Recognition of major DNA adducts of enantiomeric cisplatin analogs by HMG box proteins and nucleotide excision repair of these adducts

We examined HMG domain protein recognition of major 1,2-GG intrastrand DNA crosslinks, formed by two bifunctional enantiomeric analogs of antitumor cis-diamminedichloroplatinum(II) (cisplatin), and removal of these crosslinks during in vitro nucleotide excision repair (NER) reactions. Electrophoretic mobility shift assays show that domains A and B of HMGB1 protein bind to (2R,3R)-diaminobutanedichloroplatinum(II)-generated crosslinks with a higher affinity than to those generated by (2S,3S)-diaminobutanedichloroplatinum(II). The crosslinks of both enantiomers are removed by NER with a similar efficiency; however, HMG1B protein significantly inhibits removal of the (2R,3R)-diaminobutaneplatinum(II) adduct, but not that of the (2S,3S) enantiomer. Thus, HMG domain proteins discriminate among different conformations of the 1,2-GG intrastrand crosslinks of the two enantiomeric analogs of cisplatin, which results in different NER of these crosslinks. This observation may provide insight into the mechanisms underlying antitumor activity of cisplatin and its analogs.     

Interaction of 2-aminopyrimidine with dichloro-[1-alkyl-2-(naphthylazo) imidazole]palladium(II) complexes : Kinetic and mechanistic studies

Background  

The anticancer properties of cisplatin and palladium(II) complexes stem from the ability of the cis-MCl₂ fragment to bind to DNA bases. However, cisplatin also interacts with non-cancer cells, mainly through bonding molecules containing -SH groups, resulting in nephrotoxicity. This has aroused interest in the design of palladium(II) complexes of improved activity and lower toxicity. The reaction of DNA bases with palladium(II) complexes with chelating N,N^/donors of the cis-MCl₂ configuration constitutes a model system that may help explore the mechanism of cisplatin's anticancer activity. Heterocyclic compounds are found widely in nature and are essential to many biochemical processes. Amongst these naturally occurring compounds, the most thoroughly studied is that of pyrimidine. This was one of the factors that encouraged this study into the kinetics and mechanism of the interaction of 2-aminopyrimidine (2-NH₂-Pym) with dichloro-{1-alkyl-2-(α-naphthylazo)imidazole}palladium(II) [Pd(α-NaiR)Cl₂, 1] and dichloro-{1-alkyl-2-(β-naphthylazo)imidazole}palladium(II) [Pd(β-NaiR)Cl₂, 2] complexes where the alkyl R = Me (a), Et (b), or Bz (c).     

FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo

BACKGROUND: Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity. RESULTS: Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. CONCLUSIONS: We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.     

Synthesis and structure of a 1-D copper(II) coordination polymer bridged both by oxamido and carboxylate: in vitro anticancer activity and reactivity toward DNA and protein BSA

A 1-D copper(II) coordination polymer formulated as {[Cu₂(bdpox)(dabt)](NO₃)·H₂O}_n, where H₃bdpox and dabt denote N-benzoate-N′-[3-(diethylamino)propyl]oxamide and 2,2′-diamino-4,4′-bithiazole, respectively, was synthesized and characterized by elemental analyses, molar conductance measurement, IR and electronic spectra studies, and single-crystal X-ray diffraction. The crystal structure analysis reveals that copper(II) ions are bridged by both cis-oxamido and carboxylato groups to form a 1-D coordination polymer with corresponding Cu···Cu separations of 5.2420(10) and 5.1551(8) Å. The endo- and exo-copper(II) ions of the cis-oxamido-bridge are located in distorted square-planar and square-pyramidal geometries, respectively. There is a 2-D hydrogen bonding network in the crystal. The in vitro anticancer activities suggest that the copper(II) complex is active against selected tumor cell lines. The reactivities toward herring sperm DNA and bovine serum albumin (BSA) reveal that the copper(II) complex can interact with DNA by intercalation and effectively quench the intrinsic fluorescence of BSA via a static mechanism. The influence of hydrophobicity of the substituents in bridging ligands on DNA and protein binding properties and the in vitro anticancer activities of such copper(II) polymers is discussed.     

