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.     

Mechanism of mitochondrial DNA replication in mouse L-cells: asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence

Examination of in vivo long-labeled, pulse-labeled and pulse-chase-labeled mitochondrial DNA has corroborated and extended the basic elements of the displacement model of replication. Mitochondrial DNA molecules are shown to replicate an average of once per cell doubling in exponentially growing cultures. Analysis of the separate strands of partially replicated molecules indicates that replication is highly asynchronous with heavy-strand synthesis preceding light-strand synthesis. Native and denatured pulse-labeled replicating molecules exhibit sedimentation properties predicted by the displacement model of replication. Pulse-label incorporated into molecules isolated in the lower band region of ethidium bromide/cesium chloride gradients is found primarily in heavy daughter strands. Pulse-label incorporated into molecules isolated in the upper band region is found primarily in light daughter strands. The results of a series of pulse-chase experiments indicate that the complete process of replication requires approximately 120 minutes. Both daughter molecules are shown to segregate in an open circular form. They are then converted to closed circular molecules having a superhelix density near zero. After closure, the 7 S heavy-strand initation sequence is synthesized, and this process is accompanied by nicking, unwinding and closing of at least one of the parental strands resulting in the formation of the D-loop structure. The 7 S heavy-strand initiation sequence of the D-loop structure is not stable and turns over with a half-life of 7·9 hours. We suggest that all in vivo forms of parental closed circular mitochondrial DNA have superhelix densities of near zero, and that the previously observed superhelix density of closed circular mitochondrial DNA, σ~ ?0·02, results from the loss of the 7 S heavy-strand initiation sequence from D-loop mitochondrial DNA molecules during isolation.     

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.     

Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line.

A non-integrated form of Epstein-Barr virus DNA was purified from the Burkitt lymphoma-derived human lymphoid cell line Raji by CsCl density gradient centrifugation and neutral glycerol gradient centrifugation. This intracellular form of the virus DNA sediments at a rate typical of a covalently closed circular DNA molecule of the size of the virus genome in both neutral and alkaline solution. Treatment with low doses of X-rays leads to a discontinuous conversion of the molecules to a form with the sedimentation properties of open circular DNA (a circular duplex molecule containing one or more single-strand breaks). The direct observation of large circular DNA molecules by electron microscopy further confirms the covalently closed circular duplex structure of part of the intracellular viral DNA. Such circular molecules were not detected in corresponding DNA fractions from Epstein-Barr virus-negative human lymphoid cell lines. In ethidium bromide/CsCl density gradient centrifugation experiments, the purified non-integrated virus DNA behaves as twisted, covalently closed DNA circles with the same initial superhelix density as polyoma virus DNA. The latter additional purification technique permits the isolation of intracellular Epstein-Barr virus DNA in > 90% pure form from non-producer cells. The molecular weight of the circular virus DNA from Raji cells, determined by contour length measurements, is the same within experimental error as that of the linear DNA from virus particles.     

Superhelical DNA with local substructures. A generalization of the topological constraint in terms of the intersection number and the ladder-like correspondence surface

The presence of certain local structural elements in superhelical DNA, such as cruciforms and denatured loops, complicates the topological and geometric analysis of these molecules. In particular, the duplex axis is often difficult to define. In consequence, the usual conservation condition, Lk = Tw + Wr, is often inapplicable as formulated in terms of the winding of either strand of the DNA about the duplex axis. We present here a more general formulation of the topological conservation condition in terms of a model in which the two strands of DNA are regarded as twisting about one another, and in which one of the two strands is considered to writhe. We define a ladder-like correspondence surface, which connects the two strands nd is independent of whether or not a unique duplex axis is locally available. These considerations lead to the definition of a new topological property of superhelical DNA, the intersection number, In. This quantity describes the complexity of a local structural element; in the case of a cruciform, for example, the intersection number is a measure of the number of duplex turns removed from the major segment of the DNA by the cruciform formation. In more general terms, the topological constraint applicable to closed circular DNA is given by Lk(W,C) + In(S,C) = Tw(W,C) + Wr (C), where W and C represent the two strands of the DNA and S is the ladder-like correspondence surface that connects the two strands.     

Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S1 Nuclease

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.     

Maturation of replicating simian virus 40 DNA molecules in isolated nuclei by continued bidirectional replication to the normal termination region.

Mature SV40 DNA synthesized for different periods of time either in isolated nuclei or in intact cells was highly purified and then digested with restriction endonucleases in order to relate the time of synthesis of newly replicated viral DNA to its location in the genome. Replication in nuclei supplemented with a cytosol fraction from uninfected cells was a faithful continuation of the bidirectional process observed in intact cells, but did not exhibit significant initiation of new replicons. SV40 DNA replication in cells at 37 degrees C proceeded at about 145 nucleotides/min per replication fork. In the absence of cytosol, when DNA synthesis was limited and joining of Okazaki fragments was retarded, bidirectional SV40 DNA replication continued into the normal region where separation yeilded circular duplex DNA molecules containing one or more interruptions in the nascent DNA strands. In the presence of cytosol, this type of viral DNA was shown to be a precursor of covalently closed, superhelical SV40 DNA, the mature from of viral DNA.     

