Radiosensitization of DNA by gold nanoparticles irradiated with high-energy electrons

Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19-27 (2008). Thin films of pGEM-3Zf(-) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.     

Involvement of Coprinus endonuclease in preparing substrate for in vitro recombination

A functional recombination assay involving the tetracycline mutant plasmids, pUW1 and pUW4, was used to assess (i) the nature of the DNA substrates needed and (ii) the involvement of Coprinus endonuclease in preparing substrate, for the RecA-directed recombination process. A gapped circular plasmid and a linear or a nicked circular plasmid are efficient substrate combinations in this system to achieve a 160-fold increase in the in vitro recombination frequency over the control levels. The Coprinus endonuclease obtained from early meiotic prophase can produce such substrates. The recombination frequency obtained with the combination of gapped pUW1 plasmids initially relaxed by the Coprinus endonuclease and linear pUW4 plasmids produced by the site-specific BamHI digest is 10-fold lower than that obtained when both substrates are digested by BamHI. The results suggest that the Coprinus endonuclease creates random nicks on plasmid DNA. Glyoxal gel electrophoretic analysis was used to confirm this random nicking activity of Coprinus endonuclease.     

Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5′-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.     

Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.     

Endonuclease activity with acid and alkaline pH values in rat blood serum during whole body gamma-irradiation

Using the method of electrophoresis in agar gel of superhelical plasmid DNA, which served as a substrate for determining endonuclease activity, the authors showed that the same radiation dose influenced in a different way the activity of acid and alkaline DNAases in rat blood serum. At early times (3-6 h) following irradiation, the activity of acid nucleases increased whereas that of alkaline DNAases decreased.     

Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity

Mammalian base excision repair (BER) is mediated through at least two subpathways designated ‘single-nucleotide’ (SN) and ‘long-patch’ (LP) BER (2-nucleotides long/more repair patch). Two forms of DNA substrate are generally used for in vitro BER assays: oligonucleotide- and plasmid-based. For plasmid-based BER assays, the availability of large quantities of substrate DNA with a specific lesion remains the limiting factor. Using sequence-specific endonucleases that cleave only one strand of DNA on a double-stranded DNA substrate, we prepared large quantities of plasmid DNA with a specific lesion. We compared the kinetic features of BER using plasmid and oligonucleotide substrates containing the same lesion and strategic restriction sites around the lesion. The K_m for plasmid DNA substrate was slightly higher than that for the oligonucleotide substrate, while the V_max of BER product formation for the plasmid and oligonucleotide substrates was similar. The catalytic efficiency of BER with the oligonucleotide substrate was slightly higher than that with the plasmid substrate. We conclude that there were no significant differences in the catalytic efficiency of in vitro BER measured with plasmid and oligonucleotide substrates. Analysis of the ratio of SN BER to LP BER was addressed using cellular extracts and a novel plasmid substrate.     

Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity

We designed a novel type of cationic lipid, lipids with a cationic polar group in the polyamidoamine dendron, because these dendron-bearing lipids are expected to form complexes with plasmid DNA and achieve efficient transfection of cells by synergy of endosome buffering and membrane fusion with the endosome, both of which are useful for the promotion of the transfer of plasmid DNA from endosome to cytosol. Four kinds of lipids with polyamidoamine dendrons of first to fourth generations, DL-G1, DL-G2, DL-G3, and DL-G4, were synthesized. The lipid with a dendron of a higher generation exhibited greater ability to form lipoplexes with plasmid DNA, as estimated by agarose gel electrophoresis. While the DL-G1 lipoplex did not transfect CV1 cells, the lipoplexes containing the DL-G2, DL-G3, or DL-G4 could induce transfection of the cells, and their activity was elevated with increasing generation of the dendron. Addition of dioleoylphosphatidylethanolamine (DOPE), which is known to increase fusion ability of a lipid membrane, into the lipoplexes greatly enhanced their transfection activity. In addition, the comparison with DC-Chol-containing lipoplex, which is widely used as a nonviral vector, showed that the DL-G3-DOPE lipoplex exhibits more efficient transfections. These findings imply that these dendron-bearing lipids may form the basis for a novel family of cationic lipids for efficient gene delivery.     

