Enhanced DNA damage induced by secondary electron emission from a tantalum surface exposed to soft x rays

Both thick and thin films of pGEMR-3Zf- plasmid DNA deposited on a tantalum foil were exposed to soft X rays (effective energy of 14.8 keV) for various times in air under a relative humidity of 45% (Gamma approximately 6, where Gamma is the number of water molecules per nucleotide) and 84% (Gamma approximately 21), respectively. For a thick film, the DNA damage was induced chiefly by X-ray photons. For a thin film of DNA, X-ray-induced secondary electrons emitted from the tantalum result in a substantial increase in DNA damages. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis and laser scanning. The exposure curves for the formation of nicked circular (single-strand break, SSB), linear (double-strand break, DSB), and interduplex crosslink forms 1 and 2 were obtained for both thick and thin films of DNA. The secondary electron enhancement factor for SSBs, DSBs and crosslinks of the thin film of DNA were derived to be 3.8 +/- 0.5, 2.9 +/- 0.7 and 7 +/- 3 at Gamma approximately 6 and 6.0 +/- 0.8, 7 +/- 1 and 3.9 +/- 0.9 at Gamma approximately 21, respectively. This study provides a molecular basis for understanding the enhanced biological effects at interfaces during diagnostic X-ray examination and radiotherapy.     

Auger electron-induced double-strand breaks depend on DNA topology

From a structural perspective, the factors controlling and the mechanisms underlying the toxic effects of ionizing radiation remain elusive. We have studied the consequences of superhelical/torsional stress on the magnitude and mechanism of DSBs induced by low-energy, short-range, high-LET Auger electrons emitted by (125)I, targeted to plasmid DNA by m-[(125)I]iodo-p-ethoxyHoechst 33342 ((125)IEH). DSB yields per (125)I decay for torsionally relaxed nicked (relaxed circular) and linear DNA (1.74+/-0.11 and 1.62+/-0.07, respectively) are approximately threefold higher than that for torsionally strained supercoiled DNA (0.52+/-0.02), despite the same affinity of all forms for (125)IEH. In the presence of DMSO, the DSB yield for the supercoiled form remains unchanged, whereas that for nicked and linear forms decreases to 1.05+/-0.07 and 0.76+/-0.03 per (125)I decay, respectively. DSBs in supercoiled DNA therefore result exclusively from direct mechanisms, and those in nicked and linear DNA, additionally, from hydroxyl radical-mediated indirect effects. Iodine-125 decays produce hydroxyl radicals along the tracks of Auger electrons in small isolated pockets around the decay site. We propose that relaxation of superhelical stress after radical attack could move a single-strand break lesion away from these pockets, thereby preventing further breaks in the complementary strand that could lead to DSBs.     

Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.     

Synthesis and DNA cleavage studies of novel quinoline oxime esters

New 2-chloro-3-formyl quinoline oxime esters were synthesized by the reaction of 2-chloro-3-formyl quinoline oximes with various benzoyl chlorides in the presence of triethyl amine and dichloromethane at 0°C. The DNA photo cleavage studies of some new oxime esters were investigated by neutral agarose gel electrophoresis at different concentrations (40μM and 80μM). Analysis of the cleavage products in agarose gel indicated that few of quinoline oxime esters (3d-i) converted into supercoiled pUC19 plasmid DNA to its nicked or linear form.     

Raman spectroscopy of supercoiled and nicked ColE1 plasmid

Native supercoiled and nicked ColE1 DNA were examined using laser Raman spectroscopy. ColE1 contains 6646 base pairs (bp) and, when supercoiled, approximately 47 negative supercoils. An analytical buoyant density gradient centrifugation technique developed by Burke and Bauer was scaled to preparative quantities, and used to isolate the supercoiled plasmid fraction from its nicked counterpart. This procedure allowed enriched fractions of the supercoiled plasmid to be extracted without the use of the optical contaminant ethidium bromide. The intensities of several Raman bands were altered between the spectra of the two topological forms. Notably absent were any changes in bands arising from cytosine and guanine vibrations. The observed changes are interpreted in terms of the polymorphic structures which have been observed in many DNA structural studies. The results of this study suggest that accommodation of supercoiling takes place chiefly in A-T base pairs and backbone moieties, without substantial modification of G-C base-pair structure. Premelting effects may account for the observed changes, including a slight shift to lower frequency of a band known to be responsive to base-pair disruption. Heteronomous ribose sugar pucker is evident in both supercoiled and nicked plasmid species. No gross conformational transitions were detected for native supercoiled DNA, and consequently, subtle rearrangements appear sufficient to absorb the supercoiling deformations.     

