High DNA Cleavage Activity Research Articles

Green synthesis, characterization and biological activity of silver nanoparticles of Hypnum cupressiforme Hedw extract

Mosses are one of the oldest land plants that constitute the second largest group of the plant kingdom, are found in almost every region, have known therapeutic effects, are rich in secondary metabolites, can withstand harsh climatic conditions and thirst for a long time. Volatile extracts of Hypnum cupressiforme Hedw. were analyzed by gas chromatography-mass spectrometry in this study, and the presence of 18 different phytocompounds was determined as secondary metabolites. Using these extracts, silver nanoparticles (AgNPs) were synthesized using a green synthesis method. The AgNPs that were created were characterized. The extracts and AgNPs were tested for antibacterial, antibiofilm, antioxidant, mutagenic, and DNA-cleavage activity. The tests revealed no evidence of mutagenicity. Antibiofilm and antibacterial activity of AgNPs against P. vulgaris, P. aeruginosa, and E. faecalis were demonstrated. Both oxidatively and hydrolytically, H. cupressiforme the extract was found to have higher antioxidant properties and high DNA cleavage activity.

Characterization of a new IN-I-PpoI fusion protein and a homology-arm containing transgene cassette that improve transgene expression persistence and 28S rRNA gene-targeted insertion of lentiviral vectors.

Targeting transgene integration into a safe genomic locus would be very important for gene therapy. We have generated lentivirus vectors containing the ribosomal RNA-recognising I-PpoI endonuclease fused to viral integrase, and transgene cassettes with target site homology arms to enhance insertion targeting. These new vectors were characterised with respect to the persistence of transgene expression, insertion targeting efficiency and chromosomal integrity of the transduced cells. The aim was to find an optimally safe and effective vector for human gene therapy. Fusion protein vectors with high endonuclease activity were the most effective in the accurate targeting of transgene insertion. The homology construct increased the insertion targeting efficiency to 28% in MRC-5 cells. However, karyotyping analysis showed that the high endonuclease activity induced the formation of derivative chromosomes in as many as 24% of the analysed primary T lymphocytes. The persistence of transgene expression was excellent in homology arm-containing fusion protein vectors with reduced endonuclease activity, and these fusion proteins did not cause any detectable chromosomal rearrangements attributable to the endonuclease activity. We thus conclude that instead of the fusion protein vectors that carry a highly active endonuclease, our vectors with the ability to tether the lentivirus preintegration complex to benign loci in the genome without high ribosomal DNA cleavage activity are better suited for lentivirus-based gene therapy applications.

Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity.

Prokaryotic Argonaute proteins (pAgos) widely participate in hosts to defend against the invasion of nucleic acids. Compared with the CRISPR-Cas system, which requires a specific motif on the target and can only use RNA as guide, pAgos exhibit precise endonuclease activity on any arbitrary target sequence and can use both RNA and DNA as guide, thus rendering great potential for genome editing applications. Hitherto, most in-depth studies on the structure-function relationship of pAgos were conducted on thermophilic ones, functioning at ∼60 to 100°C, whose structures were, however, determined experimentally at much lower temperatures (20–33°C). It remains unclear whether these low-temperature structures can represent the true conformations of the thermophilic pAgos under their physiological conditions. The present work studied three pAgos, PfAgo, TtAgo and CbAgo, whose physiological temperatures differ significantly (95, 75 and 37°C). By conducting thorough experimental and simulation studies, we found that thermophilic pAgos (PfAgo and TtAgo) adopt a loosely-packed structure with a partially-melted surface at the physiological temperatures, largely different from the compact crystalline structures determined at moderate temperatures. In contrast, the mesophilic pAgo (CbAgo) assumes a compact crystalline structure at its optimal function temperature. Such a partially-disrupted structure endows thermophilic pAgos with great flexibility both globally and locally at the catalytic sites, which is crucial for them to achieve high DNA-cleavage activity. To further prove this, we incubated thermophilic pAgos with urea to purposely disrupt their structures, and the resulting cleavage activity was significantly enhanced below the physiological temperature, even at human body temperature. Further testing of many thermophilic Agos present in various thermophilic prokaryotes demonstrated that their structures are generally disrupted under physiological conditions. Therefore, our findings suggest that the highly dynamical structure with a partially-melted surface, distinct from the low-temperature crystalline structure, could be a general strategy assumed by thermophilic pAgos to achieve the high DNA-cleavage activity.