Phasmids. Properties and the use in genetic engineering. II. Packing in vitro. Lysogeny of Escherichia coli K-12

Circular monomeric lambda DNA molecules were used as a substrate for packaging reaction in vitro. For obtaining lambda DNA in circular monomeric form only, Escherichia coli recA plasmid bearing cells were used. This hybrid DNA molecule which we designated phasmid lambda pMYF11, is the pBR322 plasmid in which lambda 47.1 DNA was introduced in vitro. The phasmid can exist in the plasmid form or as a non-defective phage. The efficiency of packaging reaction in vitro proved to be similar for monomeric circular and linear form of phasmid DNA molecules. The cI- variant of the phasmid is not able to exist as a plasmid even in the cells containing homoimmune prophage. Still, cI+ phasmid variants capable of lysogenizing arise with low frequency, as a result of recombination between the resident cI+ prophage and infecting cI- phasmid.     

Quantification of the effect of substrate concentration on the conjugal transfer rate of the TOL plasmid in short-term batch mating experiments

Batch mating experiments with Pseudomonas putida PAW 1 (TOL) as a donor and Pseudomonas aeruginosa PAO 1162 as a recipient strain were performed to quantify the effect of the substrate concentration in the mating medium on the observed plasmid transfer rate coefficient. The impact of the substrate concentration in the mating medium was highly correlated with the growth history of the donor strain. When the donor strain was harvested in exponential growth phase, no impact was observed; when the donor strain was taken from the stationary phase, however, a strong impact of the substrate concentration was measured: a 10-fold reduction in the substrate concentration decreased the observed plasmid transfer rate by 55%.     

Quaternary complexes composed of plasmid DNA/protamine/fish sperm DNA/stearic acid grafted chitosan oligosaccharide micelles for gene delivery

Quaternary complexes with condensed core of plasmid DNA, protamine, fish sperm DNA and shell of stearic acid grafted chitosan oligosaccharide (CSO-SA), were prepared. The CSO-SA could self-assemble to form nano-sized micelles in aqueous solution and demonstrated excellent internalization ability of tumor cells. Dynamic light scattering (DLS) measurement and transmission electrostatic microscope (TEM) images showed that quaternary complexes had spherical shape with about 25 nm number average diameter, and the size of quaternary complexes was smaller than that of CSO-SA micelles and CSO-SA micelles/plasmid DNA binary complexes. The transfection efficiencies of quaternary complexes on HEK293 and MCF-7 cells increased with incubation time, and were significantly higher than that of CSO-SA micelles/plasmid DNA binary complexes. The optimal transfection efficiency of quaternary complexes on HEK293 and MCF-7 cells measured by flow cytometer after 96 h was 23.82% and 41.43%, respectively. Whereas, the transfection efficiency of Lipofectamine? 2000 on HEK293 and MCF-7 cells after 96 h was 32.45% and 33.23%, respectively. The data of luciferease activity measurement showed that the optimal ratio of plasmid DNA:fish sperm DNA:protamine:CSO-SA was 1:1:5:5. The results indicated that the present quaternary complexes were potential non-viral gene delivery system.     

Identification of a potent decatenating enzyme from Escherichia coli

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.     

Partition of unit-copy miniplasmids to daughter cells. II. The partition region of miniplasmid P1 encodes an essential protein and a centromere-like site at which it acts

The stable maintenance of the unit-copy lambda-P1:5R miniplasmid is dependent on adjacent but separable replication (rep) and partition (par) regions of DNA derived from its P1 plasmid parent. The par region consists of an approximately 2.5 X 10(3) base-pair (kb) segment of DNA of which the terminal kb contains the plasmid incompatibility determinant incB. Two of the 14 lambda-P1:5R partition-defective point mutants isolated are amber (nonsense) mutants, showing that a plasmid-encoded protein is essential for proper partition. All of the Par- point mutants are complemented by the wild-type par region in trans. The complementing activity was shown to be an Mr 44,000 protein encoded by the end of the par region distal to incB. Deletion analysis showed that the incB sequence is essential in cis to the plasmid in order that the plasmid be receptive to the par protein. Thus incB appears to be the target site for par protein activity. We propose that the protein binds to incB, forming a complex that is recognized as a substrate for the cellular partition apparatus. The ability of a cloned incB sequence to compete for the par protein or for the cellular partition apparatus accounts for its activity as an incompatibility determinant. The existence of a plasmid-encoded par protein suggests a specific model for equipartition.     

Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus

Intramuscular immunization with a naked DNA plasmid expressing the Plasmodium yoelii circumsporozoite protein (pPyCSP) protects mice against challenge with P. yoelii sporozoites. This protection can be improved either by coadministration of a plasmid expressing murine GM-CSF (pGMCSF) or by boosting with recombinant poxvirus expressing the PyCSP. We now report that combining these two strategies, by first mixing the priming dose of pPyCSP with pGMCSF and then boosting with recombinant virus, can substantially increase vaccine effectiveness. Not only were immune responses and protection improved but the pPyCSP dose could be lowered from 100 microg to 1 microg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-microg doses of pPyCSP plus 1 microg pGMCSF with mice primed by 1-microg doses of pPyCSP alone, the former were better protected (60% vs 0) and had higher concentrations of Abs (titers of 163, 840 vs 5, 120 by indirect fluorescent Ab test against sporozoites), more ex vivo CTL activity (25% vs 7% specific lysis), and more IFN-gamma-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-gamma spot-forming cells/106 cells). Priming with plasmid vaccine plus pGMCSF and boosting with recombinant poxviruses strongly improves the immunogenicity and protective efficacy of DNA vaccination and allows for significant reduction of dose.     

Plasmid RK2 ParB Protein: Purification and Nuclease Properties

The parCBA operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown. In this study ParB was overexpressed by cotranslation with ParC in Escherichia coli by using a plasmid construct that contained the parC and parB genes under the control of the T7 promoter. Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography. Sizing-column analysis indicated that ParB exists as a monomer in solution. Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures. ParB also exhibits 5'-->3' exonuclease activity. This ParB activity on a 5'-end-labeled, double-stranded DNA substrate produces a 3', 5'-phosphorylated dinucleotide which is further cleaved to a 3', 5'-phosphorylated mononucleotide. The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined.     

Induced mutagenesis of plasmid as well as chromosomal genes inserted into plasmid DNA. II. The mutagenic action of chemical factors

To proceed the works on induced mutagenesis in plasmids, mutagenic effects of chemicals on the DNA of RSF2124 plasmid mediating colicine E1 biosynthesis and resistance to ampicillin, were studied. After exposure to mutagens, plasmid DNA was used to transform Escherichia coli C600 rk-mk-cells. The lethal effect was estimated from inactivation of the ampicillin marker, the mutagenic effect being measured by the appearance of mutants unable to synthesize colicine (Col-). The reaction of the plasmid DNA with a mutagen was stopped by 10-fold dilutions of aliquots in TEN buffer, followed by dialysis in 10 mH CaCl2 for 24 h. To select the most efficient mutagens for plasmid DNA, the compounds were predominantly tested which are known to be effective in other systems (transforming and transfecting DNA, microbial viruses). An a result, all chemicals tested by their activity were classified into 4 groups: inducing more than 100 fold increase (hydroxylamine, O-methylhydroxylamine); inducing 10 fold increase (UV-irradiation, lambda = 254 nm; W-mutagenesis, gamma-irradiation, nitrous acid, mitomycin C); inducing less than 10fold increase (indirect UV-mutagenesis, nitrous acid, beta-chloroethyldiethylamine hydrochloride, nitrosoguanidine); no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, O-beta-diethylhydroxylamine).     

Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. InSaccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats TG_1–3 of 300–350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG_1–3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids. Published: February 17, 2003     

Developmental-stage-specific plasmid supercoiling in Chlamydia trachomatis

Chlamydia trachomatis elementary body (EB) and reticulate body (RB) developmental stages have polymorphic plasmid DNA. Several plasmid forms separated by gel electrophoresis were identified as topoisomers by treatment with topoisomerase I. Among these topoisomers was one form unique to EBs and one form unique to RBs. The unique EB plasmid topoisomer was characterized as highly supercoiled, on the basis of band migrations by gel electrophoresis and its appearance by electron microscopy. The unusual physical state of this topoisomer was probably mediated, in part, by DNA-specific structural proteins. The unique RB plasmid topoisomer was a supercoiled form of lower superhelical density than the other identified topoisomers. Developmental-stage-specific differences in super-helical density of plasmid DNA suggest cause-and-effect relationships between DNA topology and metabolic activity in RBs and metabolic quiescence in EBs.     