Densitometric quantification of DNA topoisomers in ethidium bromide-stained agarose gels and chemiluminescence-detected X ray films

This study investigates the contribution of deformational strain imposed by topological interconversions of DNA in ethidium bromide-binding on agarose gels. Closed-circular plasmid DNAs were nicked using UV exposure and the DNA bands were quantified by densitometry. The results show that the closed circular DNA binds the same amount of the dye as its nicked counterpart. The relationship between the band intensity on X-ray films of chemiluminescence-detected Southern blots and DNA concentration was shown to be linear.     

Method to eliminate linear DNA from mixture containing nicked circular, supercoiled, and linear plasmid DNA

Preparations of circular plasmid DNA in either supercoiled or nicked circular form often are contaminated with undesired linear DNA fragments arising from shearing/degradation of chromosomal DNA or linearization of plasmid DNA itself. We report a simple enzymatic method, using a combination of λ exonuclease and RecJ_f, for the selective removal of linear DNA from such mixtures. λ exonuclease digests one strand of linear duplex DNA in the 5′ to 3′ direction, whereas RecJ_f, a single-strand-specific exonuclease, digests the remaining complementary single strand into mononucleotides. This combination of exonucleases can remove linear DNA from a mixture of linear and supercoiled DNA, leaving the supercoiled form intact. Furthermore, the inability of λ exonuclease to initiate digestion at nicks or gaps enables the removal of undesired linear DNA when nicked circular DNA has been enzymatically prepared from supercoiled DNA. This method can be useful in the preparation of homogeneous circular plasmid DNA required for therapeutic applications and biophysical studies.     

The effect of superhelical density on the yield of single-strand breaks in gamma-irradiated plasmid DNA.

Using agarose gel electrophoresis, the formation of DNA single-strand breaks (SSBs) by 137Cs gamma irradiation was quantified in negatively supercoiled topological isomers of plasmid pUC18. The G value for SSB formation falls slightly from 1 x 10(8) to 8 x 10(-9) SSB Gy-1 Da-1 as the superhelical density varies from 0.00 to -0.08. This result is not in agreement with recent observations by others which suggest that increasing the negative superhelical density of plasmid DNA increases its sensitivity to X irradiation.     

Electrophoretic mobility of supercoiled,catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.     

Crosslinking of the complementary strands of DNA by UV light: dependence on the oligonucleotide composition of the UV irradiated DNA

UV light crosslinks the complementary strands of DNA. The interstrand crosslinks may contribute to the biological and pathological effects that UV irradiation is known to bring about. Here alkaline agarose gel electrophoresis was used to assess the crosslinked fraction of 31 selected restriction fragments of six viral and plasmid DNA molecules exposed to UVC light irradiation. As many as 17 independent experiments were performed with the particular DNA fragments to get sufficiently precise data suitable for quantitative analyses. The data were used to determine how the crosslinked fraction depended on the dinucleotide, trinucleotide and tetranucleotide contents of the irradiated DNA fragments. This analysis demonstrated that DNA conformation and/or flexibility, rather than the local double helix thermostability, governed the phenomenon of crosslinking. For example, (GA).(TC) suppressed the crosslink formation in DNA more than any dinucleotide composed of only G and C. In addition, (CTAG).(CTAG) promoted crosslinking much more than any other tetranucleotide, including e.g. (TATA).(TATA), whereas the closely related (CATG).(CATG) belonged among the tetranucleotides that most suppressed the UV light induced crosslinks between the complementary strands of DNA. The present data reproduced crosslinking of the analyzed 31 restriction fragments with a correlation coefficient exceeding 0.90. This result will be useful to predict crosslinking along the whole human genome.     

DNA cleavage by homo- and heterotetranuclear Cu(II) and Mn(II) complexes with tetrathioether-tetrathiol moiety

Novel homotetranuclear Cu(II) and heteronuclear Cu(II)-Mn(II) complexes with tetrathioether-tetrathiol moiety have been prepared and their DNA relaxation activities with plasmid pCYTEXP (5kb) were electrophoretically established. The cleavage products analyzed by neutral agarose gel electrophoresis indicated that the interaction of the metal complexes with supercoiled plasmid DNA yielded linear, nicked or degraded DNA. The relaxation activities of both homo- and heterotetranuclear (SK4) complexes are time- and concentration-dependent. The findings suggest that SK4 with potent nucleolytic activity is a good nuclease substitute in the presence of cooxidant. Furthermore, the observation of induction of DNA into smaller fragments by SK4 is also significant.     

