Size and base composition of RNA in supercoiled plasmid DNA

The average size and base composition of the covalently integrated RNA segment in supercoiled ColE(1) DNA synthesized in Escherichia coli in the presence of chloramphenicol (CM-ColE(1) DNA) have been determined by two independent methods. The two approaches yielded similar results, indicating that the RNA segment in CM-ColE(1) DNA contains GMP at the 5' end and comprises on the average 25 to 26 ribonucleotides with a base composition of 10-11 G, 3 A, 5-6 C, and 6-7 U.     

Sequence-dependent energetics of the B-Z transition in supercoiled DNA containing nonalternating purine-pyrimidine sequences.

The likelihood that a given DNA sequence will adopt the Z conformation in negatively supercoiled DNA depends on the energy difference between the B form and the Z form for that sequence relative to other sequences in the same molecule. This energy can be viewed simply as a sum of energies for the nearest-neighbor interactions within the sequence plus the energy required to stabilize the B-Z boundaries. Knowledge of these energetic terms would be of value in predicting when sequences become left-handed in response to negative superhelicity. Here we present an approach that can be used to determine the free-energy changes associated with all the nearest-neighbor interactions that can occur in Z-DNA. Synthetic stretches of d(C-G)n containing one or two transversions were cloned into plasmids, and the extent of the B-Z transition as a function of negative superhelicity was determined for each insert by two-dimensional agarose gel electrophoresis. By subjecting the data to statistical mechanical analysis, it was possible to evaluate the energetic penalty resulting from each base-pair (bp) substitution. Guanine to cytosine transversions cost 2.4 kcal (1 cal = 4.18 J)/(mol X bp), whereas guanine to thymine transversions cost 3.4 kcal/(mol X bp), to stabilize in the Z conformation. We have used these numbers, along with energetic values determined by others for the B-Z transition, to predict that certain strictly nonalternating purine and pyrimidine sequences may adopt the Z form readily.     

Lupus-inducing drugs alter the structure of supercoiled circular DNA domains.

We analyzed the effects of procainamide (PROC), hydralazine (HYD), N-acetylprocainamide (NAPA), and L-canavanine (CAN) on circular supercoiled plasmids as models for chromosomal loop domains. The supercoil-dependent B-Z equilibrium in recombinant plasmids was used as an indicator of structural changes induced in circular DNA. Two-dimensional gel electrophoresis showed that PROC and HYD strongly inhibited supercoil-induced Z-DNA formation, whereas NAPA caused less pronounced changes in the B-Z equilibrium, and CAN had no effect. Gel retardation assays showed that the binding of a Z-DNA-specific autoimmune antibody to a Z-DNA-containing plasmid was strongly perturbed by HYD, but not influenced by CAN. Both PROC and NAPA showed moderate inhibition of antibody binding. Our results demonstrate the different potentials of these 4 drugs to interact with DNA and to alter the tertiary topology of DNA domains. It is conceivable that the in vivo capacity of PROC and HYD to induce antinuclear antibodies may be related to their ability to influence structural features in chromosomal DNA domains or nucleosomes, thus liberating antigenic structural epitopes in DNA and/or DNA-associated proteins.     

Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.

A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction.     

Intramolecular DNA triplexes in supercoiled plasmids.

A series of inserts with oligopurine.oligopyrimidine mirror repeat sequences was investigated at the base pair level with specific chemical probes (OsO4 and diethylpyrocarbonate) to evaluate the in vitro existence of intramolecular triplexes. Two parent inserts in recombinant plasmids with (GAA)9 and (AG)12 sequences and three mutant inserts (containing transitions or transversions) revealed that base pair changes at one location affected the chemical reactivity 13 base pairs away. The specificity and nature of these reactions, as well as the thermal stability of the complexes, provide direct evidence for the existence of a triplex with a portion of the pyrimidine-rich strand folded back and Hoogsteen-paired in the major groove of the Watson-Crick duplex. The biological implications of this unorthodox DNA structure are discussed.     

Diethyl pyrocarbonate: a chemical probe for secondary structure in negatively supercoiled DNA.

