Mechanism Of DNA Cleavage Research Articles

Base‐specific Photocleavage of DNA Induced by Pazelliptine Sensitization: Study of the Mechanism by Time‐resolved Absorption and Fluorescence

AbstractThe intercalating antitumoral drug pazelliptine (PZE) is able to photosensitize the formation of single‐ and double‐strand breaks in supercoiled plasmid DNA and selective photocleavage at guanine residues is observed. In order to understand the mechanisms of DNA cleavage mediated by the photoexcited drug, singlet and triplet excited‐state processes in PZE complexed with poly(dA‐dT)‐poly(dA‐dT), poly(dG‐dC)‐poly(dG‐dC) and calf thymus DNA have been investigated by means of single photon counting fluorescence decay and transient absorption techniques. For each complex, three different binding sites have been identified, due to the existence of different geometric structures of the drug in the ground state. For one type of binding site, a proton transfer reaction occurs in the singlet excited state whatever the nucleic acid environment. In contrast, the relaxation dynamics for the other two sites are found to depend widely upon the type of polynucleotide in which the drug has been intercalated. From the results of this study, we suggest that the photodynamic action of PZE does not originate from excitation of the drug in the environment of G‐C base pairs but is initiated from its triplet state that reacts by electron transfer with the adenine bases. The specificity of cleavage could be the result of subsequent reactions leading to guanine oxidation.

Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11.

In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 A resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

Mechanism of DNA cleavage due to green cobalt(III)-bleomycin hydroperoxide irradiated by visible light

The origin of the DNA cleavage reaction due to green cobalt(III)-bleomycin hydroperoxide irradiated by light (366 nm) was developed; the importance of the activation of the peroxide ion by transferring an electron from the occupied orbital to the σ *-orbital of the peroxide ion was pointed out.

Mechanistic and Synthetic Aspects of the C-1′ Radicals in Modified Nucleosides

The direct and indirect production of C-1′ radicals has been achieved through the synthesis of modified nucleosides. Product studies and spectroscopic investigations have been used to study the structure and fate of C-1′ radical species under anoxic or aerobic conditions. The results are of fundamental importance in understanding the mechanism of DNA cleavage via C-1′ radicals. The mechanistic schemes of some very recent synthetic applications have also been revisited.

In vitro plasmid DNA cleavage by chromium(V) and -(IV) 2-hydroxycarboxylato complexes.

The ability of relatively stable Cr(V) and Cr(IV) complexes with 2-hydroxycarboxylato ligands [2-ethyl-2-hydroxybutanoate(2-) = ehba; (1R,3R,4R,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylate(2-) = quinate = qa] to induce single-strand breaks in plasmid DNA has been studied under a wide range of reaction conditions. The Cr(V) complex, Na[CrVO(ehba)2], causes substantial DNA cleavage at pH 4.0-8.0 [[Cr(V)]0 = 0.010-0.75 mM, phosphate buffer, and 37 degrees C]. The DNA cleavage is inhibited by the presence of excess ligand, by exclusion of O2, or by addition of organic compounds, such as alcohols, carboxylic acids, or DMSO, but it is not affected by traces of catalytic metals [Fe(III) or Cu(II)] or by addition of catalase. The Cr(IV)-qa complexes, unlike the Cr(V) complexes, are able to cleave DNA in the presence of the ligand in a large excess [[Cr(IV)]0 = 0.50 mM, [qa] = 20-100 mM, pH 3.5-6.0, and 37 degrees C]. This is the first direct evidence for DNA cleavage induced by well-characterized Cr(IV) complexes. The proposed mechanism for DNA cleavage includes the following: (i) partial aquation of the bis-chelated Cr(V) and -(IV) complexes with the formation of reactive monochelated forms, (ii) binding of the Cr(V) and -(IV) monochelates to the phosphate backbone of DNA, (iii) one- or two-electron oxidations at the deoxyribose moieties of DNA by Cr(V) and -(IV), and (iv) cleavage of the resulting DNA radicals or cations with or without participation of O2. The patterns of DNA damage by Cr(V) and -(IV) can include strand breaks, generation of abasic sites, and the formation of Cr(III)-DNA complexes.

