Stoichiometry of DNA strand scission and aldehyde formation by bleomycin

A colorimetric assay of DNA breakage by bleomycin has been standardized and indicates that strand scission is stoichiometric with the formation of a single equivalent of an aldehyde compound consisting of base plus deoxyribose carbons 1' to 3'. Both strand scission and aldehyde formation require the presence of O2. An alternate DNA lesion inflicted by bleomycin, alkali labilization, is O2-dependent, as is the accompanying release of free bases.     

Conformational changes in the oligonucleotide duplex d(GCGTTGCG) x d(CGCAACGC) induced by formation of a cis-syn thymine dimer. A two-dimensional NMR study

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.     

Oxidative strand scission of nucleic acids by a multinuclear copper(II) complex

The compound [Cu(2)(II)(D(1))(H(2)O)(2)](ClO(4))(4).2H(2)O [D(1)=binucleating ligand with tris(2-pyridylmethyl)amine (TMPA) moieties linked in the 5-pyridyl position by a -CH(2)CH(2)- bridge] mediated efficient oxidative cleavage of pBR322 plasmid DNA under reducing conditions. A mononuclear analogue, [Cu(TMPA)(H(2)O)](ClO(4))(2), was less effective at linearizing supercoiled (Form I) plasmid DNA as compared to the binuclear complex. A new method for quenching the copper-dependent reactions has been developed to avoid plasmid scission by the binuclear complex and the standard gel loading buffer. EDTA was not sufficient for retarding copper reaction, but diethyldithiocarbamic acid was capable of inhibiting all reactivity. Investigation of oxidative cleavage of double-helical oligonucleotides by [Cu(2)(II)(D(1))(H(2)O)(2)](ClO(4))(4) confirmed the enhanced reactivity of the binuclear over the mononuclear complex and provided mechanistic insights into the nature of the reaction. Cleavage of DNA required both the binuclear complex and a reductant and likely proceeded through an O(2)-derived intermediate that does not include a diffusible hydroxyl radical. The greater efficiency of the binuclear complex relative to the mononuclear analogue is consistent with their relative abilities to activate dioxygen.     

DNA strand scission by iron complexes of meso-tetra(N-methylpyridyl)porphines

The DNA strand scission activities of three positional isomers of Fe(III) meso-tetra(N-methylpyridyl)porphine (Fe(III)TnMPyP, where n = 2, 3 or 4) have been investigated using PM2 DNA as a substrate. A significant degree of strand scission activity was noted in the presence of oxygen without the addition of a reducing agent. This activity was probably due to the presence of reducing agents in the agarose gels used to separate the DNA forms, as higher levels were recorded with reducing agents added to the strand scission mixture. The relative order of strand scission activity in the absence of added reducing agents was found to be Fe(III)T2MPyP greater than Fe(III)T4MPyP greater than Fe(III)T3MPyP. Comparative studies were also made with Fe(II)bleomycin. High concentrations of some reducing agents inhibited strand scission. Oxygen was required to produce optimal strand scission activity for all three porphyrins. It was also noted from spectroscopic measurements that the reduced porphyrins were degraded in the presence of oxygen. Studies with a series of potential strand scission inhibitors suggest that hydrogen peroxide and possibly peroxy radicals are intermediates in the reaction mechanism, while diffusible hydroxyl radicals appear to be excluded. However, superoxide radicals cannot be ruled out.     

Preparation and duplex-to-single strand transition of the fully complementary duplex of d(G4).d(C4) in aqueous solution

The d(G4) and d(C4) molecules in the single stranded state were synthesized by the phosphotriester method and purified. The full duplex of tetramer d(G4).d(C4) was prepared by expending about a month. The duplex-to-single strand transition was observed by UV-spectroscopy. A standard hypochromic effect was observed, which is different from some experimental results reported previously.     

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.     