NMR relaxation study of crosslinked cis-1,4-polybutadiene

Proton relaxation measurements have been used to investigate the effects of crosslinking on the segmental motion in cis-1,4-polybutadiene samples. The temperature dependence of proton spin–lattice relaxation time T₁ and spin–spin relaxation time T₂ at 60 and 24.3 MHz are reported in cis-1,4-polybutadiene (PB) samples with different crosslink density including uncrosslinked PB and samples with 140, 40, and 14 repeat units between crosslinks. In addition, spin-lattice relaxation times in rotating coordinate frame, T_1p, have also been determined. The relaxation data are interpreted in terms of the effects of crosslinks on segmental chain motions. Because of their sensitivity to low-frequency motion, T₂ data are of major interest. At temperatures well above the T₁ minimum the small T₂ temperature dependence resembles solidlike behavior reflecting the nonzero averaging of dipolar interactions due to anisotropic motion of the chain segments between crosslinks. The magnitude of T₂ at 60°C is found to be proportional to the average mass between crosslinks.     

Synthesis,structure and in vitro antiproliferative activities of oxamido‐bridged dicopper(II) complexes: A comparative study of experimental evidence and molecular docking of DNA/protein binding

Two μ‐oxamido‐bridged dicopper(II) complexes, namely [Cu₂(hmpoxd)(H₂O)(phen)](ClO₄) ( 1 ) and [Cu₂(papo)(H₂O)(phen)](ClO₄)·2H₂O ( 2 ), where H₃hmpoxd and H₃papo represent N‐(2‐hydroxy‐5‐methylphenyl)‐N′‐[3‐(dimethylamino)propyl]oxamide and N‐(2‐hydroxylphenyl)‐N′‐(3‐aminopropyl)oxamide, respectively, and phen represents 1,10‐phenanthroline, were synthesized. Single‐crystal X‐ray crystallography and other methods revealed that the two copper(II) ions in complex 1 are bridged by the cis‐hmpoxd^3? with Cu···Cu separation of 5.1896(7) Å, in which the inner (Cu1) and outer (Cu2) copper(II) atoms are located in square‐planar and square‐pyramidal geometries, respectively. To evaluate the effects of bridging ligand hydrophobicity on DNA/protein binding and potential anticancer activities, comparative studies of the reactivity towards herring sperm DNA and protein bovine serum albumin (BSA) as well as cytotoxicity of complex 1 with our previously reported complex 2 were conducted theoretically and experimentally. The results indicate that the two complexes can interact interactively with DNA, and bind to BSA via the binding sites Trp213 for 1 and Trp134 for 2 . Interestingly, the in vitro anticancer activities and DNA/protein binding affinities consistently follow the order of 1 > 2 .     

Synthesis of Oligonucleotides containing the cis-Interstrand Crosslink Produced by Mitomycins in their Reaction with DNA

Mitomycin C (MC) an antitumor drug and decarbamoylmitomycin C (DMC), a derivative of MC lacking the carbamoyl moiety, are DNA alkylating agents which can form DNA interstrand crosslinks (ICLs) between deoxyguanosine residues located on opposing DNA strands. MC forms primarily deoxyguanosine adducts with a 1“-R stereochemistry at the guanine-mitosene bond (1”-α, trans) whereas DMC forms mainly adducts with a 1“-S stereochemistry (1”-β, cis). The crosslinking reaction is diastereospecific: trans-crosslinks are formed exclusively at CpG sequences, while cis-crosslinks are formed only at GpC sequences. Until now, oligonucleotides containing 1“-β-deoxyguanosine adducts or ICL at a specific site could not be synthesized, thus limiting the investigation of the role played by the stereochemical configuration at C1′′ in the toxicity of these compounds. Here, a novel biomimetic synthesis to access these substrates is presented. Structural proof of the adducted oligonucleotides and ICL were provided by enzymatic digestion to nucleosides, high resolution mass spectral analysis, CD spectroscopy and UV melting temperature studies. Finally, a virtual model of the 25-mer 1”-β ICL synthesized was created to explore the conformational space and structural features of the crosslinked duplex.     

Photophysical and Duplex‐DNA‐Binding Properties of Distamycin Dimers Based on 4,4′‐ and 2,2′‐Dialkoxyazobenzenes as the Core

Distamycin‐based tetrapeptide ( 1 ) was covalently tethered to both ends of the central dihydroxyazobenzene moiety at either the 2,2′ or 4,4′ positions. This afforded two isomeric, distamycin–azobenzene–distamycin systems, 2 (para) and 3 (ortho), both of them being photoisomerizable. Illumination of these conjugates in solution at approximately 360 nm induced photoisomerization and the time course of the process was followed by UV/Vis and ¹H NMR spectroscopy. The kinetics of the thermal reversion at various temperatures of cis to trans isomers of the conjugates obtained after photoillumination were also examined. This afforded the respective thermal‐activation parameters. Both the molecular architecture and the location of the substituent around the core azobenzene determined the rate and activation‐energy barrier for the cis‐to‐trans back‐isomerization of these conjugates in solution. Duplex–DNA binding of the conjugates and the changes in DNA‐binding efficiency upon photoisomerization was also examined by CD spectroscopy, thermal denaturation studies, and a Hoechst displacement assay. The conjugate 2 showed higher DNA‐binding affinity and a greater change in the DNA‐binding efficiency upon photoisomerization compared with its 2,2′‐disubstituted counterpart. The experimental findings were substantiated by using molecular‐docking studies involving each conjugate with a model duplex d[(GC(AT)₁₀CG)]₂ DNA molecule.     