Purine residues located within regions of DNA that have the potential to form left-handed Z-helical structures are modified preferentially by diethyl pyrocarbonate; this hyperreactivity is dependent on the degree of negative superhelicity of the circular DNA molecules. As negative superhelical density increases, guanosines in a 32-base-pair alternating G-C sequence and adenosines (but not guanosines) in a 64-base-pair alternating A-C/G-T sequence become 5- to 10-fold more reactive to diethyl pyrocarbonate. The negative superhelical densities at which enhanced reactivity occurs are similar to those reported for the point at which left-handed helices form within plasmids carrying these DNA sequences. Probing of negatively supercoiled pBR322 with diethyl pyrocarbonate reveals a hyperreactive region 31 base pairs in length of which only 9 base pairs are a perfect alternating purine and pyrimidine sequence; the reactivity of purines within this sequence indicates that purines in the anti conformation, or guanosines in the syn conformation with neighboring 3' thymidines, are not hyperreactive in the Z-DNA form.     

Nonrandom distribution of repeated DNA sequences with respect to supercoiled loops and the nuclear matrix.

The DNA in a eukaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. We have analyzed the DNA sequences that are closest to the matrix attachment points for their relative content of specific repeated sequences. Sequences were enriched (mouse satellite, human Alu family) or depleted (mouse EcoRI repeat, monkey alpha component), depending on the specific sequence and species examined. These results can be understood in terms of a nonrandom arrangement of DNA sequences with respect to nuclear DNA loops.     

Stimulation of recombination between homologous sequences on plasmid DNA and chromosomal DNA in Escherichia coli by N-acetoxy-2-acetylaminofluorene.

A plasmid containing a wild-type lac operon and a tetracycline-resistance gene was covalently modified by N-acetoxy-2-acetylaminofluorene and used to transform two series of Lac- Escherichia coli cell types. Each set contained wild-type and repair-deficient mutants. One set of cells contained a lacY mutation and the other a deletion of the entire lac operon. Survival and mutagenesis of the plasmid were measured as a function of the N-acetoxy-2-acetylaminofluorene concentration. The results indicate that when no homologous sequences are present in the chromosomal DNA, mutations occur at a low frequency: at 10% survival the frequency was 1-2 X 10(-4) mutants per transformant. When homologous sequences, the lacY allele, are present in the chromosomal DNA, Lac- plasmids are found at a high frequency in a recA-dependent, lexA-independent fashion: at 10% survival the frequency was 5-10 X 10(-2) mutants per transformant. Southern blot analysis of the restriction enzyme profiles of the resulting plasmid and host-cell DNA sequences showed recombinational transfer of host sequences to the N-acetoxy-2-acetylamino-fluorene-treated plasmid had occurred. When the host chromosomes contained Lac+ homologous sequences no mutants were found, indicating that the results were not caused by error-prone recombination.     

Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation.

A Lys-84----Cys mutant staphylococcal nuclease was selectively linked to the 5' and/or 3' terminus of a thiol-containing polypyrimidine oligonucleotide via a disulfide bond. The oligonucleotide-staphylococcal nuclease adduct is capable of binding to a homopurine-homopyrimidine region of Watson-Crick duplex DNA by the formation of a triple-helical structure. Upon the addition of Ca2+, the nuclease cleaves DNA at sites adjacent to the homopurine tract. Specific double-strand cleavage occurred predominantly at A + T-rich sites to the 5' side of the homopurine tract for both the 5'-derivatized and the 5',3'-diderivatized nucleases; the 3'-derivatized nuclease gave no cleavage. The cleavage pattern is asymmetric and consists of multiple cleavage sites shifted to the 5' side on each strand, centered at the terminal base pair of the binding site. Microgram amounts of plasmid pDP20 DNA (4433 base pairs) containing a homopurine-homopyrimidine tract were selectively cleaved by a semisynthetic nuclease with greater than 75% efficiency at room temperature within 1 hr. Cleavage reaction conditions were optimized with respect to pH, temperature, reaction times, and reaction components. Semisynthetic nucleases of this type should provide a powerful tool in chromosomal DNA manipulations.     

Dynamics of the B-to-Z transition in supercoiled DNA.

The sequence (dC-dG)16, inserted into the polylinker of plasmid pUC8, adopts a left-handed Z-DNA conformation at "natural" supercoil density. The radioactively labeled monoclonal antibody Z-D11, which has a very high affinity for this DNA conformation, provides a convenient sensitive tool to measure selectively the amount of Z-DNA. Chloroquine reversibly changes the supercoil density of plasmid DNA and thereby the equilibrium between right- and left-handed double-helical DNA. The time-dependent formation or disappearance of Z-DNA was measured by using the antibody either as a fast indicator of Z-DNA or as an additional effector of the B-to-Z equilibrium. In the middle of the transition, a relaxation time of about 1 hr is observed in 0.1 M NaCl at 22 degrees C. The kinetic data are compatible with an all-or-none transition between the two conformations. The overall rate constant for Z-DNA formation, kBZ, decreases with the square of the chloroquine concentration, while the reverse one, kZB, increases with about the fourth power.     