Base-specific Photocleavage of DNA Induced by Pazelliptine Sensitization: Study of the Mechanism by Time-resolved Absorption and Fluorescence

The intercalating antitumoral drug pazelliptine (PZE) is able to photosensitize the formation of single- and double-strand breaks in supercoiled plasmid DNA and selective photocleavage at guanine residues is observed. In order to understand the mechanisms of DNA cleavage mediated by the photoexcited drug, singlet and triplet excited-state processes in PZE complexed with poly(dA-dT)-poly(dA-dT), poly(dG-dC)-poly(dG-dC) and calf thymus DNA have been investigated by means of single photon counting fluorescence decay and transient absorption techniques. For each complex, three different binding sites have been identified, due to the existence of different geometric structures of the drug in the ground state. For one type of binding site, a proton transfer reaction occurs in the singlet excited state whatever the nucleic acid environment. In contrast, the relaxation dynamics for the other two sites are found to depend widely upon the type of polynucleotide in which the drug has been intercalated. From the results of this study, we suggest that the photodynamic action of PZE does not originate from excitation of the drug in the environment of G-C base pairs but is initiated from its triplet state that reacts by electron transfer with the adenine bases. The specificity of cleavage could be the result of subsequent reactions leading to guanine oxidation.

On the Mechanism of DNA Cleavage by Fullerenes Investigated in Model Systems: Electron Transfer from Guanosine and 8-Oxo-Guanosine Derivatives to C60

On the Mechanism of DNA Cleavage by Fullerenes Investigated in Model Systems: Electron Transfer from Guanosine and 8-Oxo-Guanosine Derivatives to C₆₀

Interleukin 7 receptor control of T cell receptor gamma gene rearrangement: role of receptor-associated chains and locus accessibility.

VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid cells. Although the molecular mechanisms of DNA cleavage and ligation have become more clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)alpha-/- murine thymocytes, it has been shown that rearrangement of the T cell receptor (TCR)-gamma locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7Ralpha to rearrangement of the TCR-gamma locus requires the gammac receptor chain and the gammac-associated Janus kinase Jak3. Production of sterile transcripts from the TCR-gamma locus, a process that generally precedes rearrangement of a locus, was greatly repressed in IL-7Ralpha-/- thymocytes. The repressed transcription was not due to a lack in transcription factors since the three transcription factors known to regulate this locus were readily detected in IL-7Ralpha-/- thymocytes. Instead, the TCR-gamma locus was shown to be methylated in IL-7Ralpha-/- thymocytes. Treatment of IL-7Ralpha-/- precursor T cells with the specific histone deacetylase inhibitor trichostatin A released the block of TCR-gamma gene rearrangement. This data supports the model that IL-7R promotes TCR-gamma gene rearrangement by regulating accessibility of the locus via demethylation and histone acetylation of the locus.

The role of metals in catalysis by the restriction endonuclease BamHI.

Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+ or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI-DNA complex, determined in the presence of Mn2+ and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV.

Mechanism of double-strand DNA cleavage effected by iron-bleomycin.

We have observed that in the absence of hydrogen peroxide the Fe(III)-bleomycin (BLM) complex exhibits high DNA cleavage efficiency, converting supercoiled Form I DNA (pBR322 or phix174) to Form II (nicked, relaxed circular); the present study may give an important clue to elucidate the fact that iron-bleomycin mediated double-strand DNA cleavage requires at least one molecule of oxygen (O2) over the amount required to form 'activated bleomycin."

Structural and functional analysis of the homing endonuclease PI-sceI by limited proteolytic cleavage and molecular cloning of partial digestion products.

PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the catalytic activity, we performed limited proteolytic digestion experiments with various proteases. Two protease-resistant fragments spanning the N- and C-terminal halves of the nuclease were identified using different proteases which cleave the protein in the same region. Each fragment contains one of the two conserved LAGLIDADG motifs. The products of the limited proteolytic digests were shown to remain associated and to exhibit specific DNA binding but to be inactive in DNA cleavage. Different from what is observed with native PI-SceI, only one complex is formed as shown in an electrophoretic mobility shift assay. Expression clones for the N- and C-terminal protein fragments obtained by tryptic digestion were constructed, and the proteins PI-SceI-N and PI-SceI-C were purified. Only PI-SceI-N exhibits DNA binding activity. Bending experiments with PI-SceI-N, a mixture of PI-SceI-N and PI-SceI-C, as well as the products of the limited tryptic digest show that a DNA substrate with the full length recognition sequence is bent by 45;. This degree of bending is also observed with a DNA containing only the right side of the recognition sequence, corresponding to one of the DNA cleavage products of PI-SceI. Our results demonstrate that the N-terminal half of PI-SceI which lacks one of the two LAGLDADG motifs is able to bind to DNA specifically and to induce one of the distortions observed to occur in the process of DNA binding by PI-SceI. These results are discussed in light of the recently solved crystal structure of PI-SceI and used to refine a model for the mechanism of DNA binding and cleavage by PI-SceI.

The mechanism of DNA cleavage by the type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis.

Three different mechanisms have been proposed to describe DNA cleavage by the type II restriction endonuclease EcoRV, which differ in the number and function of metal ions directly involved in catalysis and the different roles assigned to amino acid residues in the active sites and a phosphate group of the substrate. There are only four acidic amino acid residues close to the scissile bond: the essential Asp74 and Asp90, the non-essential Glu45, and Asp36. We show here that Asp36 can be exchanged for alanine, with only minor effects on the cleavage rate of the nearby phosphodiester bond, excluding that Asp36 could be directly involved in catalysis. Hence, the two versions of the two-metal-ion mechanism are not compatible with the experimental data, because too few ligands for two metal ions are present near the active site of EcoRV. Our result, thus, supports the one-metal-ion mechanism for EcoRV. We suggest that Asp36 has an allosteric effect by which specific contacts between one strand of the DNA and one subunit of the enzyme trigger the activation of one catalytic center. Given the similar structures of the active sites of EcoRV, EcoRI, BamHI, PvuII and FokI, as well as the occurrence of a characteristic catalytic motif in several other restriction enzymes, we conclude that these enzymes most likely share a similar mechanism of DNA cleavage, whose characteristic feature is the involvement of only one Mg2+ ion in catalysis.

Does the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?

Two models for the catalytic mechanism of the restriction endonuclease EcoRV exist which differ in the number and function of metal ions proposed to be directly involved in catalysis. In one model, two metal ions bound by Glu45, Asp74, and Asp90 are assumed to have a direct catalytic function; in the other, only one metal ion bound by Asp74 and Asp90. We show here that in the presence of Mn2+, the catalytic activity of an EcoRV-E45A mutant is only slightly reduced (1.8-fold) as compared to wild type EcoRV and that the single-turnover rate constant of DNA cleavage by E45A is reduced only 39-fold, whereas the D74A and D90A mutants are catalytically inactive under all conditions. These findings make an important catalytic function of Glu45, like binding of an essential divalent metal ion, unlikely. In addition, we have analyzed the dependence of the DNA cleavage rate by EcoRV and EcoRV mutants on the concentration of Mg2+ and Mn2+. We found for the wild type enzyme a sigmoidal dependence of the rate of DNA cleavage on the concentration of Mg2+ or Mn2+, indicative of at least two metal ions involved in DNA binding and catalysis. This, however, does not mean that EcoRV follows a two-metal-ion mechanism in DNA cleavage, because also for the E45A mutant a sigmoidal dependence of the rate of DNA cleavage on the Mg2+ concentration was found, making metal ion binding to the E45/D74 site unlikely. In contrast, the Y219C mutant shows a hyperbolic dependence. In agreement with results obtained earlier, these findings demonstrate binding of a Mg2+ ion at a site influenced by Tyr219, an amino acid residue that is far away from the active site. Metal binding at this site does not have a catalytic role but rather supports specific DNA binding. We conclude that on the basis of our data a two-metal-ion mechanism of DNA cleavage is unlikely for EcoRV and that the complex metal ion effects observed are due to metal ion binding at sites that are not directly involved in catalysis.

