Experimental and modelling studies on the DNA cleavage by elsamicin A.

The ability of elsamicin A, an antitumour antibiotic, to cleave DNA in the presence of ferrous iron and reducing agents, has been analysed using experimental and theoretical approaches. Experimentally, the antibiotic causes DNA breakage in the presence of ferrous ions and a reducing agent. The DNA-cleaving activity appears to be partially blocked by the action of superoxide dismutase and catalase. These results indicate that the elsamicin aglycone moiety (chartarin) can be involved in the production of free radicals. We have performed a broad theoretical study based in the quantum-mechanical framework, which allow us to determine the redox properties of elsamicin that lead to the generation of radical species. Our results clearly show that elsamicin acts as a true catalyst in the production of superoxide radicals. Moreover, it is suggested that the oxidation/reduction mechanism of the aglycone moiety of elsamicin (a lactone), leading to DNA breakage, is different from the mechanism followed by other well-known anti-cancer drugs, whose chromophore is a quinone.     

Switch-function of amino sugar on elsamicin A, a DNA binding antitumor antibiotic

There are a considerable number of DNA binding natural products equipped with amino sugar residues. The amino sugar of elsamicin A significantly takes part in DNA binding and antitumor activity. In addition, we found that an acetylation of the amino group on elsamicin A sugar portion plays an important switch-function for the activity of elsamicin A. The biological implication of this switch has been discussed.     

Product analyses in DNA strand scission by antitumor antibiotic elsamicin A.

Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3'-/5'-phosphate and 3'-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4' hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4' carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA.     

DNA cleavage function of seryl-histidine dipeptide and its application

Summary. Previously published evidences highlighted the effect of transglutaminase (TG, EC 2.3.2.13) activation on the reduction of the in vitro adhesive and invasive behaviour of murine B16-F10 melanoma cells, as well as in vivo. Here, we investigated the influence of spermidine (SPD) incorporation by TG into basement membrane components i.e. laminin (LN) or Matrigel (MG), on the adhesion and invasion of B16-F10 melanoma cells by these TG/SPD-modified substrates. The adhesion assays showed that cell binding to the TG/SPD-modified LN was reduced by 30%, when compared to untreated LN, whereas the reduction obtained using TG/SPD-modified MG was 35%. Similarly, tumor cell invasion by the Boyden chamber system through TG/SPD modified LN or MG was respectively reduced by 45%, and by 69%. Evaluation of matrix metalloproteinase (gelatinases MMP-2 and MMP-9) activities by gel-zymography showed that MMP-2 activity was unaffected, while MMP-9 activity was reduced by about 32% using TG/SPD-modified substrate. These results strongly suggest that the observed antiinvasive effect of TG activation in the host may be ascribed to the covalent incorporation of polyamines, which led to the post-translational modification of some components of the cell basement membrane. This modification may interfere with the metastatic property of melanoma cells, affecting the proteolytic activity necessary for their migration and invasion activities. Authors’ address: Simone Beninati, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, I-00133 Rome, Italy     

Selective inhibition of restriction endonuclease cleavage by DNA intercalators

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.     

A conformational switch controls the DNA cleavage activity of lambda integrase

The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.     

Map of chartreusin and elsamicin binding sites on DNA

Three DNA restriction fragments designated tyrT, 102-mer and 70-mer, have been used as substrates for footprinting studies using DNase I in the presence of the structurally similar antibiotics chartreusin and elsamicin A. The sequence-selective binding sites of the antibiotics can be mapped in regions which are rich in guanine + cytosine. Chartreusin and elsamicin appear to recognize and bind preferentially to sequences containing a CpG step. Regions containing a TpG step also seem to be a good binding site. The binding of elsamicin to these sites appears to be more concentration-dependent. A comparative analysis is performed of the sizes and locations of the different binding sites, aimed to infer whether the different biological effects of chartreusin and elsamicin A can be correlated to differences in their sequence-selective binding to DNA.     

DNA cleavage by hydroxy-salicylidene-ethylendiamine-iron complexes.

Bis(hydroxy)salen.Fe complexes were designed as self-activated chemical nucleases. The presence of a hy-droxyl group on the two salicylidene moieties serve to form a hydroquinone system cooperating with the iron redox system to facilitate spontaneous formation of free radicals. We compared the DNA binding and cleaving properties of the ortho -, meta- and para -(bishydroxy) salen.Fe complexes with that of the corresponding chelate lacking the hydroxyl groups. DNA melting temperature studies indicated that the para complex exhibits the highest affinity for DNA. In addition, this para compound was considerably more potent at cleaving supercoiled plasmid DNA than the regio-isomeric ortho - and meta -hydroxy-salen.Fe complexes, even in the absence of a reducing agent, such as dithiothreitol used to activate the metal complex. The DNA cleaving activity of the para isomer is both time and concentration dependent and the complexed iron atom is absolutely essential for the sequence uniform cleavage of DNA. From a mechanistic point of view, electron spin resonance measurements suggest that DNA contributes positively to the activation of the semi-quinone system and the production of ligand radical species responsible for subsequent strand scission in the absence of a reducing agent. The para -hydroxy-salen.Fe complex has been used for detecting sequence-specific drug-DNA interactions. Specific binding of Hoechst 33258 to AT sequences and chromomycin to GC sequences were shown. The para -bis(hydroxy)salen.Fe derivative complements the tool box of footprinting reagents which can be utilised to produce efficient cleavage of DNA.     

