Mechanism Of DNA Cleavage Research Articles

Seven-coordinated cobalt(II) complexes with 2,6-diacetylpyridine bis(4-hydroxybenzoylhydrazone): synthesis, characterisation, DNA binding and cleavage properties

Synthesis and characterisation of three seven-coordinated cobalt(II) complexes of 2,6-diacetylpyridine bis(4-hydroxybenzoylhydrazone) (H4L) ligand, [Co(H2L)(H2O)2] (1), [Co(H4L)(N3)2] (2) and [Co(H4L)(NCS)2] (3) are described. The structures of the complexes were characterised by elemental analysis, IR, UV–vis and magnetic susceptibility measurement. The molecular structure of the [Co(H4L)(NCS)2] (3) was also determined by X-ray crystallography. Single crystal X-ray revealed that the Co(II) complex (3) has a pentagonal–bipyramidal coordination geometry, with pentadentate N3O2 ligand in the equatorial plane of the bipyramid and two isothiocyanato groups in the axial area. Interaction of the cobalt(II) complexes with CT-DNA was investigated by absorption titration method and viscosity measurements. Cleavage activity of the complexes with pBR 322 plasmid DNA was evaluated by agarose gel electrophoresis in presence and absence of an oxidative agent, and the mechanism of DNA cleavage was investigated. The results suggest that the cobalt(II) complexes bind effectively and they exhibit nuclease activity, which has strong dependence on the concentration of complex and reaction time, both in presence and absence of hydrogen peroxide.

Synthesis, structure, DNA binding and cleavage activity of a new copper(II) complex of bispyridylpyrrolide

A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl] (PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space group Pccn, a = 0.9016(3) nm, b = 1.0931(4) nm, c = 2.5319(8) nm, and V = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(II) complex with calf thymus DNA (CT-DNA) was investigated by electronic absorption, circular dichroism (CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled pBR322 DNA in the presence of ascorbate (H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.

Crystal structure of human Fanconi-associated nuclease 1.

Crystal structure of human Fanconi-associated nuclease 1.

DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis.

The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6–7 nucleotides) downstream of the asymmetric recognition sequence 5′-GCCGC-3′. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.

Mechanism and regulation of meiotic recombination initiation.

Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence.

Sequence-specific DNA glycosylase found in a restriction-modification system

Restriction-modification systems consist of genes that encode a restriction enzyme and a cognate modification methyltransferase. It was believed that restriction enzymes are sequence-specific endonucleases that introduce double-strand breaks at specific sites by catalyzing the cleavages of phosphodiester bonds. R.PabI is a type II restriction enzyme from a hyperthermophilic archaea Pyrococcus abyssi that recognizes 5'-GTAC-3' sequence and cleaves DNA duplexes without the addition of a divalent cation. The structural and mutational analyses of R.PabI in our previous work showed that R.PabI forms a homodimer and has a novel DNA-binding fold called a "half-pipe," which consists of a highly curved anti-parallel β-sheet. Because the structure of R.PabI shares no structural similarity to any other protein, the structural basis of the sequence-recognition and DNA-cleavage mechanisms remained unclear. In this study, we report the crystal structure of R.PabI in complex with a DNA duplex containing the R.PabI recognition sequence. The structure of the R.PabI-DNA complex shows that R.PabI unwinds a DNA duplex at a 5'-GTAC-3' site and flips the guanine and adenine bases out of the DNA helix to recognize the sequence. The electron-density map of the R.PabI-DNA complex shows that R.PabI releases adenine bases from the R.PabI recognition sequence. Biochemical assays using HPLC and MALDI-TOF MS spectrometry also support the observation that R.PabI releases adenine bases by hydrolysis. These results show that R.PabI is not an endonuclease but a sequence-specific adenine DNA glycosylase. R.PabI is the first example of a restriction enzyme that shows DNA glycosylase activity. Mutational analysis reveals the active site of the adenine DNA glycosylase activity of R.PabI. The two opposing apurinic/apyrimidinic (AP) sites generated by R.PabI are cleaved by heat promoted β elimination and/or by endogenous AP endonucleases of host cells to introduce a double-strand break.

Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella.

Prokaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of > 600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, overexpression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification.

