+ as the "nonproductive" metal ions. Of the two productive metal ions, Mg2+ specifically promotes synthesis of the correct CCA, whereas Mn2+ preferentially accelerates synthesis of the noncognate CCC and poly(C). Thus, despite evolution of structural diversity of two classes, both classes use metal ions to determine catalysis and specificity. These results provide critical insights into the catalytic mechanism of CCA synthesis to allow the two classes to be related to each other, and to members of the larger family of DNA and RNA polymerases.     

The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining

The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg(2+) is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3'-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.     

Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease.

The rate constant for the phosphoryl transfer step in site-specific DNA cleavage by EcoRV endonuclease has been determined as a function of pH and identity of the required divalent metal ion cofactor, for both wild-type and T93A mutant enzymes. These measurements show bell-shaped pH-rate curves for each enzyme in the presence of Mg2+ as a cofactor, indicating general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The kinetic data support a model for phosphoryl transfer based on wild-type and T93A cocrystal structures, in which the ionizations of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group. The model concurs with recent observations of two metal ions bound in the active sites of the type II restriction endonucleases BamHI and BglI, suggesting the possibility of a similar catalytic mechanism functioning in many or all members of this enzyme family.     

Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.     

Mapping metal ions at the catalytic centres of two intron-encoded endonucleases.

Divalent metal ions play a crucial role in forming the catalytic centres of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yielded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites. Specific hydroxyl radical-induced cleavage was observed within, and immediately after, two conserved LAGLIDADG motifs in both proteins and at sites at, and near, the scissile phosphates of the corresponding DNA substrates. Titration of Fe2+-containing protein-DNA complexes with Ca2+ ions, which are unable to support endonucleolytic activity, was performed to distinguish between the individual metal ions in the complex. Mutations of single amino acids in this region impaired catalytic activity and caused the preferential loss of a subset of hydroxyl radical cleavages in both the protein and the DNA substrate, suggesting an active role in metal ion coordination for these amino acids. The data indicate that the endonucleases cleave their DNA substrates as monomeric enzymes, and contain a minimum of four divalent metal ions located at or near the catalytic centres of each endonuclease. The metal ions involved in cleaving the coding and the non-coding strand are positioned immediately after the N- and C-terminally located LAGLIDADG motifs, respectively. The dual protein/nucleic acid footprinting approach described here is generally applicable to other protein-nucleic acid complexes when the natural metal ion can be replaced by Fe2+.     

Leaving group stabilization by metal ion coordination and hydrogen bond donation is an evolutionarily conserved feature of group I introns.

To understand the behavior of group I introns on a biologically fundamental level, we must distinguish those traits that arise as the products of natural selection (selected traits) from those that arise as the products of neutral drift (non-selected traits). In practice, this distinction relies on comparing the similarities and differences among widely divergent introns to identify conserved traits. Here we address whether the strategies used by the eukaryotic group I intron from the Tetrahymena ciliate to stabilize the leaving group during splicing are maintained in the group I intron from the widely divergent Azoarcus bacterium. A substrate analogue containing a 3'-phosphorothiolate linkage, in which a sulfur atom replaces the bridging 3'-oxygen atom of the scissile phosphate, reacts 20-fold slower in the Azoarcus reaction than the corresponding unmodified substrate in the presence of Mg(II) as the only divalent cation. However, Mn(II) relieves this negative effect such that the 3'-S-P bond cleaves 21-fold faster than does the 3'O-P bond. Other thiophilic divalent metal ions such as Co(II), Cd(II), and Zn(II) similarly support cleavage of the S-P bond. These results indicate that a metal ion directly coordinates to the leaving group in the transition state of the Azoarcus ribozyme reaction. Additionally, the 3'-sulfur substitution eliminates the approximately 10(3)-fold contribution of the adjacent 2'-OH to transition state stabilization. Considering that sulfur accepts hydrogen bonds weakly compared to oxygen, this result suggests that the 2'-OH contributes to catalysis by donating a hydrogen bond to the 3'-oxygen leaving group in the transition state, presumably acting in conjunction with the metal ion to stabilize the developing negative charge. These same catalytic strategies of metal ion coordination and hydrogen bond donation operate in the Tetrahymena ribozyme reaction, suggesting that these features of catalysis have been conserved during evolution and thus extend to all group I introns. The two ribozymes also exhibit quantitative differences in their response to 3'-sulfur substitution. The Azoarcus ribozyme binds and cleaves the phosphorothiolate substrate more efficiently relative to the natural substrate than the Tetrahymena ribozyme under the same conditions, suggesting that the Azoarcus ribozyme better accommodates the phosphorothiolate at the active site both in the ground state and in the transition state. These differences may reflect either a less tightly knit Azoarcus structure and/or spatial deviations between backbone atoms in the two ribozymes that arise during divergent evolution, analogous to the well-documented relationship between protein sequence and structure.     