Synthesis, structural characterization, and density functional theory calculations of the two new Zn (II) complexes as antibacterial and anticancer agents with a neutral flexible tetradentate pyrazole‐based ligand

Two new mono‐ and dinuclear zinc (II) complexes with a formula of [Zn (LN)OMeCl] (1) and [Zn2(LN)(N3)2Cl2] (2) [where LN = 1,1,3,3‐tetrakis(3,5‐dimethyl‐1‐pyrazolyl)propane)] have been synthesized in methanol as a solvent and fully characterized by the single crystal X‐ray diffraction, elemental analysis, FT‐IR, UV–Vis, density functional theory (DFT) calculations, antibacterial, and anticancer properties. In both of them, zinc ion is tetrahedrally coordinated to two nitrogen atoms from one of the chelating bidentate bis(3,5‐dimethylpyrazolyl)methane units of the LN ligand and one terminal chlorine. The forth coordination site was occupied by methoxy group in 1 and azide co‐ligand in complex 2. In contrast to 1, in the structure of 2 two metal centers are bridged by the tetradentate chelating LN ligand to form a dinuclear complex. Interestingly, the environment around the central atom in these complexes are mimicked with the core structure of the zinc metalloproteinases, Thermolysin {[NNO]ZnX} and Carbonic anhydrase {[NNN]ZnX} for complexes 1 and 2, respectively. Data analysis of the antibacterial activities demonstrated that the gram positive species showed more sensitivity toward the target title agents. Although, both complexes exhibit antibacterial activities and kill bacteria at low concentration, but complex 1 has higher DNA cleavage activity than 2. Additionally, the cytotoxicity of the free ligand and its complexes was evaluated against the human carcinoma cell line (Caco‐2) by the MTT method and the IC50 values were calculated for all of them. Due to the presence of pyrazole rings in the structure of the ligand and its complexes, these materials have a substantial potential to inhibit cancer cell metastasis. The energetic stabilization due to donor‐acceptor interactions in terms of the natural bond orbital (NBO) analyses and nonlinear optical properties were also examined by using the DFT calculations.

In vitro and in vivo antitumoral activity of a ternary copper (II) complex

Recently, a high number of copper derivatives has been evaluated as DNA-targeting metallodrugs, due to the lower toxicity and its potential to cleave DNA. Several strategies have been testing to develop metal compounds effective against tumour cells. In this work, the ternary copper (doxycycline)-(1,10-phenanthroline) complex [Cu(dox)(phen)]2+ was especially designed as an antitumoral drug, previously showing high cytotoxicity and DNA cleavage activity. We aimed to further investigate the in vitro cytotoxic activity in both tumoral and non-tumoral cells, in vitro genotoxic potential, and in vivo antitumor activity using BALB/C mouse injected with sarcoma S180 and Ehrlich cell lines. Our results indicated that this compound exhibits a moderate genotoxic potential, with selective growth inhibition of tumor cells, especially the murine melanoma B16F10. Its main mechanism of action seems to be through ROS generation. We have further shown a significant reduction of the implanted tumor size in the animal model, suggesting that this compound has great antitumoral potential against many tumor types. [Cu(dox)(phen)]2+ is selectively cytotoxic for melanoma B16F10 and showed high chemotherapeutic potential in vivo against implanted sarcoma S180 and Ehrlich ascites tumours.

Construction and characterization of adenoviral vectors for the delivery of TALENs into human cells

Transcription activator-like effector nucleases (TALENs) are designed to cut the genomic DNA at specific chromosomal positions. The resulting DNA double strand break activates cellular repair pathways that can be harnessed for targeted genome modifications. TALENs thus constitute a powerful tool to interrogate the function of DNA sequences within complex genomes. Moreover, their high DNA cleavage activity combined with a low cytotoxicity make them excellent candidates for applications in human gene therapy. Full exploitation of these large and repeat-bearing nucleases in human cell types will benefit largely from using the adenoviral vector (AdV) technology. The genetic stability and the episomal nature of AdV genomes in conjunction with the availability of a large number of AdV serotypes able to transduce various human cell types make it possible to achieve high-level and transient expression of TALENs in numerous target cells, regardless of their mitotic state. Here, we describe a set of protocols detailing the rescue, propagation and purification of TALEN-encoding AdVs. Moreover, we describe procedures for the characterization and quantification of recombinant viral DNA present in the resulting AdV preparations. The protocols are preceded by information about their underlying principles and applied in the context of second-generation capsid-modified AdVs expressing TALENs targeted to the AAVS1 “safe harbor” locus on human chromosome 19.