High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs.

With the goal of developing techniques for DNA insertional mutagenesis in zebrafish, we established procedures for rapidly obtaining and injecting large numbers of fertilized eggs. Using either of two plasmid constructs, we injected uncut DNA into fertilized eggs at the one- or two-cell stage. Fish hatched from injected eggs were raised to sexual maturity, and the frequency of transgenic founder fish was determined by pair-mating the fish and testing DNA extracted from pools of their 16-hr-old offspring by the polymerase chain reaction (PCR) and then Southern analysis. Eggs injected with one of two different plasmids yielded no transgenic fish, but 7-25% (19 of 115 overall) of the eggs injected with the other plasmid transmitted the injected sequences to their offspring (F1). Of seven lines studied further, all were able to pass the foreign DNA sequences to the next (F2) generation. Inheritance in the F2 generation was Mendelian in the five lines tested. PCR and Southern analysis indicated that the plasmid sequences were present in multiple copies, probably tandemly arranged. Two founder fish carried more than one independent integration of the plasmid sequences. The line studied in more detail was a mosaic carrying two independently segregating copies of the transgene in one germ cell and a third copy in another germ-line precursor cell. The ability to obtain and inject large numbers of zebrafish eggs combined with a high frequency of germ-line integration may be steps toward the goal of being able to perform insertional mutagenesis with this organism.     

Homologous pairing in stretched supercoiled DNA

By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (≈2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing in vivo. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions.     

Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo.

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.     

Rapid "footprinting" on supercoiled DNA.

A DNase protection technique is described and applied to the interaction of three lac control proteins with supercoiled lac DNA. The technique uses end-labeled oligonucleotide primers to probe specific DNA regions as an alternative to protocols requiring restriction endonuclease cleavage or blotting. Thus DNA may be probed with high resolution in its native state. It is demonstrated that the introduction of supercoiling into DNA accelerates the rate of lac ps promoter binding by RNA polymerase but does not alter the positions at which polymerase, c-AMP-binding protein, or lac repressor bind to lac DNA.     

Dynamics of site juxtaposition in supercoiled DNA

Juxtaposition kinetics between specific sites in supercoiled DNA is investigated at close to physiological ionic conditions by Brownian dynamics simulations. At such conditions, supercoiled DNA is interwound, and the probability of spatial site juxtaposition is much higher than in relaxed DNA. We find, however, that supercoiling does not correspondingly increase the rate of juxtaposition at these physiological conditions. An explanation to this unexpected finding emerges on analysis of the juxtaposition dynamics. We note that although a particular site i(1) in supercoiled DNA is often in close proximity (juxtaposed) to another site i(2), the change of i(2) occurs very slowly and depends largely on internal slithering of opposite segments of the DNA superhelix. Such slithering results in long correlations between successive values of i(2); these correlations increase the average time of juxtaposition between two DNA sites. Random collisions between sites located on different superhelix branches-although increasing in importance with DNA size-contribute less substantially to site juxtaposition at high salt than slithering for DNA up to 6 kb in length.     

Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or IB enzymes. We used real-time single-molecule micromechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type IB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type IB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type IB molecules but by using a completely different structural and mechanistic framework.     

ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB.

The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAB complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA. UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency. Our results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision. A possible role of supercoiled domains in the incision process is discussed.     

DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid.

Ltk- aprt- mouse L cells were transformed to the tk+ phenotype with 10 ng of the herpes simplex virus-1 thymidine kinase (tk) gene and 20 micrograms of pBR322 or simian virus 40 (SV40) DNA. DNAs from five cloned cell lines show restriction endonuclease fragments that hybridize to both tk and pBR322 or SV40 DNA. In all of the cell lines some of these fragments also contain cellular DNA sequences. The use of carrier DNAs with defined sequences has enabled us to demonstrate that the joining of carrier and selectable gene sequences occurs in mouse cells. In one case we have been able to use the ampicillin resistance marker of pBR322 to "rescue" a recombinant plasmid. An analysis of the junction between pBR322 and tk in this plasmid suggests that a small area of homology (16 of 19 base pairs) might be involved in the recombination process.