Does a single metal ion bridge the A-9 and scissile phosphate groups in the catalytically active hammerhead ribozyme structure?

We have constructed a model structure that we believe represents the strongest possible physically and chemically reasonable representation of a hypothesized catalytically active hammerhead ribozyme structure in which a single divalent metal ion bridges the A9 and scissile phosphate groups. It has been proposed that such a structure arises from a conformational change in which the so-called ground-state structure (as observed by X-ray crystallography) rearranges in such a way that the pro-R oxygen atoms of both the A9 and scissile phosphate groups are directly coordinated by a single divalent metal ion in the transition-state of the hammerhead ribozyme cleavage reaction. We show that even the small subset of possible model structures that are consistent with these requirements, and that are stereochemically and sterically reasonable, are contradicted by experimental evidence. We also demonstrate that even a minimal subset of assumptions, i.e. that stems I and II are helical and that the two phosphate groups are coordinated by a divalent metal ion in the standard octahedral geometry, are sufficient to lead to this contradiction. We therefore conclude that such a mechanism of hammerhead ribozyme catalysis is untenable, at least in its present formulation.     

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P

Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes.     

The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites

Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.     

Ca2+ binding in the active site of HincII: implications for the catalytic mechanism

The 2.8 A crystal structure of the type II restriction endonuclease HincII bound to Ca(2+) and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglII, and BglI, the observed calcium cation is directly ligated to the pro-S(p) oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-R(p) oxygen of the phosphate group 3' of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3'-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca(2+)-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na(+) ions.     

Investigation of restriction enzyme cofactor requirements: a relationship between metal ion properties and sequence specificity

Restriction enzymes are important model systems for understanding the mechanistic contributions of metal ions to nuclease activity. These systems are unique in that they combine distinct functions which have been shown to depend on metal ions: high-affinity DNA binding, sequence-specific recognition of DNA, and Mg(II)-dependent phosphodiester cleavage. While Ca(II) and Mn(II) are commonly used to promote DNA binding and cleavage, respectively, the metal ion properties that are critical to the support of these functions are not clear. To address this question, we assessed the abilities of a series of metal ions to promote DNA binding, sequence specificity, and cleavage in the representative PvuII endonuclease. Among the metal ions tested [Ca(II), Sr(II), Ba(II), Eu(III), Tb(III), Cd(II), Mn(II), Co(II), and Zn(II)], only Mn(II) and Co(II) were similar enough to Mg(II) to support detectable cleavage activity. Interestingly, cofactor requirements for the support of DNA binding are much more permissive; the survey of DNA binding cofactors indicated that Cd(II) and the heavier and larger alkaline earth metal ions Sr(II) and Ba(II) were effective cofactors, stimulating DNA binding affinity 20-200-fold. Impressively, the trivalent lanthanides Tb(III) and Eu(III) promoted DNA binding as efficiently as Ca(II), corresponding to an increase in affinity over 1000-fold higher than that observed under metal-free conditions. The trend for DNA binding affinity supported by these ions suggests that ionic radius and charge are not critical to the promotion of DNA binding. To examine the role of metal ions in sequence discrimination, we determined specificity factors [K(a)(specific)/K(a)(nonspecific)] in the presence of Cd(II), Ba(II), and Tb(III). Most interestingly, all of these ions compromised sequence specificity to some degree compared to Ca(II), by either increased affinity for a noncognate sequence, decreased affinity for the cognate sequence, or both. These results suggest that while amino acid-base contacts are important for specificity, the properties of metal ion cofactors at the catalytic site are also critical for sequence discrimination. This insight is invaluable to our efforts to understand and subsequently design sequence-specific nucleases.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

Role of divalent metal ions in the hammerhead RNA cleavage reaction.