Oxidative stress relaxation and the measurement of sol fraction in rubber vulcanizates

A simple method to determine the chain scission mechanism of the oxidative degradation of rubber vulcanizates is proposed. The method involves the measurement of oxidative stress decay and the change in sol fraction, which allow us to distinguish whether scission occurs randomly along the main chain, near crosslinks or of crosslinks. The applicability of this method was well established using natural rubber vulcanizates as the reference samples. The chain scission of cis-1,4-polyisoprene vulcanizaties was proved to take place randomly along the main chain irrespective of their crosslink structure. On the other hand, the chain scission of dicumyl peroxide cured cis-1,4-polybutadiene takes place selectively near crosslinks. It is suggested that the unusual behavior of cis-1,4-polybutadiene vulcanizates is due to the characteristic structure of the crosslinks.     

Separation and quantification of histone H1 subtypes and high-mobility-group proteins by reversed-phase liquid chromatography: protein levels in rat tissues during postnatal development

The rapid separation and quantification of histone H1 subtypes and high-mobility-group (HMG) chromatin proteins by reversed-phase liquid chromatography on a butylsilica-based column is described. The proteins were fractionated by means of a multi-step acetonitrile gradient containing 0.1% trifluoroacetic acid. This system is capable of resolving the four main HMG proteins (1, 2, 14 and 17), HMG I, protein P1 with HMG 18 and HMG 19 (in one peak) and five histone H1 subtypes in a single 33-min analysis. This method was used to study levels of these chromosomal proteins in nuclei of rat liver, spleen, testis and thymus during postnatal development from 1 to 20 weeks of age. Although no clear tissue specificity of the HMG proteins was apparent, there were significant differences in the relative amounts of these proteins in different tissues. The relative amount of HMG 1 increased from 1 to 12 weeks of age and decreased thereafter, whereas those of HMG 14 and HMG 17 remained almost unchanged. Marked quantitative differences were observed in the five histone H1 subtypes in different tissues. The largest changes in their levels during development were found in the liver and the smallest changes in the thymus. The changes in the spleen and testis were intermediate. These results suggest that the changes in the relative amounts of histone H1 subtypes and HMG proteins observed during postnatal development of the rat may result from differences in the structure of chromatin in these tissues and thus reflect the activity of molecular mechanisms involved in replication and differentiation of the cells.     

Synthesis and spectral studies on platinum complexes with mono- and bidentate N-donor organic ligands

Platinum(II) complexes of types PtLX₂, PtL₂X₂, PtLX″ and the Pt(IV) complexes PtLXY (where L = mono- or bidentate organic ligand containing nitrogen donor atoms; X = Cl or Br; X′ = oxalate or malonate and Y = Br) have been synthesized and characterized from their elemental analysis, IR and X-ray photoelectron spectral data. The Pt 4f_7/2 binding energies indicate that 1,8-naphthalene-diamine ligand is a better donor of electron density to the metal than other ligands studied here. The Cl 2p_3/2 binding energies in the square planar Pt(II) complexes are observed in the range 198.8 ± 0.8 eV. The ν (PtCl) vibrations (ca 335 and 320 cm^?1) corresponding to two cis-Cl ligands were observed in the IR spectra.The extent of the interaction between cis-dichloro-bis-(theophylline)platinum(II) with calf thymus DNA has beenstudied. The UV difference spectra resulting from aquated Pt^II(theoph)₂-DNA interaction exhibit bands at 282 and 292 nm attributable to the change in the electron distribution of the base moieties induced by binding with platinum and due to the loss of base stacking. Melting profiles for the DNA samples treated with Pt-complex showed decrease in the melting temperature. Binding of the guanine residues of the DNA, involving probably (N7)-0(6) positions to the metal is implied.     