Characterization of HU‐331 as a cannabinoid quinone inhibitor of topoisomerase II (549.6)

Topoisomerases are essential enzymes that are involved in DNA metabolism. Due to the double‐stranded DNA cleavage mechanism, topoisomerase II is an effective target for anticancer drugs, such as doxorubicin. However, doxorubicin causes cardiac toxicity and can induce DNA damage resulting in secondary cancers. A cannabinoid‐based quinone HU‐331 has been identified as a potential anticancer drug that demonstrates more potency in cancer cells with less off‐target toxicity than doxorubicin. Reports indicate that HU‐331 does not promote cell death via apoptosis, cell cycle arrest, caspase activation, or DNA strand breaks. However, the precise mechanism of action is poorly understood. We proposed that HU‐331 inhibits human topoisomerase IIα function by inhibiting the ATPase domain of the enzyme. We employed biochemical assays to study the mechanism of action of HU‐331 against purified topoisomerase IIα. These assays examined DNA cleavage, ligation, relaxation, and ATPase function of topoisomerase IIα. Our results demonstrate that HU‐331 inhibits topoisomerase IIα‐mediated DNA relaxation at micromolar levels. We find that HU‐331 does not induce DNA strand breaks, decreasing the risk of secondary cancers. When added prior to the DNA substrate, HU‐331 blocks DNA cleavage and relaxation activities of topoisomerase IIα. Our results also show that HU‐331 inhibits the ATPase function of topoisomerase IIα. Preliminary binding studies also indicate that HU‐331 decreases the ability of topoisomerase IIα to bind DNA. We have thus far concluded that HU‐331 impacts the catalytic activity of topoisomerase IIα by inhibiting the ATPase function. These results provide a promising foundation for the potential of HU‐331 as an anticancer drug. Future studies will explore the ability of HU‐331 to abrogate cleavage and relaxation activities. We will also further clarify the impact of HU‐331 on the enzyme:DNA interaction.

Mechanism of DNA Cleavage Catalysed by the System Comprising an Unsaturated Tetraaza Macrocycle and La(III) and Ce(III) Ions

In order to further the design of efficient artificial nuclease models for hydrolytic cleavage of DNA, the catalytic system made from an unsaturated tetraaza macrocycle and La(III) and Ce(III) ions was constructed and used as catalyst in the cleavage of plasmid PUC19 DNA. The interactions of these catalytic systems with plasmid PUC19 DNA has been investigated by UV-Vis absorption spectroscopy, fluorescence spectroscopy, cyclic voltammetry and gel electrophoresis. The results show that the combination of the catalytic system with DNA involves electrostatic binding through the phosphodiester linkages of DNA. The gel electrophoresis results also showed that supercoiled PUC19 plasmid DNA (cccDNA) was converted to the nicked form of DNA (ocDNA) under a nitrogen atmosphere. On the basis of these results, a mechanism for the catalytic cleavage of DNA is proposed.

Elucidating Restriction Endonucleases Reaction Mechanisms via Dwell-Time Distribution Analysis

We have developed a Total Internal Reflection Fluorescence (TIRF) microscopy based assay that allows us to simultaneously measure the length of the catalytic cycle for hundreds of restriction endonuclease molecules in one experiment. We stably attach thousands of short duplex DNA molecules, each labeled with a single quantum dot semiconductor nanocrystal, to a passivated glass surface within a flow channel. The disappearance of a quantum dot indicates that its DNA tether has been cleaved. We introduce restriction endonuclease molecules into the channel in the absence of magnesium, which permits binding to, but not cleavage of the surface immobilized DNA substrate. When buffer containing magnesium is introduced into the flow channel, DNA cleavage by the pre-bound restriction endonuclease molecules is initiated. This synchronization allows us to measure the lag time between the introduction of magnesium and the completion of DNA cleavage for the entire population of enzymes. Analysis of the dwell-time distributions can provide insights into the DNA cleavage mechanism. Our observations suggest that EcoRV, a dimeric Type II restriction endonuclease that cleaves the palindromic sequence GAT↓ATC (where ↓ is the cut site), requires two kinetic steps to complete duplex cleavage after prebinding. However, dwell-time distributions suggest that BcnI, which is active as a monomer and cleaves the pseudopalindromic sequence 5'-CC↓SGG-5' (where S stands for C or G), requires more than four kinetic steps to complete duplex cleavage. Furthermore, experiments performed with strand-specific DNA substrates suggest that the number of steps indicated by the dwell-time distribution depends on which strand of the recognition site must be cleaved to result in quantum dot release. By designing additional substrates that mimic the various intermediate states, we plan to dissect the mechanism by which BcnI cleaves each strand of the intact restriction site.