Effect of 2-chloroethylnitrosoureas on plasmid DNA including formation of strand breaks and interstrand cross-links

Plasmid [3H]pBR 322 was incubated with various alkylating agents including chlorozotocin, N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), N-ethyl-N-nitrosourea (Enu) and dimethylsulfate (DMS). Formation of DNA strand breaks was followed by separation of the various forms of DNA on agarose gels and liquid scintillation counting of the bands. All alkylating agents examined were capable of rapidly producing strand breaks in time and concentration dependent fashion. Bands migrating as relaxed circular and supercoiled forms of the plasmid disappeared, and extensive alkylation resulted in formation of a band that migrated faster than the linear form of DNA. Electron microscopy of this band showed that it consisted of relaxed circles. Prolonged storage of alkylated plasmid resulted in fragmentation of the DNA, possibly due to strand scission at apurinic sites. A new neutral denaturation technique was developed, which allowed for the detection of DNA interstrand cross-links with minimal effects on other potentially labile sites of the alkylated DNA. The level of alkylation was quantitated by incubating [3H]pBR 322 with [2-chloroethyl-U-14C]chlorozotocin and was shown to be independent of DNA concentration but have a linear relationship with drug concentration. Linear and relaxed circular forms of the plasmid were alkylated to a somewhat higher extent than supercoiled DNA. Alkylation of pBR 322 with defined superhelical densities showed no preferential loss in DNA with a specific superhelical density, indicating that alkylation-induced unwinding is independent of superhelicity under the experimental conditions used.     

Analysis of laser-induced plasmid DNA photolysis by capillary electrophoresis

Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 μmol/l ethidium bromide (EB) and then exposed to 110 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.     

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.     

Circular intermediates with missing nucleotides in the conversion of supercoiled or nicked circular to linear duplex DNA catalyzed by two species of BAL 31 nuclease

The extracellular nucleases from Alteromonas espejiana BAL 31 can catalyze the endonucleolytic and/or exonucleolytic hydrolysis of duplex DNA in response to a variety of alterations, either covalent or noncovalent, in DNA structure. The nuclease can exist as at least two kinetically and molecularly distinct protein species. The two species that have been studied, called the 'fast' (F) and 'slow' (S) nucleases, both readily convert negatively supercoiled DNAs to linear duplex molecules and accomplish this conversion through the formation of a circular duplex intermediate containing usually a single interruption in one strand. It is further shown that most of these intermediates contain gaps arising from the removal in a processive manner of one or more nucleotide residues after the introduction of the initial strand break (nick). Considering only the intermediates with gaps, the average number of missing residues is 6.3 +/- 0.5 and 2.8 +/- 0.3, respectively, for DNA acted upon by the F and S enzymes independently of the extent of conversion of supercoiled DNA. The nicks and gaps are bounded by 3'-hydroxyl and 5'-phosphoryl termini. When singly nicked circular DNA is used as the substrate, conversion to the linear duplex form occurs predominantly through a gapped circular intermediate with the same average numbers, within experimental error, of missing nucleotides for the respective nuclease species as found when supercoiled DNA is the substrate. The conversion to linear duplex DNA is much slower when nicked circular DNA is the substrate compared to that found when supercoiled DNA is the starting material.     

Tris对高LET碳重离子辐照下质粒DNA链断裂的保护作用

利用100 keV/μm碳离子束(初始能量为290 MeV/u)照射溶解于纯水、10 mmol/L Tris、1 mmol/L EDTA及TE 缓冲液中的pUC19质粒DNA.通过琼脂糖凝胶电泳技术分析了不同溶液中各种形态DNA分子所占份额,并计算得到不同剂量下平均每个质粒分子中单链断裂(SSB)及双链断裂(DSB)的数目.发现Tris通过抑制SSB和DSB的产生对碳重离子辐照下的质粒DNA有明显的保护作用,而EDTA能够加剧SSB的产生而抑制DSB的形成.     

An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of Celosia cristata

An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled pBlueScript SK+ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower protein concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA.     

Hydrolysis of DNA by a Dipeptides Containing Histidine

Three ligands which contain histidine and conjugated by a flexible linker, have been characterized and evaluated as DNA cleavage agents. The cleavage activity of metal complexes were evaluated by monitoring the conversion of supercoiled plasmid DNA (pUC19) (Form I) to nicked circular DNA (Form II) by agarose gel electrophoresis. The results showed that the cleavage activity of Cu(II) complexes was enhanced compared with histidine. Specially, at a high reaction concentration (0.2 mM), Cu(II) complexes can cleave the plasmid DNA with some selectivity.     

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.