Parallel analysis of oligodeoxyribonucleotide (oligonucleotide) interactions. I. Analysis of factors influencing oligonucleotide duplex formation.

A novel method for the analysis of oligonucleotide-oligonucleotide interactions is described. Oligonucleotides of different sequence are synthesised in situ as stripes on the surface of a glass slide (see accompanying paper). Multiple hybridizations are then carried out on each oligonucleotide simultaneously to determine the dependence of oligonucleotide duplex formation on duplex length, base composition, hybridisation solvent and sequence complexity.     

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.     

Synthesis and characterization of a d(ApG) platinated nonanucleotide duplex.

The nonamer 5'd(CTCAGCCTC) 3' 1 has been reacted with cis-diamminediaquaplatinum(II) in water at pH 4.2. The major reaction product was shown by enzymatic digestion and 1H NMR to be the d(ApG)cis-Pt(NH3)2 chelate [cis-Pt(NH3)2[d(CTCAGCCTC)-N7(4),N7(5)]] 1-Pt. When mixed with its complementary strand 2, 1-Pt forms a B DNA type duplex 3-Pt with a Tm of 35 degrees C (versus 58 degrees C for the unplatinated duplex). The NMR study of the exchangeable protons of 3-Pt revealed that the helix distortion is localized on the CA*G*-CTG moiety (the asterisks indicating the platinum chelation sites) with a strong perturbation of the A*(4)T(15) base pair related to a large tilt of A*(4).     

Strand breaks in whole plasmid dna produced by the decay of (125)I in a triplex-forming oligonucleotide.

DNA strand breaks produced by the decay of (125)I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the (125)I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of (125)I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the (125)I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.     

Site-specific strand breaks in RNA produced by (125)I radiodecay

Decay of ¹²⁵I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of ¹²⁵I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3′-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when ¹²⁵I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that ¹²⁵I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.     

N.m.r. and c.d. studies of the DNA fragments d(TATATATA) and d(TATATA) in solution

DNA fragments d(TATATATA) and d(TATATA) were studied in low-salt aqueous solutions and found to coexist in more than one conformer. 1H-n.m.r. demonstrates that single-stranded and double-stranded states are involved in the conformational coexistence. Circular dichroism spectroscopy indicates a global B-DNA stacking of bases in the fragments. 31P-n.m.r. resonances of the TpA and ApT phosphodiester bonds are substantially separated in the spectra of both d(TATATATA) and d(TATATA) duplexes to suggest an alternating architecture of their backbones. In fact, the oligonucleotide duplexes are much more alternating than the corresponding polynucleotide under the same solution conditions. The alternating character of the d(TATATATA) double helix is further enhanced in molar caesium fluoride solutions. The oligonucleotide isomerization into X-DNA is, however, accompanied by gel formation, which makes high resolution n.m.r. measurements impossible.     

Synapsis, strand scission, and strand exchange induced by the FLP recombinase: analysis with half-FRT sites.

We have used a previously described cross-linking assay and half-FRT site substrates to examine the requirements for synapsis, strand exchange, and strand scission. The cross-linking assay showed that the minimum functional FRT site needed for synapsis contains two inverted FLP-binding elements surrounding an 8-bp core. This indicates that four FLP molecules interact with four binding elements in a synaptic complex. The analysis using half-sites showed that the enzyme can catalyze efficient strand exchange between a half-site and the intact FRT site. The reaction occurred only if the half-site had at least 2 bp but no more than 4 bp of the adjoining core sequence. The exchange occurred exclusively at the regions of limited core homology between the respective half-site and the FRT site. The absence of strand exchange between an intact site and a half-site bearing regions of core nonhomology indicates that 1 bp of homology is not sufficient for the formation of stable recombinant structures. Qian et al. (X.-H. Qian, R. B. Inman, and M. M. Cox, J. Biol. Chem. 265:21779-21788, 1990) have recently shown that the FLP protein can catalyze the formation of dimeric, trimeric, and tetrameric complexes with half-FRT sites. We show that only half-sites that contained at least 2 bp of adjacent core could form stable dimer products and be cleaved by the enzyme. Stable dimers were formed between a noncleavable half-site and a cleavable half-site, suggesting that only a single cleavage event is needed for the formation of the dimer.     