Enthalpy-driven nuclease-like activity and mechanism of peptide-chlorambucil conjugates.

We report the results of attaching the anticancer drug chlorambucil (CLB) to two high-affinity DNA binding peptides: Met-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyM-10) and Gln-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyQ-10). These CLB-peptide conjugates cleave DNA very effectively and sequence-selectively without the use of chemicals, heat, or UV irradiation. Polyacrylamide gel electrophoresis identifies the sites where CLB-HyM-10 and CLB-HyQ-10 attack a complementary pair of 5'-(32)P-labeled duplexes derived from pBR322 in the absence of piperidine or other chemical additives. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has confirmed the preferential cleavage sites as well as a novel stepwise cleavage mechanism of sequence-selective DNA cleavage. Resembling restriction endonucleases, the CLB-peptide conjugates appear to be capable of producing double strand DNA breaks. Circular dichroism studies show that CLB-HyM-10 and CLB-HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven. The maintenance of a conserved negative binding free energy in DNA-conjugate interactions is a crucial feature of the universal enthalpy-entropy compensation phenomenon. The strongly enthalpy-driven binding of CLB-peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.

Adsorptive transfer voltammetry applied to the study of chromium-induced DNA damage in the presence of curcumin

The interaction between double-stranded (ds) calf-thymus DNA and chromium in the presence of curcumin (CC) was studied by differential pulse adsorptive transfer voltammetry using carbon paste electrode (CPE). Curcumin–Cr complex generated changes in calf thymus DNA. The mechanism for DNA cleavage by curcumin–Cr complex appears to involve both the hydroxyl radical as well as singlet oxygen. The characteristic peak of dsDNA, due to the oxidation of guanine residues, drastically decreased. The increased DNA damage by curcumin–Cr complex was observed in the presence of various concentrations of chromium(VI).

Photoinduced DNA cleavage by atomic oxygen precursors in aqueous solutions

Reactive oxygen species are known to induce DNA strand cleavage and have been explored as treatments for cancer. The development of aqueous-soluble dibenzothiophene-S-oxide (DBTO) derivatives has made it possible to investigate the mechanism of DNA cleavage by these photoactivatable precursors of atomic oxygen. In addition to the release of atomic oxygen, DBTO can also undergo other processes such as α-cleavage. An objective of this work was to establish whether the extent of strand scission could be attributed to a direct reaction between atomic oxygen and DNA. To accomplish this aim, the extent of strand cleavage upon irradiation of three different DBTO derivatives was measured by the conversion of circular pUC19 plasmid (Form I) to nicked (Form II) as monitored by gel electrophoresis. The interaction of the sulfoxides with DNA was systematically studied by optical melt and fluorescence anisotropy experiments. Thiols are susceptible to rapid oxidation by atomic oxygen, and thus, glutathione was used as a ROS scavenger to determine if DNA cleavage was induced by the release of atomic oxygen. The results from these experiments indicated atomic oxygen was at least partially responsible for the observed strand scission.

DNA-binding, photocleavage studies of ruthenium(II) complexes with 2-(2-quinolinyl) imidazo[4,5-f][1,10]phenanthroline

Two new ruthenium complexes with [Ru(L)2(qip)]2+ (L=bpy (2,2′- bipyridine), phen (1,10-phenanthroline); qip=2-(2-quinolinyl)imidazo[4,5-f][1,10]phenanthroline), have been synthesized and characterized by elemental analysis, ES-MS, 1H NMR. The binding properties of two complexes towards CT-DNA were investigated by various optical methods and viscosity measurements. The experiment results suggested that both Ru(II) complexes can intercalate into DNA base pairs. Strong quenching in emission intensity of two Ru(II) complexes were observed upon addition of Ag+ in the absence and presence of CT-DNA. Furthermore, the two complexes can promote cleavage of pBR322 DNA under irradiation at 365nm, and complex 2 exhibits a stronger DNA-photocleavage efficiency than complex 1. The mechanism of DNA cleavage suggests that singlet oxygen (1O2) is likely to be the cleaving agent.

Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency

Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10IE204Q. Detailed characterization of plasmids from 64 colonies screened from the library of ∼200 000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis.