Fe (Ii)-Chelates Based on Redox-Active Pyridoxal-Betaines as C-Centered Radicals Causing Single- and Double-Strand Scissions to Dna

The ability of 1-[N-Ethoxycarbonylmethylpridoxylidenium]-2-[2-pyridyljhydrazine bromide code name-[L2-9 = L⁺, X^-]-FE(II) chelate [L2-9-Fe(II)] to induce breaks both in the 43kb linear double-strandphage DNA, and in the 4363 base pair supercoiled pBR322 plasmid DNA is herein described. Neither the free ligand nor FE(II) alone demonstrated any effect on the DNA. The cleaving ability is shown to occur instantaneously under strictly anaerobic conditions, either in the presence or absence of the enzyme catalase. It is also shown to be dose dependent. Thus, atDNA: L2-9-Fe(II) molar ratio of 3.7: 1.0, the linear DNA is randomly cleaved into fragments ranging from 23. Ikb to 4.3kb, whereas at approximately 1:1 molar ratio, the range extends down to 2.5kb fragments. By contrast, at 1:2.7 [plasmid DNA]: chelate-Fe(II). molar ratio, a single-strand nick was observed, and a double strand break was noted at a 1:50 ratio ([plasmid DNA]: chelate-Fe(II). A multi-stage redox cycling involving a carbon-centered (L, X^-)-Fe(III) radical capable of transferring an electron to the DNA to form high unstable [DNA]^- anion-radical is invoked to explain the degradation of the chain macromolecule. Possible modes for regeneration of the chelate-Fe(III) radical both at the cell-free and at the cell levels are proposed.     

Unpaired bases in the B--Z junction of the supercoiled plasmid

The restriction analysis has been used to establish that O-beta-diethylaminoethylhydroxylamine (OHA) produces modification of unpaired cytidines in the polylinker region adjacent to the Z-insert (dG-dC)10. (dG-dC)10 in the negatively supercoiled plasmid pGC20. The length of the transition region between B- and Z-portions of DNA is not less than 36 bps. The reaction of OHA with the unpaired cytidines in the B-Z junction is a fixing one and produces no secondary despiralling of the neighboring regions. The reaction with DNA proceeds much slower than the one with monomers and single-strand polynucleotides. The structural nonuniformity has been observed, which is manifested in the alternating B and "non-B" form DNA in the B-Z junction. It is suggested that these junctions may contain nucleotide sequences which are stable to violation of the B structure during the change in superhelical density of DNA.     

Induced mutagenesis of plasmid and chromosomal genes inserted into plasmid DNA. I. The mutagenic action of radiation

This paper describes the results of treating plasmid DNA in vitro with mutagens, to obtain mutations both in plasmid genes and chromosomal genes comprised within the plasmid, thus avoiding disorganization characteristic of in vivo mutagenesis. The model system is represented by DNA of RSF2124 responsible for colicine E1 synthesis and resistance to ampicillin. Col- mutants were looked for after exposure to UV- and gamma-irradiation. The lethal effect was estimated as inactivation of the ampicillin resistance marker. After reisolation from mutant transformant of the plasmid DNA, the novel character and resistance to ampicillin proved to retain in the course of subsequent transformations and passages of transformed colonies, suggesting the mutational nature of the changes. Exposure of RSF2124 to short-wave UV-irradiation (lambda = 254 nm) produced a pronounced mutagenic effect: the relative quantity of Col- mutants under optimal conditions of mutagenesis increased about 10 times. In the case of W-reactivation (additional UV-irradiation of C600 wild type cells) of lethal lesions, a 95% reliable increase in mutagenic effect was observed. Significant enhancement of mutagenesis (about 4-fold) was detected when only recipient cells were exposed to low doses of UV (the so-called indirect UV mutagenesis). Thus, with regard to W- and indirect UV mutagenesis, the plasmid DNA behaves like DNA of temperate phages which suggests their evolutionary relationship. Treatment of plasmid DNA with acridine orange prior to UV, only protected from lethal lesions. Gamma-irradiation (60Co) at the dose producing 100-fold inactivation, increased the yield of Col- mutants by one order of magnitude. The presence of RSF2124 plasmid in a cell does not affect its UV sensitivity.