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.     

Ability of viral topoisomerase II to discern the handedness of supercoiled DNA: bimodal recognition of DNA geometry by type II enzymes

Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIalpha and Escherichia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase IIalpha and topoisomerase IIbeta to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under- and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domain during DNA cleavage.     

Topoisomerase II from Chlorella virus PBCV-1 has an exceptionally high DNA cleavage activity

Chlorella virus PBCV-1 topoisomerase II is the only functional type II enzyme known to be encoded by a virus that infects eukaryotic cells. However, it has not been established whether the protein is expressed following viral infection or whether the enzyme has any catalytic features that distinguish it from cellular type II topoisomerases. Therefore, the present study characterized the physiological expression of PBCV-1 topoisomerase II and individual reaction steps catalyzed by the enzyme. Results indicate that the topoisomerase II gene is widely distributed among Chlorella viruses and that the protein is expressed 60-90 min after viral infection of algal cells. Furthermore, the enzyme has an extremely high DNA cleavage activity that sets it apart from all known eukaryotic type II topoisomerases. Levels of DNA scission generated by the viral enzyme are approximately 30 times greater than those observed with human topoisomerase IIalpha. The high levels of cleavage are not due to inordinately tight enzyme-DNA binding or to impaired DNA religation. Thus, they most likely reflect an elevated forward rate of scission. The robust DNA cleavage activity of PBCV-1 topoisomerase II provides a unique tool for studying the catalytic functions of type II topoisomerases.     

Site-specific DNA cleavage by Chlorella virus topoisomerase II

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.     

Topoisomerase II from Chlorella virus PBCV-1. Characterization of the smallest known type II topoisomerase

Type II topoisomerases, a family of enzymes that govern topological DNA interconversions, are essential to many cellular processes in eukaryotic organisms. Because no data are available about the functions of these enzymes in the replication of viruses that infect eukaryotic hosts, this led us to express and characterize the first topoisomerase II encoded by one of such viruses. Paramecium bursaria chlorella virus 1 (PBCV-1) infects certain chlorella-like green algae and encodes a 120-kDa protein with a similarity to type II topoisomerases. This protein was expressed in Saccharomyces cerevisiae and was highly active in relaxation of both negatively and positively supercoiled plasmid DNA, catenation of plasmid DNA, and decatenation of kinetoplast DNA networks. Its optimal activity was determined, and the omission of Mg(2+) or its replacement with other divalent cations abolished DNA relaxation. All activities of the recombinant enzyme were ATP dependent. Increasing salt concentrations shifted DNA relaxation from a normally processive mechanism to a distributive mode. Thus, even though the PBCV-1 enzyme is considerably smaller than other eukaryotic topoisomerase II enzymes (whose molecular masses are typically 160-180 kDa), it displays all the catalytic properties expected for a type II topoisomerase.     

Impact of the C-terminal domain of topoisomerase IIalpha on the DNA cleavage activity of the human enzyme

The enzymatic function of the C-terminal domain of eukaryotic topoisomerase II is not well defined. This region of the enzyme is highly variable and hydrophilic and contains nuclear localization signals and phosphorylation sites. In contrast to eukaryotic topoisomerase II, type II enzymes from chlorella virus completely lack the C-terminal domain. These viral enzymes are characterized by a robust DNA cleavage activity, high coordination between their two active site tyrosyl residues, and reduced sensitivity to anticancer drugs. As a first step toward characterizing the contribution of the C-terminal domain of human topoisomerase IIalpha to enzyme function, the protein was truncated at amino acid 1175, which corresponds to the C-terminal residue of Paramecium bursaria chlorella virus-1 topoisomerase II as determined by BLAST sequence alignment. Although the overall catalytic activity of the resulting enzyme, hTop2alphaDelta1175, was lower than that of full-length topoisomerase IIalpha, the mutant protein displayed a double-stranded DNA cleavage activity that was approximately 2-3-fold higher. While the DNA breaks created by hTop2alphaDelta1175 were primarily double stranded, cuts generated by topoisomerase IIalpha were primarily single stranded. Thus, the enhanced cleavage observed for hTop2alphaDelta1175 appears to be due, at least in part, to an increase in active site coordination. Finally, hTop2alphaDelta1175 displayed a distinctly lower susceptibility to anticancer agents than did topoisomerase IIalpha, despite the fact that it showed a similar binding affinity for etoposide. Therefore, the C-terminal domain of human topoisomerase IIalpha appears to play significant roles in modulating the DNA cleavage/ligation reaction of the enzyme and its response to anticancer agents.     

DNA cleavage and religation by human topoisomerase II alpha at high temperature.