Evolutionary Comparison of the Mechanism of DNA Cleavage with Respect to Immune Diversity and Genomic Instability

It is generally assumed that the genetic mechanism for immune diversity is unique and distinct from that for general genome diversity, in part because of the high efficiency and strict regulation of immune diversity. This expectation was partially met by the discovery of RAG1 and -2, which catalyze V(D)J recombination to generate the immune repertoire of B and T lymphocyte receptors. RAG1 and -2 were later shown to be derived from a transposon. On the other hand, activation-induced cytidine deaminase (AID), which mediates both somatic hypermutation (SHM) and the class-switch recombination (CSR) of the immunoglobulin genes, evolved earlier than RAG1 and -2 in jawless vertebrates. This review compares immune diversity and general genome diversity from an evolutionary perspective, shedding light on the roles of DNA-cleaving enzymes and target recognition markers. This comparison revealed that AID-mediated SHM and CSR share the cleaving enzyme topoisomerase 1 with transcription-associated mutation (TAM) and triplet contraction, which is involved in many genetic diseases. These genome-altering events appear to target DNA with non-B structure, which is induced by the inefficient correction of the excessive supercoiling that is caused by active transcription. Furthermore, an epigenetic modification on chromatin (histone H3K4 trimethylation) is used as a mark for DNA cleavage sites in meiotic recombination, V(D)J recombination, CSR, and SHM. We conclude that acquired immune diversity evolved via the appearance of an AID orthologue that utilized a preexisting mechanism for genomic instability, such as TAM.

Mn(II) complexes with sulfonamides as ligands.: DNA interaction studies and nuclease activity

Sulfonamides derived from 8-aminoquinoline react with Mn(II) and Mn(III) salts to form Mn(II) complexes; the Mn(III) species are reduced to the divalent state in the presence of 1,10 phenanthroline and bipyridine. Their molecular structure, determined by single crystal X‐ray diffraction, show that all the complexes present a distorted octahedral geometry, in which the deprotonated sulfonamide acts as a bidentate ligand. UV–visible spectroscopy and changes in the melting temperature (Tm) of calf thymus DNA show a strong interaction of these complexes with DNA. The significant hypochromicity of the charge transfer transition at 370nm without an appreciable change in wavelength and the minor changes in the relative viscosity of calf thymus DNA have been attributed to an interaction between the surface of DNA and the complexes. [Mn(qbsa)2(MeOH)2], [Mn(qbsa)2(phen)], [Mn(qtsa)2(H2O)2] and [Mn(qtsa)2(phen)] (where qbsa=N-quinolin-8-yl-bencenesulfonamide, qtsa=N-quinolin-8-yl-p-toluenesulfonamide and qnsa=N-quinolin-8-yl-naftalenesulfonamide) exhibit a prominent nuclease activity and the mechanism of DNA cleavage is investigated.

Efficient DNA Cleavage Induced by RhIII(TMPyP)5+ in the Presence of Ascorbic Acid

The ability of Rh(III) tetrakis-N-methylpyridyl porphyrin, RhIII(TMPyP)5+, to interact with and cleave DNA was, investigated by UV-visible, luminescence, circular dichroism (CD), electron spin resonance (ESR) and gel electrophoresis methods in the presence or absence of ascorbic acid. UV-absorption data showed that RhIII(TMPyP)5+ is capable of interacting with DNA, as indicated by the appearance of a red shift and hypochromicity of the Soret band. The CD data revealed that RhIII(TMPyP)5+ was capable of binding to DNA via an external binding mode. The RhIII(TMPyP)5+ showed fluorescence and phosphorescence at room temperature. The phosphorescence increased in the presence of DNA and this could be attributed to the shielding of the metal-porphyrin by DNA. Gel electrophoresis studies revealed that RhIII(TMPyP)5+ was only able to cleave DNA in the presence of the reducing agent ascorbic acid. ESR data indicated the formation of RhII(TMPyP)5+ by reduction of RhIII(TMPyP)5+ with ascorbic acid. The involvement of Rh(III)/Rh(II) species in catalytic DNA cleavage and a possible DNA cleavage mechanism is discussed.

DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences

The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.

The Structure of an Archaeal Viral Integrase Reveals an Evolutionarily Conserved Catalytic Core yet Supports a Mechanism of DNA Cleavage in trans

The first structure of a catalytic domain from a hyperthermophilic archaeal viral integrase reveals a minimal fold similar to that of bacterial HP1 integrase and defines structural elements conserved across three domains of life. However, structural superposition on bacterial Holliday junction complexes and similarities in the C-terminal tail with that of eukaryotic Flp suggest that the catalytic tyrosine and an additional active-site lysine are delivered to neighboring subunits in trans. An intramolecular disulfide bond contributes significant thermostability in vitro.