A common chemical mechanism used for DNA cleavage by copper(II) activated by thiols and ascorbate is distinct from that for copper(II): hydrogen peroxide cleavage

By use of a [32P] end-labelled 163 base pair linear fragment of duplex DNA isolated from tyrTDNA, the apparent sequence selectivity previously reported for DNA cleavage by CuII:thiol systems was shown to be identical to the sequence selectivity in scission shown by the CuII:ascorbate system. On this basis, a general mechanism of DNA cleavage is proposed for CuII with reducing agents including thiols as well as reducing sugars, including ascorbate and glucosamine. This mechanism involves hydroxyl radical generation and leads to both base damage and strand scission, and based on results with CuII:thiol cleavage the former occurs before the latter is detectable. Cleavage of DNA by CuII:hydrogen peroxide follows a different chemical mechanism, distinguishable by its different sequence preference.

1,2-Dithiolan-3-one 1-oxides: a class of thiol-activated DNA-cleaving agents that are structurally related to the natural product leinamycin.

Leinamycin is a recently discovered, thiol-dependent DNA-cleaving natural product. The mechanism of DNA cleavage by leinamycin is unknown. Inspired by this intriguing natural product, we have investigated the DNA-cleaving properties of three 1,2-dithiolan-3-one 1-oxides (1-3) that are structurally related to the suspected DNA-cleaving "core" of leinamycin. It was found that, similar to leinamycin, these three 1,2-dithiolan-3-one 1-oxides are thiol-dependent DNA-cleaving agents. At the concentrations of 1-3 used in these experiments (approximately 100 microM), efficient DNA cleavage is absolutely dependent on added thiol, with optimum cleavage occurring at 5-10 equiv (500 microM-1 mM) of added thiol. 2-Mercaptoethanol, glutathione, dithiothreitol, and thiophenol function with approximately equal efficiency as triggering agents for the cleavage reaction. DNA cleavage by 1-3 is not highly pH-dependent. Cleavage of DNA by these sulfur heterocycles is diminished by the removal of molecular oxygen from the reaction medium, by the radical scavengers methanol, ethanol, and mannitol, and by the enzyme catalase. Superoxide dismutase does not suppress DNA cleavage by these compounds. When diethylenetriaminepentaacetic acid is employed in these reactions as a chelator of adventitious trace metal ions, DNA cleavage is efficiently inhibited. The S-deoxy analog of 1 does not cleave DNA under conditions where 1 effects efficient thiol-mediated cleavage of DNA. These experiments indicate that, in concert with thiols, 1,2-dithiolan-3-one 1-oxides convert molecular oxygen to DNA-cleaving oxygen radicals. The marked effect of catalase further suggests that molecular oxygen is converted to hydrogen peroxide which ultimately cleaves DNA via a trace metal-dependent Fenton reaction. This work demonstrates that 1,2-dithiolan-3-one 1-oxides represent a general class of thiol-potentiated DNA-cleaving molecules.

Syntheses, structures and reactivities of designed analogues of cobalt(III)-bleomycinsz: Insight into the mechanism of sequence-specific DNA cleavage upon illumination

Three Co (III) complexes of a designed ligand PMAH that mimics the metal-binding domain of the antitumor antibioticbleomycin (BLM) have been isolated and structurally characterized. The coordination structures of the various forms of Co(III)-BLMs have been established on the basis of spectral similarities between these synthetic analogues and the corresponding Co(III)-BLMs. All three analogues, like Co(III)-BLMs, induce DNA strand scission upon UV illumination. Both DNA cleavage and spin trapping experiments demonstrate that UV irradiation of the analogues generates a C/N-based radical on the ligand framework which rapidly reacts with water to produce -OH radical near the DNA helix and causes strand scission. A similar mechanism could account for the photoactivity of the Co(III)-BLMs. Covalent attachment of DNA-binding groups to these analogues enhances the DNA-affinities and photocleavage efficiencies to a great extent. The hybrid analogues promote sequence-specific DNA photodamage at micromolar concentrations. The metallated cores of the hybrid analogues are the primary determinant of the observed sequence-specificity. Details of the mode of binding of the hybrid analogues to DNA have been explored by NMR techniques.

Sequence preference for strand cleavage of gapped duplexes by dynemicin A: possible mechanism of sequence-dependent double-stranded breaks.