A common DNA religation assay for topoisomerase II takes advantage of the fact that the enzyme can rejoin cleaved nucleic acids but cannot mediate DNA scission at suboptimal temperatures (either high or low). Although temperature-induced DNA religation assays have provided valuable mechanistic information for several type II enzymes, high-temperature shifts have not been examined for human topoisomerase IIalpha. Therefore, the effects of temperature on the DNA cleavage/religation activity of the enzyme were characterized. Human topoisomerase IIalpha undergoes two distinct transitions at high temperatures. The first transition occurs between 45 and 55 degrees C and is accompanied by a 6-fold increase in the level of DNA cleavage at 60 degrees C. It also leads to a loss of DNA strand passage activity, due primarily to an inability of ATP to convert the enzyme to a protein clamp. The enzyme alterations that accompany the first transition appear to be stable and do not revert at lower temperature. The second transition in human topoisomerase IIalpha occurs between 65 and 70 degrees C and correlates with a precipitous drop in the level of DNA scission. At 75 degrees C, cleavage falls well below amounts seen at 37 degrees C. This loss of DNA scission appears to result from a decrease in the forward rate of DNA cleavage rather than an increase in the religation rate. Finally, similar high-temperature alterations were observed for yeast topoisomerase II and human topoisomerase IIbeta, suggesting that parallel heat-induced transitions may be widespread among type II topoisomerases.     

Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells

DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds to unmodified C or 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.     

Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga.

An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.     

Base excision repair intermediates as topoisomerase II poisons

Abasic sites are the most commonly formed DNA lesions in the cell and are produced by numerous endogenous and environmental insults. In addition, they are generated by the initial step of base excision repair (BER). When located within a topoisomerase II DNA cleavage site, "intact" abasic sites act as topoisomerase II poisons and dramatically stimulate enzyme-mediated DNA scission. However, most abasic sites in cells are not intact. They exist as processed BER intermediates that contain DNA strand breaks proximal to the damaged residue. When strand breaks are located within a topoisomerase II DNA cleavage site, they create suicide substrates that are not religated readily by the enzyme and can generate permanent double-stranded DNA breaks. Consequently, the effects of processed abasic sites on DNA cleavage by human topoisomerase IIalpha were examined. Unlike substrates with intact abasic sites, model BER intermediates containing 5'- or 3'-nicked abasic sites or deoxyribosephosphate flaps were suicide substrates. Furthermore, abasic sites flanked by 5'- or 3'-nicks were potent topoisomerase II poisons, enhancing DNA scission approximately 10-fold compared with corresponding nicked oligonucleotides that lacked abasic sites. These findings suggest that topoisomerase II is able to convert processed BER intermediates to permanent double-stranded DNA breaks.     

N-acetyl-p-benzoquinone imine, the toxic metabolite of acetaminophen, is a topoisomerase II poison

Although acetaminophen is the most widely used analgesic in the world, it is also a leading cause of toxic drug overdoses. Beyond normal therapeutic doses, the drug is hepatotoxic and genotoxic. All of the harmful effects of acetaminophen have been attributed to the production of its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Since many of the cytotoxic/genotoxic events triggered by NAPQI are consistent with the actions of topoisomerase II-targeted drugs, the effects of this metabolite on human topoisomerase IIalpha were examined. NAPQI was a strong topoisomerase II poison and increased levels of enzyme-mediated DNA cleavage >5-fold at 100 microM. The compound induced scission at a number of DNA sites that were similar to those observed in the presence of the topoisomerase II-targeted anticancer drug etoposide; however, the relative site utilization differed. NAPQI strongly impaired the ability of topoisomerase IIalpha to reseal cleaved DNA molecules, suggesting that inhibition of DNA religation is the primary mechanism underlying cleavage enhancement. In addition to its effects in purified systems, NAPQI appeared to increase levels of DNA scission mediated by human topoisomerase IIalpha in cultured CEM leukemia cells. In contrast, acetaminophen did not significantly affect the DNA cleavage activity of the human enzyme in vitro or in cultured CEM cells. Furthermore, the analgesic did not interfere with the actions of etoposide against the type II enzyme. These results suggest that at least some of the cytotoxic/genotoxic effects caused by acetaminophen overdose may be mediated by the actions of NAPQI as a topoisomerase II poison.     

1,4-Benzoquinone is a topoisomerase II poison

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic metabolites, especially 1,4-benzoquinone. The cellular consequences of 1,4-benzoquinone are consistent with those of topoisomerase II-targeted drugs. Therefore, it has been proposed that the compound initiates specific leukemias by acting as a topoisomerase II poison. This hypothesis, however, has not been supported by in vitro studies. While 1,4-benzoquinone has been shown to inhibit topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage have not been reported. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of the compound on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. DNA cleavage enhancement probably was unseen in previous studies due to the presence of reducing agents in reaction buffers and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. 1,4-Benzoquinone increased topoisomerase II-mediated DNA cleavage primarily by enhancing the forward rate of scission. In vitro, the compound induced cleavage at DNA sites proximal to a defined leukemic chromosomal breakpoint and displayed a sequence specificity that differed from that of etoposide. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. The present findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of specific leukemias induced by benzene and its metabolites.     