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.     

Joint molecule formation following conjugation in wild type and mutant Escherichia coli recipients

The sedimentation coefficients of closed circular Simian virus (SV40) DNA, phage PM2 DNA and animal mitochondrial DNAs in alkaline NaCl and alkaline CsCl were found to decrease by about 5% as the initial superhelix densities decreased from 0.0 to ?0.10, corresponding to a decrease in the degree of strand interwinding from 1.0 to 0.9 net turns per ten base pairs. The small dependence of the appropriately normalized sedimentation coefficients on the degree of strand interwinding is taken to indicate that fully titrated and denatured closed circular DNA is highly supercoiled in a positive sense. This supercoiling results from the spontaneous decrease in the number of secondary turns in the no longer ordered pairs of polynucleotide strands.The measured sedimentation coefficients form a smoothly connected monotonie curve when plotted along with the sedimentation coefficients in alkali (Sebring et al., 1971) of parental closed circles derived from closed circular SV40 DNA replicating intermediates. These DNAs have degrees of strand interwinding that range from 0.6 to 0.15.The possibility raised by Paoletti &; LePecq (1971) that closed circular duplex DNAs contain positive supercoils, i.e. have degrees of strand interwinding greater than 1.0, has been ruled out in a series of ethidium bromide titrations of partially replicated mitochondrial DNA before and after removal of the progeny strand. More ethidium bromide was required in the latter case for relaxation, a result which shows that intercalated ethidium and a displacing strand act on the duplex in the same way, and that both unwind the duplex. This result requires the supercoils of naturally closed circular DNAs to be negative.     

Homologous pairing of DNA molecules promoted by a protein from Ustilago

A protein from mitotic cells of Ustilago maydis was purified on the basis of its ability to reanneal complementary single strands of DNA. The protein catalyzed the uptake of linear single strands by super-helical DNA, but only in reactions with homologous combinations of single-strand fragments and super-helical DNA from phages phi X174 and fd. No reaction occurred with heterologous combinations. The protein also efficiently paired circular single strands and linear duplex DNA molecules. The product was a joint molecule in which the circular single strand displaced one strand of the duplex. Efficient pairing depended upon ATP, and ATPase activity was found associated with the purified protein. ATP-dependent reannealing of complementary single strands was not detectable in the rec1 mutant of Ustilago, which is deranged in meiotic recombination, as complete tetrads are rare, and is defective in radiation-induced mitotic gene conversion.     

Identification of the site of interruption in relaxed circles producing during bacteriophage lambda DNA circle replication.

The DNA that accumulates in the lambda infection restricted to the early (circular) stage of replication consists of approximately two-thirds covalently closed circles and one-third relaxed circles bearing a single interruption in either strand of the duplex. The latter molecules are presumed to be a unique class in that the interruption is not repairable by DNA polymerase and ligase. Preferential radioisotopic labeling of the region immediately adjacent to the interruption, followed by hybridization to sheared fragments of the lambda chromosome with varying guanine plus cytosine content, suggested that the nick resides at the position of the mature molecular ends of the lambda chromosome. Digestion of the labeled molecules with restriction enzymes and reconstruction experiments in which Hershey circles were generated by annealing of interrupted strands isolated from the relaxed circles support this interpretation. The results indicate that the relaxed circles consist of a population containing one interruption in either of the two strands of the duplex jointly representing the two "nicks" contained in Hershey circles (in which the cohesive ends are annealed). These molecules could result from the inability of the maturation function to make the required staggered endonucleolytic cuts when the DNA substrate is a monomeric circle rather than a multimeric linear molecule. Alternatively, this interruption could be the result of an endonucleolytic cutting event critical to DNA replication.     

Hemicatenanes form upon inhibition of DNA replication

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assembled into chromatin and then into synthetic nuclei which undergo one round of regulated replication. During a study of restriction endonuclease cut plasmid replication intermediates (RIs) by the Brewer–Fangman 2D gel electrophoresis technique, we have observed the formation of a strong spike of X-shaped DNA molecules in extracts that otherwise yield very little or no RIs. Formation of these joint molecules is also efficiently induced in replication-competent extracts upon inhibition of replication fork progression by aphidicolin. Although their electrophoretic properties are quite similar to those of Holliday junctions, 2D gels of doubly cut plasmids show that these junctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points. Neutral–neutral–alkaline 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely structure to account for these observations. The mechanism of formation of these novel joint DNA molecules and their biological implications are discussed.     

Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.     