An investigation on new ruthenium(ii) hydrazone complexes as anticancer agents and their interaction with biomolecules

A new set of ruthenium(II) hydrazone complexes [Ru(H)(CO)(PPh3)2(L)] (1) and [RuCl2(DMSO)2(HL)] (2), with triphenyl phosphine or DMSO as co-ligands was synthesized by reacting benzoyl pyridine furoic acid hydrazone (HL) with [Ru(H)(Cl)(CO)(PPh3)3] and [RuCl2(DMSO)4]. The single crystal X-ray data of complexes 1 and 2 revealed an octahedral geometry around the ruthenium ion in which the hydrazone is coordinated through ON and NN atoms in complexes 1 and 2 respectively. The interaction of the compounds with calf thymus DNA (CT-DNA) has been estimated by absorption and emission titration methods which indicated that the ligand and the complexes interacted with CT-DNA through intercalation. In addition, the DNA cleavage ability of these newly synthesized ruthenium complexes assessed by an agarose gel electrophoresis method demonstrated that complex 2 has a higher DNA cleavage activity than that of complex 1. The binding properties of the free ligand and its complexes with bovine serum albumin (BSA) protein have been investigated using UV-visible, fluorescence and synchronous fluorescence spectroscopic methods which indicated the stronger binding nature of the ruthenium complexes to BSA than the free hydrazone ligand. Furthermore, the cytotoxicity of the compounds examined in vitro on a human cervical cancer cell line (HeLa) and a normal mouse embryonic fibroblasts cell line (NIH 3T3) revealed that complex 2 exhibited a superior cytotoxicity than complex 1 to the cancer cells but was less toxic to the normal mouse embryonic fibroblasts under identical conditions.

Synthesis and Biological Activity of Some New Pyrazole Copper(II) Complexes

As a part of systematic investigation of synthesis and biologically active compounds of pyrazole derivatives containing transition metal, several new pyrazole copper(II) complexes 3a−f were synthesized from pyrazole sodium salts 2a−f, which were produced from spiro‐pyrazoles 1a−f and sodium hydride by a ring‐opening reaction. All the synthesized compounds were characterized by spectroscopic analysis. Pyrazole copper(II) complexes 3a−d and 3f exhibited high DNA cleavage activity in vitro. Furthermore, compounds 3a−f were tested for their growth inhibitory activity in A549 lung cancer, B16F10 murine melanoma, and HeLa human uterine carcinoma cells. Compounds 3c,d displayed moderate B16F10 and HeLa inhibitory activity levels (3c: IC50 = 45 μM in B16F10 cells and 34 μM in HeLa cells, 3d: IC50 = 50 μM in B16F10 cells and 32 μM in HeLa cells).

Reduced expression of MUTYH with suppressive activity against mutations caused by 8‐hydroxyguanine is a novel predictor of a poor prognosis in human gastric cancer

The MUTYH gene encodes a DNA glycosylase that can initiate the excision repair of adenine mispaired with 8-hydroxyguanine (8OHG) and is responsible for a susceptibility to multiple colorectal adenomas and carcinomas. To determine whether the MUTYH gene is involved in gastric carcinogenesis, we first examined the expression level of MUTYH in gastric cancer. The reduced expression of MUTYH mRNA transcript was detected in both gastric cancer cell lines and primary gastric cancers using qRT-PCR analysis. Immunohistochemical analysis also showed a significant reduction in MUTYH protein expression in gastric cancer, compared with non-cancerous gastric epithelium (immunohistochemical score, 175.5 ± 43.0 versus 281.5 ± 24.8; p < 0.0001). Among the gastric cancers, the MUTYH expression level was significantly associated with the histopathology (p < 0.0001) and the pT stage (p < 0.001). The outcome of patients with gastric cancer exhibiting low MUTYH expression was significantly worse than the outcome of patients with gastric cancer exhibiting high MUTYH expression (p = 0.0007, log-rank test) and a multivariate analysis revealed that reduced MUTYH expression was an independent predictor of a poor survival outcome among the gastric cancer patients (hazard ratio, 1.865; 95% confidence interval, 1.028-3.529; p = 0.0401). We next compared the functional effects of MUTYH on gastric cancer cells, based on their MUTYH expression levels. MUTYH-over-expressing stable clones of the gastric cancer cell line AGS showed: (a) higher DNA cleavage activity towards adenine:8OHG mispair-containing substrates; (b) higher suppressive activity against mutations caused by 8OHG in a supF forward mutation assay; and (c) higher suppressive activity for cellular proliferation than empty vector-transfected AGS clones. These results suggested that MUTYH is a suppressor of mutations caused by 8OHG in gastric cells and that its reduced expression is associated with a poor prognosis in gastric cancer.

