Monte Carlo simulation of strand-break induction on plasmid DNA in aqueous solution by monoenergetic electrons

The radiation-induced process of strand breaks on pBR322 plasmid DNA in aqueous solution for different energy electrons was studied by Monte Carlo simulation. Assumptions of induction mechanisms of single- and double-strand breaks (SSBs and DSBs) used in the simulation are that SSB is induced by OH or H reaction with DNA and that DSB is induced by two SSBs on the opposite strands within 10 bp. Dose-response relationships of SSBs and DSBs were demonstrated for monoenergetic electrons of 100 eV, 10 keV, 1 keV and 1 MeV, and the yields of SSB and DSB were calculated. The dose-response relationships of SSBs and DSBs can be fitted by linear and linear-quadratic functions, respectively. The ratio of quadratic to linear components of DSB induction changes due to the electron energy. A high contribution of the linear component is observed for 1 keV electrons in the dose range below 160 Gy. The yields of SSBs and DSBs for all examined electron energies lie well within the experimental data when the probability of strand-break induction by OH and H is assumed to be around 0.1-0.2. The yield of SSBs has a minimum at 1 keV, while the yield of DSBs has a maximum at 1 keV in the examined energies. The strand breaks are formed most densely for 1 keV electrons.     

Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.     

Cooperativity in nucleosomes assembly on supercoiled pBR322 DNA.

Many studies have shown that in reconstituted chromatin model systems, containing only purified DNA and histone octamer, nucleosomes can adopt well defined locations with respect to DNA nucleotide sequence. Recently, nucleosome-nucleosome interactions were suggested as one of the factors underlying preferential nucleosomes positioning. In the present paper this aspect has been studied by topological analysis and electron microscopy visualization of minichromosomes reconstituted at different histone/DNA ratios. Both methods suggest that cooperativity plays a role in nucleosomes formation. A linear cooperative model in which nucleosomes are formed on discrete sites with cooperative interactions occurring only between nearest neighbours allows to calculate the cooperative constant. The reported results show that basic interactions, which are of relevance in the process of chromatin folding, are present also in very simple model system.     

Monte Carlo implementation of supercoiled double-stranded DNA

Metropolis Monte Carlo simulation is used to investigate the elasticity of torsionally stressed double-stranded DNA, in which twist and supercoiling are incorporated as a natural result of base-stacking interaction and backbone bending constrained by hydrogen bonds formed between DNA complementary nucleotide bases. Three evident regimes are found in extension versus torsion and force versus extension plots: a low-force regime in which over- and underwound molecules behave similarly under stretching; an intermediate-force regime in which chirality appears for negatively and positively supercoiled DNA and extension of underwound molecule is insensitive to the supercoiling degree of the polymer; and a large-force regime in which plectonemic DNA is fully converted to extended DNA and supercoiled DNA behaves quite like a torsionless molecule. The striking coincidence between theoretic calculations and recent experimental measurement of torsionally stretched DNA (Strick et al., Science. 271:1835, 1996; Biophys. J. 74:2016, 1998) strongly suggests that the interplay between base-stacking interaction and permanent hydrogen-bond constraint takes an important role in understanding the novel properties of elasticity of supercoiled DNA polymer.     

Nucleosome "phasing" and cruciform structures in circular supercoiled pBR322 DNA

Cruciform structures have been detected in pBR322 supercoiled DNA, both in its naked state and when complexed with histone octamer, using S1 endonuclease cleavage and EcoRI restriction. An inspection of the DNA sequence shows that the S1-hypersensitive sites are very near to AT-rich regions of pBR322 genome. A nucleosome "phasing" in these regions, as found on AT-rich regions of SV40 DNA (15), has been shown by restriction enzymes analysis. On the basis of these results it can be proposed that cruciform structures protrude on the nucleosome surface. This model explains the reason why these structures, which need high superhelical density, can exist in supercoiled DNA partially relaxed by nucleosome formation.     

Ozonolysis of supercoiled pBR322 DNA resulting in strand scission to open circular DNA.

Treatment of supercoiled pBR322 DNA with ozone resulted in the conversion of closed circular DNA to open circular DNA. Restriction analysis of the resulting open circular DNA showed that ozonolysis in the absence of salt caused single strand cleavage at specific sites.     

Efficient transformation of Serratia marcescens with pBR322 plasmid DNA

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.     

The site-specific deletion in plasmid pBR322

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII. The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis. Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule. THe deletion was not controlled by the recA system of the host bacteria.     

Characterization of hydroxyl free radical mediated damage to plasmid pBR322 DNA

We have investigated hydroxyl free radical mediated damage to pBR322 DNA produced by ascorbate/iron and oxygen in a phosphate-buffered in vitro system. An observed lag phase in DNA nicking suggests a multi-target model of hydroxyl free radical attack on DNA. In the present report we further examine the model system and show that there is a "heat labile" component of the ascorbate/iron system which can be completely restored by the readdition of ascorbate. These observations have allowed us to rule out the possibility that intermediates build up in the reaction and act independently of ascorbate to increase the reaction rate. We have investigated the initial rate of OH production with two OH trapping agents, salicylate and deoxyguanosine, and find that the lag in DNA nicking is not due to a corresponding lag in the production of OH as assessed by formation of the products, dihydroxybenzoic acids and 8-hydroxydeoxyguanosine, respectively. We have found that the energy of activation for DNA supercoiled nicking is 13.9 kcal/mole and for OH trapping by salicylate is 21.1 kcal/nmole. These two activation energies are sufficiently different to suggest that the rate-limiting steps of these two reactions are different. Investigation of the rate of oxygen consumption during the ascorbate/iron-mediated DNA damage showed that oxygen was not a limiting component at any point in the reaction. The addition of catalase slowed down oxygen consumption by 31% and this data taken together with our previous observations on the model implicate hydrogen peroxide as a key intermediate in DNA damage caused by hydroxyl free radical.     