Catalytic site-specific cleavage of a DNA-target by an oligonucleotide carrying bleomycin A5.

Oligonucleotide reagents have been created which are capable of catalytic site-specific cleavage of DNA-targets. The oligonucleotide reagent Blm-R-pd(CCAAACA) bearing the bleomycin A5 (Blm-RH) residue was used to degrade the DNA-target pd(TGTTTGGCGAAGGA). It has been shown that at equimolar reagent: target concentration the bleomycin oligonucleotide derivative can repeatedly cleave the target at G9, G7, T5, T4 and T3 in site-specific manner. This paper demonstrates that with a 10-fold excess of the DNA-target relative to the reagent 30% degradation of the target was observed primarily at a single position G7. The paper also shows that one reagent molecule containing bleomycin A5 residue was capable to degrade three molecules of the DNA-target. The catalytic activity of Blm-R-pd(CCAAACA) was the highest in the temperature range close to the melting temperature of the reagent-target complex, that is under conditions where the oligonucleotide reagent can form a complementary complex and easily dissociate to interact with the next molecule of the target. The number of target molecules degraded by the bleomycin reagent is limited by the degradation of the antibiotic residue itself.     

Packing features of the all-AT oligonucleotide d(AAATTT)

We describe the packing features of the oligonucleotide duplex d(AAATTT)2, as determined by X-ray diffraction. There is little information on sequences that only contain A and T bases. The present structure confirms that these sequences tend to pack as a helical arrangement of stacked oligonucleotides in a B conformation with Watson-Crick hydrogen bonding. Our results demonstrate that the virtual TA base step between stacked duplexes has a negative twist that improves base stacking. This observation is consistent with the low stability of TA base steps in B-form DNA.     

Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission

The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.     

C-1' hydrogen abstraction of deoxyribose in DNA strand scission by dynemicin A.

Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, abstracts the C-1' hydrogen of DNA deoxyribose and then the damaged DNA leads to strand breaks with the formation of 5'- and 3'-phosphate termini. The lesions of C-4' hydrogen also occur at 3' side of G.C base pairs (i. e., 5'-CT and 5'-GA), leading to 5'-phosphate and 3'-phosphoglycolate termini or 4'-hydroxylated abasic sites. The C-1' hydrogen abstraction by dynemicin A is distinct from the preferential C-5' hydrogen abstraction of calicheamicin and neocarzinostatin.     

Interaction of Lambda- and Delta-[Ru(bpy)2(pbmz)](PF6)2 with the oligonucleotide duplex d(CGCGAATTCGCG)2

The interaction of the enantiomeric complexes Lambda- and Delta-[Ru(bpy)(2)(pbmz)](PF(6))(2) (bpy=2,2'-bipyridine, pbmz=2-(2'-pyridyl)benzimidazole) with the DNA duplex d(CGCGAATTCGCG)(2) was investigated by means of 2D NMR techniques. The synthesis of the enantiomers was based on the optically pure complexes Lambda- and Delta-[Ru(bpy)(2)(py)(2)](2+) and were characterized by CD and NMR spectroscopy. NMR data indicate that both enantiomers bind weakly to the oligonucleotide, approaching from the minor groove at the centre of the helix. The perturbation of the B-DNA conformation is minor with an apparent absence of enantioselectivity. Molecular modelling calculations in conjunction with the NOE data support the suggestion that more than one binding modes are present. The imidazole amine group of the pbmz ligand is probably hydrogen bonded to the DNA phosphodiesteric backbone at the AATT step, and this may provide an explanation for the diminished enantioselectivity observed.     

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex.

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.