The structure of replicating adenovirus 2 DNA molecules

Adenovirus 2 (Ad2)-infected KB cells were exposed to a 2.5 min pulse of ³H-thymidine at 19 hr after infection. The labeled DNA molecules were separated from cell DNA and mature Ad2 DNA by sucrose gradient sedimentation and CsCI equilibrium centrifugation under conditions designed to minimize branch migration and hybridization of single strands. Electron microscopy-of fractions containing radioactivity revealed two basic types of putative replicating molecules: Ad2 length duplex DNA molecules with one or more single-stranded branches (type I) and Ad2 length linear DNA molecules with a single-stranded region extending a variable distance from one end (type II). Length measurements, partial denaturation studies and 3′ terminal labeling experiments were consistent with the following model for Ad2 DNA replication. Initiation of DNA synthesis occurs at or near an end of the Ad2 duplex. Following initiation, a daughter strand is synthesized in the 5′ to 3′ direction, displacing the parental strand with the same polarity. This results in the formation of a branched replicating molecule (type I). Initiations at the right and left molecular ends are approximately equal in frequency, and multiple initiations on the same replicating molecule are common. At any given displacement fork in a type I molecule, only one of the two parental strands is replicated. Two nonexclusive mechanisms are proposed to account for the replication of the other parental strand. In some cases, before completion of a round of displacement synthesis initiated at one end of the Ad2 duplex, a second initiation will occur at the opposite end. In these doubly initiated molecules, both parental strands serve as templates for displacement synthesis. Two type II molecules are generated when the oppositely moving displacement forks meet. Alternatively, displacement synthesis may proceed to the end of the Ad2 duplex, resulting in the formation of a daughter duplex and a parental single strand. Replication of the displaced parental strand is then initiated at or near its 3′ terminus, producing a type II molecule. Daughter strand synthesis proceeds in the 5′ to 3′ direction in type II molecules generated by either mechanism, and completion of synthesis results in the formation of a daughter duplex.     

DNA endonuclease activities associated with melanoma cell chromatin

Chromatin-associated DNA endonucleases, extracted from Cloudman mouse melanoma cell nuclei, were separated on isoelectric focusing into seven fractions in two widely separated groups pH 3.4–5.4 and 7.5–9.3, each active on calf thymus DNA. All fractions in the former group, pI's 3.4, 4.4 and 5.4, produced at least one single-strand scission per molecule on circular duplex phage PM2 DNA, and transformed circular single-stranded phage fd DNA into linear strands of uniform length. In the second group there was no detectable activity against PM2 DNA, but two fractions pI's 7.5 and 8.0 were active on fd DNA as above, whereas the other two, pI's 8.5 and 9.0 transformed fd DNA into a number of different sized, discrete segments. These results indicate that, even allowing for possible enzymatic identity of some of the isoelectrically separated forms, at least three different DNA endonucleases are associated with mouse melanoma cell chromatin.     

Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction

Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.     

Nuclease S1 cleavage and the primary structure of mitochondrial DNA.

The single-strand-specific nuclease S1 from Aspergillus oryzae rapidly converts superhelical mitochondrial DNA (African Green Monkey cells, Vero ATCC; CCL 81) into nicked circular DNA. These nicked mitochondrial DNA molecules contain two nicks, one in each strand. The phosphodiester backbones are cleaved during this reaction at or near sites that are alkali-labile. In a second slow reaction the circular mitochondrial DNA is converted into a linear duplex DNA. Permutation tests indicate that this linear DNA represents a nonpermutated collection of DNA molecules. These results suggest that two of the alkai-labile sites in the phosphodiester backbones of the mitochondrial chromosome are closely spaced on opposite strands and at specific positions.     

Replication of kinetoplast DNA of Crithidia acanthocephali. I. Density shift experiments using deuterium oxide

The protozoan Crithidia acanthocephali contains, within a modified region of a mitochondrion, a mass of DNA known as kinetoplast DNA (kDNA). This DNA consists mainly of an association of approximately 27,000 covalently closed 0.8-mum circular molecules which are apparently held together in a definite ordered manner by topological interlocking. After culturing of C. acanthocephali cells for 25 generations in medium containing 75% deuterium oxide, both nuclear DNA (rhonative, nondeuterated=1.717 g/cm3) and kDNA (rhonative, nondeuterated=1.702 g/cm3) increased in buoyant density by 0.012 g/cm3. The replication of the two DNAs was studied by cesium chloride buoyant density analysis of DNAs from exponentially growing cells taken at 1.0, 1.4, 2.0, 3.0, and 4.0 cell doublings after transfer of cells from D2O- containing medium into medium containing only normal water. The results obtained from analysis of both native and denatured nuclear DNAs indicate that this DNA replicates semiconservatively. From an analysis of intact associations of kDNA, it appears that this DNA doubles once per generation and that the newly synthesized DNA does not segregate from parental DNA. Fractions of covalently closed single circular molecules and of open circular and unit length linear molecules were obtained from associations of kDNA by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium gradient centrifugation. Buoyant density profiles obtained from these fractions indicate that: (a) doubling of the kDNA results from the replication of each circular molecule rather than from repeated replication of a small fraction of the circular molecules; (b) replication of kDNA is semiconservative rather than conservative, but there is recombination between the circles at an undefined time during the cell cycle.     

Naturally occurring cross-links in yeast chromosomal DNA.

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 X 10(8) were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of cross-links in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equilibrium banding in CsCI. Experiments with larger E. coli DNA molecules (Mn = 5.2 X 10(8)) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are small number of cross-links, one to four, permolecule.