A hammerhead self-cleaving domain composed of two oligoribonucleotides was used to study the role of divalent metal ions in the cleavage reaction. Cleavage rates were measured as a function of MgCl2, MnCl2, and CaCl2 concentration in the absence or presence of spermine. In the presence of spermine, the rate vs metal ion concentration curves are broader, and lower concentrations of divalent ions are necessary for catalytic activity. This suggests that spermine can promote proper folding of the hammerhead and one or more divalent ions are required for the reaction. Six additional divalent ions were tested for their ability to support hammerhead cleavage. In the absence of spermine, rapid cleavage was observed with Co2+ while very slow cleavage occurred with Sr2+ and Ba2+. No detectable specific cleavage was observed with Cd2+, Zn2+, or Pb2+. However, in the presence of 0.5 mM spermine, rapid cleavage was observed with Zn2+ and Cd2+, and the rate with Sr2+ was increased, indicating that while these three ions could not promote proper folding of the hammerhead they were able to stimulate cleavage. These results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead. Additionally, an altered divalent metal ion specificity was observed when a unique phosphorothioate linkage was inserted at the cleavage site. The substitution of a sulfur for a nonbridging oxygen atom substantially reduced the affinity of an important Mg2+ ion necessary for efficient cleavage. In contrast, the reaction proceeds normally with Mn2+, presumably due to its ability to coordinate with both oxygen and sulfur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific cleavages of DNA by ascorbate in the presence of copper ion or copper chelates

The DNA-cleavage specificity of ascorbate in the presence of copper ion is analyzed with end-labeled pBR322 DNA fragments. The nonenzymatic reaction of Cu(II)/ascorbate and DNA shows certain degrees of cleavage preference toward purine-containing short segments in the labeled DNA under mild conditions (at 0 degrees C and 10 min). The segments of pyrimidine clusters are least susceptible to cleavage. The DNA scission cannot be detected in the absence of metal ions, and is greatly diminished in the presence of EDTA and metal-chelating peptide. It is more specific than the nuclease-like scission activity induced by cuprous-phenanthroline complex. This scission activity in relation to the antiviral and antitumor activities of vitamin C reported in the literature deserves a crucial consideration.     

Chlorella virus Marburg topoisomerase II: high DNA cleavage activity as a characteristic of Chlorella virus type II enzymes

Although the formation of a covalent enzyme-cleaved DNA complex is a prerequisite for the essential functions of topoisomerase II, this reaction intermediate has the potential to destabilize the genome. Consequently, all known eukaryotic type II enzymes maintain this complex at a low steady-state level. Recently, however, a novel topoisomerase II was discovered in Paramecium bursaria chlorella virus-1 (PBCV-1) that has an exceptionally high DNA cleavage activity [Fortune et al. (2001) J. Biol. Chem. 276, 24401-24408]. If robust DNA cleavage is critical to the physiological functions of chlorella virus topoisomerase II, then this remarkable characteristic should be conserved throughout the viral family. Therefore, topoisomerase II from Chlorella virus Marburg-1 (CVM-1), a distant family member, was expressed in yeast, isolated, and characterized. CVM-1 topoisomerase II is 1058 amino acids in length, making it the smallest known type II enzyme. The viral topoisomerase II displayed a high DNA strand passage activity and a DNA cleavage activity that was approximately 50-fold greater than that of human topoisomerase IIalpha. High DNA cleavage appeared to result from a greater rate of scission rather than promiscuous DNA site utilization, inordinately tight DNA binding, or diminished religation rates. Despite the fact that CVM-1 and PBCV-1 topoisomerase II share approximately 67% amino acid sequence identity, the two enzymes displayed clear differences in their DNA cleavage specificity/site utilization. These findings suggest that robust DNA cleavage is intrinsic to the viral enzyme and imply that chlorella virus topoisomerase II plays a physiological role beyond the control of DNA topology.