Escherichia coli DNA topoisomerase I catalyzed linking of single-stranded rings of complementary base sequences

Eco DNA topoisomerase I (E. coli omega protein) has been observed to catalyze the formation of double-stranded, covalently closed DNA from complementary single-stranded DNA rings, a novel reaction which is topologically forbidden without the enzyme-catalyzed breakage and rejoining of DNA backbone bonds. Incubation of a mixture of single-stranded PM2 DNA rings of complementary base sequences with omega yields a species with a sedimentation coefficient in an alkaline medium characteristic of a covalently closed circular double-stranded DNA. Buoyant density measurements in CsCl at alkaline pH also identify the product as a covalently closed duplex ring. If the omega-catalyzed reaction is stopped short of completion, highly negatively supercoiled molecules are formed which sediment more slowly in an alkaline medium than the final duplex product. As the reaction proceeds the mean sedimentation rate of the intermediates increases. This is in agreement with the expectation that the linking number between the two complementary rings increases gradually during the course of the reaction from zero to that of a relaxed covalently closed circular DNA duplex. The possible role of DNA topoisomerases in genetic recombination is discussed.     

Ethidium bromide-mediated renaturation of denatured closed circular DNAs. The nature of denaturation-resistant fractions of bacteriophage PM2 closed circular DNA

Addition of the intercalating dye ethidium bromide (EtdBr) to a solution of alkali-denatured double-stranded closed circular PM2, ΦX174, or λb₂b₅c phage DNAs, under conditions such that the solution remains strongly alkaline, can result in the renaturation of up to 100% of the DNA upon neutralization of the solution. For a fixed time of incubation of the alkaline dye-containing solution before neutralization, there exists a minimum concentration of the dye below which no EtdBr-mediated renaturation is observed for each species of closed circular DNA examined. These minimum concentrations increase, for a given DNA, with increasing ionic strength and temperature. The kinetics of accumulation of forms renaturing upon neutralization of alkaline solutions, at fixed concentrations of dye and DNA, are dependent upon the molecular weight and superhelix density of the starting DNA. After extended periods of incubation at a fixed ionic strength and temperature, however, the profiles of percentage of DNA renatured as a function of ethidium concentration become very similar for all the closed circular DNAs tested and display a transition from an absence of dye-mediated renaturation to virtually 100% renaturation upon neutralization over a small range of dye concentration. Circular DNA containing one or more strand scissions remains strand-separated under all the conditions used to effect the renaturation of closed circular DNA. These findings indicate that configurations of closed circular DNA, in which at least some of the complementary bases are apposed, can be selectively stabilized and accumulate in the presence of ethidium in solutions containing 0.19 N hydroxide ion.     

DNA wrapping in nucleosomes. The linking number problem re-examined.

Chromatin was assembled in vitro from relaxed closed circular DNA (SV40) and core histones at histone to DNA ratios of 0.2 to 0.3 (g/g) and incubated with topoisomerase I to relax supercoils in DNA regions not constrained by protein. Addition of histones H1 + H5 to the chromatin at an ionic strength of 0.1 M, in the presence of the solubilizing agent, polyglutamic acid, and topoisomerase I, increased the magnitude of the DNA linking number change, relative to protein-free DNA. No change in the linking number distribution occurred for relaxed protein-free DNA under these conditions. Control experiments indicated that the increase in the absolute value of the DNA linking number change in the chromatin could not be attributed to an increase in the number of nucleosomes per DNA molecule. These data suggest a solution to the linking number problem associated with models of chromatin structure.     