Supercondensed structure of plasmid pBR322 DNA in an Escherichia coli DNA topoisomerase II mutant

An unusual structural component, supercondensed pBR322 DNA, has been found in plasmid pBR322 DNA samples isolated from a DNA topoisomerase II mutant of Escherichia coli, SD108 (topA+, gyrB225). The supercondensed pBR322 DNA moved faster than supercoiled pBR322 DNA as a homogeneous band in agrose gels when the DNA samples were analysed by electrophoresis. The mobility of the supercondensed DNA was not substantially affected by chloroquine intercalation. The supercondensed pBR322 DNA migrated as a high density "third DNA band" when the samples were subjected to caesium chloride/ethidium bromide gradient equilibrium centrifugation. The unusual pBR322 DNA visualized by electron microscopy was a globoid-shaped particle. These observations suggest that the pBR322 plasmid can assume a tertiary structure other than a supercoiled or relaxed structure. DNA topoisomerases may be involved in the supercondensation of plasmid DNA and chromosomal DNA.     

Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli

Summary Monomeric pBR322 DNA that had been linearized at its unique SalI site transformed wild-type Escherichia coli with 10² to 10³ times less efficiency than CCC plasmid DNA. Dose-response experiments indicated that a single linear plasmid molecule was sufficient to produce a transformant. Transformation with linearized pBR322 DNA was reduced 10 to 40 fold in recA ^–, recBC ^– or recF ^– backgrounds. In contrast, transformation with CCC DNA was unaffected by the rec status of the host. Transformation with linear pBR322 DNA was increased 3-fold in a DNA ligase-overproducing (lop11) mutant and decreased to a similar degree by transient inactivation of ligase in a ligts7 mutant.A proportion (ranging from about 9% in the wild-type to 42% in a recBC, lop11 mutant) of the transformants obtained with SalI-linearized pBR322 monomeric DNA contained deleted plasmids. Deletion rates were generally higher in rec ^– strains. Dephosphorylation of the termini on linear DNA or the creation of blunt-ended pBR322 molecules (by end-filling the SalI 5 protrusions or by cleavage with PvuII) decreased the transformation frequencywhilst increasing the deletion rate.Linear pBR322 dimeric DNA gave transformation frequencies in recA ⁺ and recA ^– strains that were reduced only 3 to 7 fold respectively relative to frequencies obtained with dimeric CCC DNA. Furthermore, in contrast to transformation with linear monomeric DNA, deletions were not observed.We propose that the majority of transformants arise, not by simple intracellular reannealing and ligation of the two cohesive SelI-termini of a linear molecule, but by intramolecular recombination. Deleted plasmids could be generated therefore during recyclization caused by recombination between short directly repeated sequences within a pBR322 monomer. We suggest that perfectly recircularized monomeric pBR322 molecules, which are found in the majority of transformants, arise primarily by intramolecular recombinational resolution of head-to-tail linear pBR322 dimers. Such linear oligomeric forms are created during preparation of linearized plasmid DNA by annealing of the SalI cohesive termini and constitute a variable proportion of the total molecules present.     

Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

Abstract We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli relA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yield per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli .     

Cloning of mitochondrial DNA from the petite negative yeast Schizosaccharomyces pombe in the bacterial plasmid pBR322

Summary The entire mitochondrial (mt) genome of the yeast Schizosaccharomyces pombe (S. pombe) was cloned in the BamHI site of the Escherichia coli plasmid pBR322. Three lines of evidence demonstrate that the complete mtDNA molecule was amplified without rearrangement or partial loss. First, restriction of the hybrid plasmid with BamHI led to the recovery of two fragments corresponding to the linearized plasmid and the BamHI-cut mtDNA. Second, restriction of cloned and native mtDNA with HindIII revealed identical fragments. Third, mitochondrial ribosomal RNA hybridized to the same HindIII fragments from cloned mtDNA and from mtDNA isolated from mitochondria.     

Transduction of multi-copy plasmid pBR322 by bacteriophage Mu

Summary The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers. Plasmid transducing particles contain DNA molecules of Mu DNA length. Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell. The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization. Possible mechanisms that may explain the origin of plasmid transducing particles are discussed.     

Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.     

High-copy-number derivatives of the plasmid cloning vector pBR322

A stable copy-number mutant of a pBR322-derived plasmid was isolated. The mutation was found to be a single G → T transversion located near the 3' end of a DNA segment coding for the regulatory RNA I. The resulting copy number for this plasmid is approx. 1000 per cell or 65 % of total cellular DNA. Several cloning vectors have been constructed from this copy-number mutant and their practical application is discussed.