Synthesis,characterization, and DNA-binding properties of copper(II), cobalt(II), and nickel(II) complexes with salicylaldehyde 2-phenylquinoline-4-carboylhydrazone

Salicylaldehyde 2-phenylquinoline-4-carboylhydrazone (H₂L), and its novel copper(II), cobalt(II), and nickel(II) complexes MHL · Cl · nH₂O [M=Cu n = 3 (1), M=Co n = 2 (2), M=Ni n = 3.5 (3)] have been synthesized and characterized by elemental analysis, molar conductivity, spectroscopic, and thermal analysis. The interaction of these complexes with calf thymus DNA was investigated by UV absorption spectroscopy, fluorescence spectroscopy, cyclic voltammetry, and viscosity measurements. The results suggest that these complexes bind to DNA via an intercalation binding mode and their affinity to DNA follows the order of 3 > 1> 2. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metal(II) complexes of bioactive aminonaphthoquinone-based ligand: synthesis,characterization and BSA binding,DNA binding/cleavage,and cytotoxicity studies

An aminonaphthoquinone ligand, L, and its metal complexes of general formula [MLCl₂] {M = Co(II), Ni(II), Cu(II) and Zn(II)} have been synthesized and characterized by analytical and spectral techniques. Tetrahedral geometry has been assigned to Ni(II) and Zn(II) complexes and square planar geometry to Co(II) and Cu(II) complexes on the basis of electronic spectral and magnetic susceptibility data. The binding of complexes with bovine serum albumin (BSA) is relatively stronger than that of free ligand and alters the conformation of the protein molecule. Interaction of these complexes with CT-DNA has been investigated using UV-Vis and fluorescence quenching experiments, which show that the complexes bind strongly to DNA through intercalative mode of binding (K_app 10⁵ M^?1). Molecular docking studies reiterate the mode of binding of these compounds with DNA, proposed by spectral studies. The ligand and its complexes cleave plasmid DNA pUC18 to nicked (Form II) and linear (Form III) forms in the presence of H₂O₂ oxidant. The in vitro cytotoxicity screening shows that Cu(II) complex is more potent against MCF-7 cells and Zn(II) complex exhibits marked cytotoxicity against A-549 cells equal to that of cisplatin. Cell imaging studies suggested apoptosis mode of cell death in these two chosen cell lines.     

Synthesis and structures of complexes ofN-2-nitroxyethylpicolinamide and 2-(2-pyridyl)-2-oxazoline with PdCl2

The reaction ofN,N′-bis(2-nitroxyethyl)pyridine-2,6-dicarboxamide with PdCl₂ afforded previously unknowncis-(N-2-nitroxyethylpicolinamide-N,N′)dichloropalladium(II) andcis-[2-(2-pyridyl)-2-oxazoline-N,N′]dichloropalladium(II), which were isolated as a cocrystallizate of the molecular compounds. Its structure was established by X-ray diffraction analysis. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1604–1606, August, 1999.     

Solvent and structural effects on the electro- and photo-reduction of cis - β -Co(trien)(RC6H4NH2)Cl]Cl2 type complexes in water/1,4-dioxane media

The electro- and photo-reduction of a series of cobalt(III) complexes of the type cis-β-[Co(trien)(RC₆H₄NH₂)Cl]Cl₂ where R=H, p-OMe, p-OEt, p-Me, p-Et, p-F and m-Me has been studied in 1,4-dioxane/water. The redox potential (E _1/2) and photo-reduction quantum yield (φ_Co(II)) data were correlated with solvent and structural parameters to shed light on the mechanism of these reactions. Correlation of E _1/2 and φ_Co(II) with macroscopic solvent parameters viz. relative permittivity indicates that the reactivity is influenced by both specific and non-specific solute-solvent interactions. The Kamlet-Taft solvatochromic comparison method was used to separate and quantify these effects. Increasing the percentage of organic co-solvent in the medium enhances both reduction processes; there exists good correlation between E _1/2 and φ_Co(II) suggesting a similar solvation in these redox processes.     

New Copper(II) Complexes Containing 2-Furoic Hydrazide and 5-Nitro-2-Furoic Hydrazide Ligands: Synthesis,Thermal, Magnetic and Spectroscopic Characterization