Nature of the Complementary Strands Synthesized in Vitro upon the Single-Stranded Circular DNA of Bacteriophage øX174 after Ultraviolet Irradiation

This paper describes experiments intended to decide whether UV lesions in DNA act as absolute blocks to chain elongation by the Escherichia coli DNA polymerase or only slow down the polymerization process. Ultraviolet (UV)-irradiated, single-stranded (SS) circular DNA of bacteriophage øX174 was used as template for the polymerase in a reaction mixture in vitro, under conditions allowing synthesis of not more than one complementary strand per template molecule. The mean length of the newly synthesized complementary strands (as determined by velocity sedimentation in alkaline CsCl gradients), as well as the over-all template activity (as measured by deoxyadenosine monophosphate [dAMP] incorporation) was found to decrease with the number of biologically lethal hits sustained by the irradiated templates. With the increase of time or temperature of reaction, the net synthesis of complementary strands increased (as a consequence of increased initiation), but their mean length remained constant. The mean length of synthesized strands was greater than would be expected if all biologically lethal hits were to block the polymerization process. The lethal hits which serve as blocking lesions are inferred to be pyrimidine dimers because it is possible to obtain synthesis of full-length complementary strands if, when heat-denatured, UV-irradiated, double-stranded replicative form (RF II) DNA of bacteriophage øX174 is used as a template, it is pretreated with yeast photoreactivating enzyme (YPRE) in presence of visible light.     

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Physical and topological properties of circular DNA

Several types of circular DNA molecules are now known. These are classified as single-stranded rings, covalently closed duplex rings, and weakly bonded duplex rings containing an interruption in one or both strands. Single rings are exemplified by the viral DNA from φX174 bacteriophage. Duplex rings appear to exist in a twisted configuration in neutral salt solutions at room temperature. Examples of such molecules are the DNA''s from the papova group of tumor viruses and certain intracellular forms of φX and λ-DNA. These DNA''s have several common properties which derive from the topological requirement that the winding number in such molecules is invariant. They sediment abnormally rapidly in alkaline (denaturing) solvents because of the topological barrier to unwinding. For the same basic reason these DNA''s are thermodynamically more stable than the strand separable DNA''s in thermal and alkaline melting experiments. The introduction of one single strand scission has a profound effect on the properties of closed circular duplex DNA''s. In neutral solutions a scission appears to generate a swivel in the complementary strand at a site in the helix opposite to the scission. The twists are then released and a slower sedimenting, weakly closed circular duplex is formed. Such circular duplexes exhibit normal melting behavior, and in alkali dissociate to form circular and linear single strands which sediment at different velocities. Weakly closed circular duplexes containing an interruption in each strand are formed by intramolecular cyclization of viral λ-DNA. A third kind of weakly closed circular duplex is formed by reannealing single strands derived from circularly permuted T2 DNA. These reconstituted duplexes again contain an interruption in each strand though not necessarily regularly spaced with respect to each other.     

Phi X 174 gene A protein does not cleave at a mouse mitochondrial DNA sequence homologous to the phi X and G4 cleavage site

The light strand origin of replication of mouse mitochondrial DNA contains a 30-nucleotide region which is 60% homologous to the 30-nucleotide conserved sequence in φX174 and G4 viral DNAs known to contain the viral gene A protein cleavage site. Gene A protein does not cleave closed circular mouse mitochondrial DNA under conditions in which φX174 closed circular DNA is cleaved.     

An international collaborative study of ''genetic drift'' in Salmonella typhimurium strains used in the Ames test

The efficiency of Weigle reactivation of ultraviolet light-irradiated single and double-stranded phi X174 DNA by wild-type and excision repair-defective E. coli hosts was determined. After limited exposure to ultraviolet light, the efficiency of Weigle reactivation by an ultraviolet light-irradiated wild-type host was greater for double-stranded phi X174 DNA than for its single-stranded counterpart. However, the efficiency of inducible recovery of the double-stranded DNA molecule decreased as its exposure to ultraviolet light increased until it became constant at a value 1.5 times less than that for single-stranded form of phi X174 DNA. The efficiency of Weigle reactivation of the single-stranded DNA molecule by the same host, however, was independent of the dose to the DNA, as were the efficiencies of reactivation for both forms of phi X174 DNA by ultraviolet light-irradiated excision repair-deficient hosts. In excision repair-defective hosts the efficiency of Weigle reactivation of double-stranded phi X174 DNA was also 1.5 times less than that for the single-stranded molecule. These results suggest that the Weigle reactivation of double-stranded phi X174 DNA is mediated in part by an excision repair process, and that this component of Weigle reactivation eventually can be saturated by ultraviolet light-induced DNA damage leaving other repair processes, such as trans-damage synthesis, responsible for the remaining inducible reactivation.     

Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1.

Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.     

phi X174-directed DNA and protein syntheses in infected minicells.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.     

φX174 gene A protein does not cleave at a mouse mitochondrial DNA sequence homologous to the φX and G4 cleavage site

The light strand origin of replication of mouse mitochondrial DNA contains a 30-nucleotide region which is 60% homologous to the 30-nucleotide conserved sequence in φX174 and G4 viral DNAs known to contain the viral gene A protein cleavage site. Gene A protein does not cleave closed circular mouse mitochondrial DNA under conditions in which φX174 closed circular DNA is cleaved.     

Characterization of an endonuclease activity associated with Friend-murine leukemia virus

An endonuclease activity shown to be associated with Friend leukemia virus has been characterized using double-stranded phi X174 DNA as substrate. In the presence of Mg2+, the endonuclease activity was able to convert supercoiled circular DNA duplexes to the relaxed form by introducing single-stranded nicks into the DNA. Most of the nicked DNA duplexes contained only one nick per strand, since unit length DNA was the predominant species obtained when the nicked DNA was analyzed by alkaline sucrose gradient centrifugation. The regions into which the nick could be introduced were evenly distributed around the circular DNA molecule. When Mn2+ was substituted for Mg2+ in the reaction mixture, the number of nicks introduced into circular DNA duplexes by the virus associated endonuclease was greatly increased. In contrast to circular duplexes, linear duplexes and single-stranded DNA functioned poorly as substrates for the virus-associated enzyme. The Friend leukemia virus-associated endonuclease activity is with respect to these characteristics very similar to the endonuclease activity associated with the p32 protein of the avian myeloblastosis virus [1]. The molecular weight of the Friend leukemia virus endonuclease was estimated by gel filtration on a Sephacryl S-200 column to be about 45 000.     

Preparation and characterization of form V DNA, the duplex DNA resulting from association of complementary, circular single-stranded DNA.

Complementary circular single strands of plasmid PβG or bacteriophage PM2 DNA but not of single-stranded φX174 DNA associate under hybridisation conditions, giving rise to a two-stranded complex. This DNA, which we call form V, has well-defined physico-chemical properties. It sediments as a sharp peak in neutral sucrose gradients; its electrophoretic mobility in agarose gels is between that of covalently closed (form I) and denatured DNA. In the electron microscope form V appears as highly folded duplex molecules indistinguishable from form I. However, increasing concentrations of ethidium bromide which lead to relaxation and recoiling of form I DNA have no comparable effect upon form V. At 260 nm form V PβG DNA has a hypochromicity of 18.6%, as compared to 23.4% in the case of PβG form II DNA and 10.5% in the case of single-stranded φX174 DNA. The thermal melting of form V is non-cooperative with gradual increase in absorbance similar to that of single-stranded DNA. The circular dichroism spectrum of form V DNA differs from that of form I, circular nicked (form II) and single-stranded φX174 DNA in that it shows a negative band at 295 nm and a shift for the main positive band from 273 to 266 nm. We propose that form V consists of right-handed Watson-Crick type double-helices which are compensated by an equal number of left-handed duplex turns and negative supercoils. Wo cannot decide whether left-handed duplex turns are stabilised by base-stacking and hydrogen bonding, as for example in the models described by Rodley et al. (1976) or Sasisekharan &; Pattabiraman (1976), or whether they are merely compensatory turns without inherent stability.     

Is DNA really a double helix?

Do the two chains of the DNA molecule coil round one another plectonemically ? If so, what is the approximate value of Lk (the linking number) for any closed, circular DNA molecule? Experiments using gel electrophoresis have shown that supercoiled DNA molecules usually migrate in a series of discrete bands. The only tenable explanation for this quantized behavior is that the molecules in one band all have the same value of Lk and that this value differs by unity from that of the adjacent bands. Various experiments in which circular DNA is unwound by known amounts show that (given this assumption) Lk for relaxed DNA is very roughly equal to $\text{N}\text{10}$ (where N is the number of base-pairs), as expected from the classical double helix.The original model for the double helix was right-handed. The experimental evidence for this feature is suggestive but not yet completely compelling.     