DNA cleavage by aromatic amines.

A series of aryl amines was found to induce cleavage of DNA. Subsequent refinement led to an efficient family of dimeric derivatives capable of cleavage at low concentration. Initial investigations suggest this is an unprecedented mode of DNA cleavage, which may be ultimately applied to the development of sequence-specific agents.     

Selective and effective cleavage of RNAs by vinyladenine-vinylamine copolymer.

Poly(uridylic acid) (poly[U]) and poly(adenylic acid) (poly[A]) are efficiently cleaved, via phosphodiester linkage hydrolysis, at pH 8, 50 degrees C by use of poly(vinyladenine-co-vinylamine) as catalyst. The catalytic activity of the copolymer surpasses the value of poly(vinylamine), indicating a significant role of the adenine residue in the copolymer for the effective catalysis.     

Selective cleavage of closely-related mRNAs by synthetic ribozymes.

In Phaseolus vulgaris L. (French bean) glutamine synthetase (GS) is encoded by four closely-related genes termed gln-alpha, gln-beta, gln-gamma and gln-delta. We have constructed and characterised in vitro a number of hammerhead ribozymes designed to cleave individual RNAs encoded by these genes. The three ribozymes, termed J1, J2 and J3, were targeted to cleave RNA at the start of the gamma and beta, and the middle of the gamma, GS open reading frames respectively. All three ribozymes successfully discriminated between the four (alpha, beta, gamma and delta) highly homologous sequences, even though the targeted sites of cleavage shared up to 18 out of 22 identical bases with other gene family members. The ribozyme-mediated cleavage reactions were Mg2+ dependent and enhanced at higher temperatures, although the J1 ribozyme retained considerable activity at physiological temperatures. Both J1 and J2 demonstrated a time-dependent cleavage of their targeted GS RNAs, although these two ribozymes differed markedly in their ability to cleave multiple substrate molecules. The rate of cleavage by J1 was found to be reduced in the presence of related GS RNAs and by total leaf poly(A) RNAs. The implications of these results for ribozyme activity in vivo are discussed.     

Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage

EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.     

Site-specific cleavage of DNA by E. coli DNA gyrase.

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.     

Selective cleavage of pyrophosphate linkages.

Pyrophosphate linkages have a number of important roles in biology and are also formed chemically with great ease. They often are unwanted products, such as in the nonenzymatic oligomerization of mononucleotides. We have found that Zr(4+)- and Th(4+)-ions catalyze the symmetrical hydrolysis of pyrophosphate linkages. Oligonucleotide analogs linked by pyrophosphate bonds are substantially degraded in the presence of these metals, even at 0 degrees C. Conditions are described which permit the decapping of a pyrophosphate capped oligonucleotide. Oligodeoxynucleotides can be decapped by this procedure without cleavage of phosphodiester linkages. Oligoribonucleotides are susceptible to partial hydrolysis and require purification by HPLC after decapping.     

DNA cleavage specificity of a group of cationic metalloporphyrins

The ability of a group of water-soluble metalloporphyrins to cleave DNA has been investigated. Incubation of Mn3+, Fe3+, or Co3+ complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2T4MPyP) with DNA in the presence of ascorbate, superoxide ion, or iodosobenzene results in DNA breakage. Comparisons between the rates of porphyrin autodestruction with the rates of strand scission of covalently closed circular PM2 DNA indicate that the porphyrins remain intact during the cleavage process. Analysis of the porphyrin-mediated strand scissions on a 139-base-pair restriction fragment of pBR322 DNA using gel electrophoresis/autoradiography/microdensitometry reveals that the minimum porphyrin cleavage site is (A X T)3. The cleavage pattern within a given site was found to be asymmetric, indicating that porphyrin binding and the strand scission process are highly directional in nature. In addition to an analysis of the mechanism of porphyrin-mediated strand breakage in terms of the DNA cleavage mechanism of methidium-propyl-iron-EDTA and Fe-bleomycin, the potential of the cationic metalloporphyrins as footprinting probes and as new "reporter ligands" for DNA is presented and 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.