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.

A trigonal-prismatic Ag(I) complex and an octahedral Cu(II) complex incorporating with oxamide Ni(II) complex ligand: Syntheses, crystal structures and oxidative nuclease activities

A mononuclear macrocyclic complex NiIIL3a (L3a=dianion of 2,3-dioxo-5,6:13,14-dibenzo-9,10-cyclohexyl-7,12-bis(methoxycarbonyl)-1,4,8,11-tetraazacyclotetradeca-7,11-diene), which shows high DNA cleavage activity in the presence of H2O2, was reported in our previous work. Considering that many systems for natural enzyme-mediated DNA cleavage contain two or more metal active sites, two new trinuclear complexes [Cu(NiL3a)2(dca)2]·2CH3OH (abbreviated as Cu(NiL3a)2) and [Ag(NiL3a)2(NO3)]·2CH3OH·0.5H2O (abbreviated as Ag(NiL3a)2) were synthesized in this work, where dca is the dicyanamide. The complexes were structurally characterized by single crystal X-ray analysis. The central Cu(II) or Ag(I) atom is linked to two [NiL3a] ligands by oxamido bridges forming a trinuclear structure. In Cu(NiL3a)2, the central Cu(II) ion is in an octahedral coordination geometry. Whereas in Ag(NiL3a)2, the central Ag(I) ion is in a rarely reported trigonal-prismatic coordination geometry. The DNA cleavage behavior of the complexes in the presence of H2O2 was studied in detail. Comparing with the NiL3a, the trinuclear complex Ag(NiL3a)2 nearly has no ability to cleave DNA, whereas Cu(NiL3a)2 is a much better DNA cleavage agent. Cu(NiL3a)2 can efficiently convert supercoiled DNA to nicked DNA with a rate constant of 0.074±0.002min−1 when 40μM Cu(NiL3a)2 and 0.6mM H2O2 are used. The cleavage mechanism between the complex Cu(NiL3a)2 and plasmid DNA is likely to involve singlet oxygen as reactive oxygen species. Circular dichroism (CD) and fluorescence spectroscopy indicate that both Cu(NiL3a)2 and NiL3a bind to DNA by a groove binding mode, and the binding between Cu(NiL3a)2 and DNA is much stronger than that between NiL3a and DNA. The present results may provide some information for the design of efficient multinuclear artificial nucleases.

ChemInform Abstract: Bifunctional 2-Naphthyl Propargylic Sulfones Exhibiting High DNA Intercalating and Alkylating Activity.

A number of novel 2-naphthyl propargylic sulfones were synthesized as nucleic base alkylating agents. Extremely high DNA cleavage activity was observed for the sulfones with a free ω-hydroxyl group in the carbon chain in contrast to the ester conjugates possessing an additional intercalating unit.

Characterization of complexation of PVP copolymer with DNA

AbstractInteractions of amphiphilic copolymer poly(N‐vinylpyrrolidone‐co‐N‐t‐Boc‐ tryptophanamido‐N′‐methacrylthiourea) [P(NVP‐co‐TrpAMT)] and poly(N‐vinylpyrrlidone) (PVP) with DNA were studied with the aim to understand the difference in their complexation efficiencies when considered as a potential non‐viral delivery vector. Fluorescence spectroscopy, ethidium bromide (EB) quenching, ζ potential measurement, and agarose gel electrophoresis were all carried out in Tris‐HCl (pH 7.4) solution to assess the degree of interaction between the (co)polymers P(NVP‐co‐TrpAMT) and DNA. All of these measurements show that the high affinity of the copolymer for DNA can be demonstrated. We also found that P(NVP‐co‐TrpAMT)‐I (PVP/PTrpAMT molar ratio was 100:2.88) exhibited a high DNA cleavage activity and induced the supercoiled form of the plasmid cleavage in the nicked and linear forms.Moreover, P(NVP‐co‐TrpAMT)‐I exhibited stronger affinity for DNA than PVP and P(NVP‐co‐TrpAMT)‐II (PVP/PTrpAMT molar ratio of 100:6.35). This phenomenon is believed to be related to the structure of TrpAMT and the hydrophobic nature of the copolymer. P(NVP‐co‐TrpAMT)‐II with higher hydrophobic segments creates a strong hydrophobic interaction and induces a steric barrier which hinders their complexation with DNA. The study reveals important information about the inner mechanisms involved in the interaction of the PVP copolymer with DNA, which aids in the design of novel efficient non‐ionic gene carriers. Copyright © 2008 John Wiley &amp; Sons, Ltd.