A double-stranded DNA cleavage mechanism by a novel enediyne type antitumor antibiotic, dynemicin A, has been investigated through sequence-dependent strand breakage of a series of duplexes containing a single nucleotide gap. We found that (1) dynemicin A breaks specifically at the 3'-shifted position by one base opposite the gap, (2) the strong cleavage is detected at 5'-Pu_Pu/3'-PyPuPy sequences, and (3) dynemicin H (aromatized form of dynemicin A) gives only a small inhibition effect (20%) on the cleavage of gapped duplex by dynemicin A. The long half-life of aromatization of dynemicin A (118 min, in the presence of DNA) obtained from HPLC analysis provides enough time for the second cleavage. The present results strongly indicate a two-step mechanism for the double-stranded DNA scission of dynemicin A. Namely, this double-stranded break is caused by two drug molecules, each of which cuts one DNA strand.

Oxidative and nonoxidative mechanisms of site-specific DNA cleavage induced by copper-containing metallothioneins.

DNA cleavage induced by metallothionein (MT) containing copper was investigated by a DNA sequencing technique. Reconstituted Cd7-MT showed no ability to cause DNA cleavage. Commercially available rabbit MT I caused DNA cleavage, suggesting that DNA cleavage is due to the metal contained in commercial Mt. Cu2Cd5-MT and Cu12-MT were prepared by the treatment of commercial rabbit MT I with [Cu(CH3CN)4]CIO4. Cu12-MT frequently induced an alteration of thymine residues, especially in the 5'-GTC-3' sequence, and piperidine treatment led to chain cleavage at the thymine residues. The site specificity was similar to that obtained with Cu(I) plus H2O2. H2O2 enhanced DNA cleavage induced by Cu12-MT. Catalase and a Cu(I)-specific chelating agent, bathocuproine, inhibited DNA cleavage. These results suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA cleavage. Commercial MT and Cu2Cd5-MT induced DNA cleavage much less than Cu12-MT, but gave particularly specific DNA cleavage. Cu2Cd5-MT induced cleavage specifically at the central guanine residue of the 5'-GGT-3' sequence. A similar cleavage pattern was obtained with commercial MT. No effect of piperidine treatment suggests that the DNA cleavage might not be due to base damage and/or liberation. The DNA cleavage was inhibited efficiently by EDTA, but not by bathocuproine and catalase. Experiments with DNA ligands, albumin, and denatured DNA suggest that commercial MT and Cu2Cd5-MT induce nonoxidative cleavage of the deoxyribose phosphate backbone through its DNA recognition. These two types of cleavage mechanisms are discussed in relation to the possible role of Cu-MT in carcinogenesis.

Insights into the Mechanism of DNA Cleavage by Dynemicin A as Revealed by DNA-Binding and -Cleavage Studies of Synthetic Analogs

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTInsights into the Mechanism of DNA Cleavage by Dynemicin A as Revealed by DNA-Binding and -Cleavage Studies of Synthetic AnalogsAndrew G. Myers, Scott B. Cohen, Norma J. Tom, David J. Madar, and Mark E. FraleyCite this: J. Am. Chem. Soc. 1995, 117, 28, 7574–7575Publication Date (Print):July 1, 1995Publication History Published online1 May 2002Published inissue 1 July 1995https://doi.org/10.1021/ja00133a043RIGHTS & PERMISSIONSArticle Views423Altmetric-Citations32LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Molecular dynamics study of the binding of elsamicin A to DNA.

Molecular mechanics (MM) and dynamics (MD) were used to examine the conformational flexibility of elsamicin A, an antitumour antibiotic, in aqueous solution. Furthermore, MM and MD simulations were performed to obtain the first three-dimensional model of the elsamicin-A-DNA complex. Comparison of MD simulations of free and DNA-bound elsamicin A provided insight into the mechanism of DNA cleavage. Moreover, the drug-DNA complexes were analyzed to investigate specific drug-DNA interactions, as well as the role of the solvent water molecules in the drug-DNA binding. On the basis of the results derived from these simulations, several issues related to the sequence-selective binding of elsamicin A to DNA are discussed.