The early melting of closed duplex DNA: analysis by banding in buoyant neutral rubidium trichloroacetate.

Aqueous RbTCA permits the buoyant banding of both native and denatured DNA at room temperature and neutral pH. A unique property of this solvent is the bouyant resolution of closed circular, underwound DNA (I) from the corresponding nicked (II) species. Conditions are reported here in which PM-2 DNA I is physically resolved from native PM-2 DNA II, the buoyant separation being 1.27 mq/ml in 3.3 M RbTCA at 25 degrees C. The separation between nicked and closed DNAs increases with temperature up to 35.5 degrees C, at which PM-2 DNA II cooperatively melts and subsequently pellets. The isothermal buoyant density of a cloed DNA increases linearly as the linking number (Lk) of the closed DNA decreases. The early melting of closed DNA may be monitored with high precision by buoyant banding in RbTCA, it being possible to detect the disruption of as few as 40 base pairs in PM-2 DNA (10,000 base pairs). The constraint that the linking number be conserved in closed DNA requires that a change in duplex winding be accompanied by a compensating change in supercoiling. We estimate the linking number deficiency of PM-2 DNA I to be 0.094 turns per decibase pair. This result permits the estimation of the EtdBr unwinding angle, phi, by comparison with alternative determinations of the linking number deficiency which depend upom the value of phi. The result obtained here is that phi = 27.7 degrees +/- 0.5 degrees and is approximately independent of temperature over the range 15 degrees-35 degrees.     

Light-scattering studies on deoxyribonucleic acid flexibility. The solution properties of a small circular deoxyribonucleic acid molecule

Previous investigations on the persistence length of DNA in solution have revealed large discrepancies between hydrodynamic results and those from light-scattering techniques which have potentially a greater resolving power. The information obtained from experiments on a small circular DNA molecule has resolved these discrepancies. The non-superhelical circular double-stranded DNA molecule from bacteriophage [unk]X174-infected cells is small enough to permit accurate light-scattering extrapolations, and its solutions have negligible anisotropy. The persistence length obtained from experimental investigations on this molecule is comparable with that obtained by hydrodynamic techniques, even with variation of the excluded-volume factor.     

Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate

Following its cleavage of double-stranded DNA, topoisomerase II is covalently bound to the 5'-termini of both nucleic acid strands. However, in order to isolate this enzyme-cleaved DNA complex in the presence of magnesium (the enzyme's physiological divalent cation), reactions must be terminated by the addition of a strong protein denaturant such as sodium dodecyl sulfate (SDS). Because of the requirement for a protein denaturant, it is unclear whether DNA cleavage in this in vitro system takes place prior to or is induced by the addition of SDS. To distinguish between these two possibilities, experiments were carried out to determine whether topoisomerase II bound DNA contains 3'-OH termini prior to denaturation. This was accomplished by using circular single-stranded phi X174 DNA as a model substrate for the enzyme. As found previously for topoisomerase II mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5'-termini of cleaved phi X174 molecules. Moreover, optimal reaction pH as well as optimal salt and magnesium concentrations was similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not salt reversible, and did not require the presence of SDS. Furthermore, cleavage products generated in the absence of protein denaturant could be labeled at their 3'-OH DNA termini by incubation with terminal deoxynucleotidyltransferase and [alpha-32P]ddATP. Finally, cleaved phi X174 molecules could be joined to a radioactively labeled double-stranded oligonucleotide by a topoisomerase II mediated intermolecular ligation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of in vitro synthesized covalently closed circular double-stranded DNA by selective denaturation and filtration

Based upon their resistance to irreversible denaturation, covalently closed circular (CCC) DNA (non-nicked, double-stranded circular molecules) can be purified by alkaline denaturation, neutralization, and filtration through a nitrocellulose membrane. This procedure offers a simple means of isolating in vitro synthesized CCC DNA molecules. The preparations of molecules obtained by this method consisted of 91-97% CCC DNA and contained no detectable inhibitors of biological activity or enzymatic digestion.