New Gadolinium Complex with Efficient Hydrolase-like Activity: A 100-Million-Fold Rate Enhancement in Diester Hydrolysis

The synthesis, structure, and hydrolase-like catalytic activity of a new mononuclear gadolinium complex [Gd(L)(NO3)(H2O)3](NO3)2 (1) are reported. A clean two-stage kinetic reaction for hydrolysis of the diester 2,4-bis(dinitrophenyl)phosphate by 1 was followed, and the rate constants were determined. A high DNA cleavage activity was also demonstrated. The active species in the hydrolytic process is proposed based on the X-ray structure, electrospray ionization mass spectrometry analysis, and kinetic and potentiometric equilibrium studies of 1.

DNA cleavage, structural elucidation and anti-microbial studies of three novel mixed ligand Schiff base complexes of copper(II)

Three new copper complexes of mixed ligands derived from Schiff bases (condensation of p-aminoacetanilide and substituted benzaldehydes) with 1,10-phenanthroline have been synthesized and characterized by elemental analysis, IR, UV-Vis, magnetic moments, conductivity and electrochemical measurements. The spectral techniques suggest that all the copper complexes exhibit octahedral geometry. The low electrical conductance of the complexes supports their neutral nature. The monomeric nature of the complexes was assessed from their magnetic susceptibility values. The in vitro biological screening effects of the investigated compounds were tested against the bacteria Escherichia coli, Staphylococcus aureus, and Salmonella typhi and the fungi Rhizopus stolonifer and Candida albicans by the serial dilution method. A comparative study of the MIC values of the Schiff bases and their copper complexes indicates that the metal complexes exhibited higher antibacterial activity than the free ligands. The DNA cleavage ability of the complexes was monitored by the gel electrophoresis technique. It was found that electron withdrawing group substituted copper complex had higher DNA cleavage activity than the other copper complexes. .

DNA Methylation Impacts the Cleavage Activity of Chlorella Virus Topoisomerase II

Topoisomerase II from Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) displays an extraordinarily high in vitro DNA cleavage activity that is 30-50 times higher than that of human topoisomerase IIalpha. This remarkable scission activity may reflect a unique role played by the type II enzyme during the viral life cycle that extends beyond the normal control of DNA topology. Alternatively, but not mutually exclusively, it may reflect an adaptation to some aspect of the viral environment that differs from the in vitro conditions. To this point, the genomes of many chlorella viruses contain high levels of N6-methyladenine (6mA) and 5-methylcytosine (5mC), but the DNA employed in vitro is unmodified. Therefore, to determine whether methylation impacts the ability of chlorella virus topoisomerase II to cleave DNA, the effects of 6mA and 5mC on the PBCV-1 and CVM-1 enzymes were examined. Results indicate that 6mA strongly inhibits DNA scission mediated by both enzymes, while 5mC has relatively little effect. At levels of 6mA and 5mC methylation comparable to those found in the CVM-1 genome (10% 6mA and 42% 5mC), the level of DNA cleavage decreased approximately 4-fold. As determined using a novel rapid quench pre-equilibrium DNA cleavage system in conjunction with oligonucleotide binding and ligation assays, this decrease appears to be caused primarily by a slower forward rate of DNA scission. These findings suggest that the high DNA cleavage activity of chlorella virus topoisomerase II on unmodified nucleic acid substrates may reflect, at least in part, an adaptation to act on methylated genomic DNA.

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.

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 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.

Bifunctional 2-naphthyl propargylic sulfones exhibiting high DNA intercalating and alkylating activity

A number of novel 2-naphthyl propargylic sulfones were synthesized as nucleic base alkylating agents. Extremely high DNA cleavage activity was observed for the sulfones with a free ω-hydroxyl group in the carbon chain in contrast to the ester conjugates possessing an additional intercalating unit.