Exocyclic DNA lesions stimulate DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

DNA adducts are mutagenic and clastogenic. Because of their harmful nature, lesions are recognized by many proteins involved in DNA repair. However, mounting evidence suggests that lesions also are recognized by proteins with no obvious role in repair processes. One such protein is topoisomerase II, an essential enzyme that removes knots and tangles from the DNA. Because topoisomerase II generates a protein-linked double-stranded DNA break during its catalytic cycle, it has the potential to fragment the genome. Previous studies indicate that abasic sites and other lesions that distort the double helix stimulate topoisomerase II-mediated DNA cleavage. Therefore, to further explore interactions between DNA lesions and the enzyme, the effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. When located within the four-base overhang of a topoisomerase II cleavage site (at the +2 or +3 position 3' relative to the scissile bond), 3,N(4)-ethenodeoxycytidine, 3,N(4)-etheno-2'-ribocytidine, 1,N(2)-ethenodeoxyguanosine, pyrimido[1,2-a]purin-10(3H)-one deoxyribose (M(1)dG), and 1,N(2)-propanodeoxyguanosine increased DNA scission approximately 5-17-fold. Enhanced cleavage did not result from an increased affinity of topoisomerase IIalpha for adducted DNA or a decreased rate of religation. Therefore, it is concluded that these exocyclic lesions act by accelerating the forward rate of enzyme-mediated DNA scission. Finally, treatment of cultured human cells with 2-chloroacetaldehyde, a reactive metabolite of vinyl chloride that generates etheno adducts, increased cellular levels of DNA cleavage by topoisomerase IIalpha. This finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.     

Cobalt enhances DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in cleavage reactions. Cobalt(II) stimulated cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.     

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.     

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.     

Chlorella virus SC-1A encodes at least five functional and one nonfunctional DNA methyltransferases

Chlorella virus SC-1A encodes at least six DNA methyltransferases (MTases): four N⁶-methyldeoxyadenine (m⁶A) MTases, M- CviSI (TGC^mA), M· CviSII C^mATG), M· CviSIII (TCG^mA) and M^mCviSIV (G^mATC), one 5-methyldeoxycytosine (m⁵C) MTase, M· CviSV (RC^mCG), and one nonfunctional m⁵C MTase, M· CviSVI, which is homologous to the MTase M· CviJI [RG^mC(T/C/G)] produced by another chlorella virus IL-3A. Genes encoding three of the SC-1A m⁶A MTases (M·CviSI, M· CviSII, and M· CviSIII) and the nonfunctional m⁵C MTase were cloned and sequenced. Neither M· CviSI nor M· CviSIII genes hybridized to genes for their respective isomethylomers, M· CviRI and M· CviBIII, from other chlorella viruses. However, the M· CviSII gene hybridized strongly to its M· CviAII isomethylomer gene from virus PBCV-1. Like the prototype chlorella virus PBCV-1, the SC-1A genome contains inverted terminal repeats, one of which is adjacent to the nonfunctional m⁵C MTase. The three cloned m⁶A MTase genes are distributed throughout the approx. 345 kb SC-1A genome.     

RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1

Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.     

Human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for DNA ligation. Use of 5' covalently activated oligonucleotide substrates to study enzyme mechanism

Despite the importance of topoisomerase II-mediated DNA ligation to the essential physiological functions of the enzyme, the mechanistic details of this important reaction are poorly understood. Because topoisomerase II normally does not release cleaved DNA molecules prior to ligation, it is not known whether all of the nucleic acid specificity of its cleavage/ligation cycle is embodied in DNA cleavage or whether ligation also contributes specificity to the enzyme. All currently available ligation assays require that topoisomerase II cleave the initial DNA substrate before rejoining can be monitored. Consequently, it has been impossible to examine the specificity of DNA ligation separately from that of scission. To address this issue, a cleavage-independent topoisomerase II DNA ligation assay was developed. This assay utilizes a nicked oligonucleotide whose 5'-phosphate terminus at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. Human topoisomerase IIalpha and enzymes with active-site mutations that abrogated cleavage activity ligated the activated nick by catalyzing the direct attack of the terminal 3'-OH on the activated 5'-phosphate. Results with different DNA sequences indicate that human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for ligation that parallels its specificity for DNA cleavage.