Two new copper(II) complexes, [Cu₂(L¹)₄(H₂O)₂](SO₄)₄· 2H₂O and [Cu(L²)₂(H₂O)₂]SO₄, were isolated containing 2-furoic hydrazide and 5-nitro-2-furoic hydrazide ligands, respectively. The complexes were characterized by thermal, magnetic and spectroscopic techniques, showing a distorted tetragonal environment around the metal ion. Compound (1), containing 2-furoic hydrazide as the ligand, appears to be dimeric in the solid state, with substituted hydrazine acting as a bidentate bridging ligand. On the contrary, a monomeric species was observed with the 5-nitro-2-furoic hydrazide ligand, probably in the cis configuration, for compound (2). Magnetic measurements for the binuclear copper(II) complex (1) were carried out at low temperatures, in the 2–300 K range, and a magnetic field of 500 G, indicating that besides an intramolecular ferromagnetic interaction between the two Cu(II) centers, for which J/k = 1.07 K, further weak antiferromagnetic interactions between adjacent dimers, with Jz/k =–0.95 K, should be taken into account. However, in MeOH/H₂O solution, evidence of equilibria involving the dimer (1) and the corresponding mononuclear cis and trans species was obtained from e.p.r. spectra.     

DNA Binding and Cleavage Activity of cis‐Cobalt Aromatic Oxime Complexes and Its Pyridine/imidazole Adducts

cis‐Cobalt complexes with salicycaldoxime(SAO), (Z)‐1‐(2‐hydroxyphenyl)ethanonoxime (HEO), (Z)‐1‐(2,5‐dihydroxyphenyl)ethanonoxime (DEO), (Z)‐1‐(2,5‐dihydroxyphenyl)(phenyl)methanonoxime (DPO) and their adducts with pyridine (Py) and imidazole (Im) were synthesized and characterized by elemental analysis, magnetic susceptibility, UV‐Vis and IR spectra. The electrochemical studies were carried by cyclic voltammeter, the peak potential separation and formal potential of complexes were independent of sweep rate or scan rate (ν) indicating a quasi reversible one‐electron redox process. Absorption studies and thermal denature studies revealed that each of these octahedral complexes is an avid binder of calf thymus DNA. The apparent binding constants for mixed ligand complexes are in order of ～10³‐10³ M^?1. Based on the data obtained in the DNA binding studies a partial intercalative mode of binding is suggested for these complexes. The nucleolytic cleavage activity of parent complexes and their pyridine adduct were carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of oxidant (H₂O₂). All the metal complexes show enhanced cleavage activity in presence of oxidant. The hydrolytic cleavage of DNA of Co(DEO)₂ and Co(DPO)₂ is evidenced from the control experiments showing discernable cleavage inhibition in the presence of the hydroxyl radical inhibitor DMSO and EDTA.     

A new 1H-pyridin-(2E)-ylidene ruthenium complex as sensitizer for a dye-sensitized solar cell

The new heteroleptic ruthenium(II) complex containing a 1H-pyridin-(2E)-ylidene (PYE) ligand was synthesized and characterized using UV/Vis, FTIR, and NMR spectroscopies, mass spectrometry, elemental analysis, and cyclic voltammetry. The photovoltaic performance of the ruthenium complex as a charge transfer photosensitizer in nc-titanium dioxide based dye-sensitized solar cell was studied and compared with cis-bis(isothiocyanato)(2,2′-bipyridyl-4,4′-dicarboxylato)(2,2′-bipyridyl-4,4′-di-nonyl)ruthenium(II) (Z907) under standard AM 1.5 sunlight. The complex CS90 gave a photocurrent density of 1.80?mA?cm^?2, 400?mV open-circuit potential, and 0.58 fill factor yielding an efficiency of 0.42% where the reference Z907 yielded an efficiency of 4.12%. The decrease in conversion efficiency observed for CS90 is attributed to a steric interaction between PYE and the TiO₂ surface that prevents optimum binding and also restricts ligand dynamics that are associated with oxidation state changes.     

Synthesis,crystal structure,and DNA cleavage activity of a dinuclear nickel(II) complex with a macrocyclic ligand

A new Ni(II) complex, namely [Ni₂(OAc)L]·ClO₄·H₂O, was synthesized by [2 + 2] cyclo-condensation between 2,6-diformyl-4-methylphenol and N,N-bis(3-aminopropyl)-4-methoxybenzylamine (amba) in the presence of nickel(II) and characterized by spectroscopy, elemental analysis, and X-ray single crystal diffraction. The interactions of the complex with DNA have been measured by spectroscopy and viscosity measurements. Absorption spectroscopic investigation reveals intercalative binding of the Ni(II) complex with DNA, with a binding constant of 2.6 × 10⁴ M⁻¹. Fluorescence spectroscopy shows that the Ni(II) complex can displace ethidium bromide and bind to DNA, with a quenching constant of 7.57 × 10³ M⁻¹. The appearance of increased CD bands near 245 and 275 nm gives evidence for effective complex DNA binding. The agarose gel electrophoresis studies show that the complex displays effective DNA cleavage activity in the absence of any external agents.