A. Richard Morgan

Native and denatured DNA, cross-linked and palindromic DNA and circular covalently-closed DNA analysed by a sensitive fluorometric procedure

Abstract Ethidium bromide intercalates duplex DNA with a 25 fold enhancement of its fluorescence. At pH 8 denatured DNA shows about 50% the fluorescence enhancement of duplex DNA due to intramolecular hydrogen-bonding. By raising the pH to 12 short intramolecular duplex regions can be selectively destabilized without altering long duplex DNA. This forms the basis for a sensitive assay for duplex DNA in the presence of denatured DNA. Cross-linked and palindromic DNA differ from normal duplex DNA by their spontaneous renaturation after a heat step with return of fluorescence. Covalently-closed circular DNA is similarly distinguished from open circular DNA.

The sense of naturally occurring superhelices and the unwinding angle of intercalated ethidium

Abstract Superhelical closed circular DNA is not only of widespread occurrence but has been extensively utilized in studying the conformation of DNA. Superhelix densities of these DNAs have routinely been calculated using the assumption that ethidium unwinds the primary helix by 12 °. This assumption has been questioned by Paoletti & LePecq (1971 a ) who proposed that ethidium winds the duplex by 13 °. In this paper we show by a combination of chemical and enzymatic means that ethidium unequivocally unwinds the duplex, but that the unwinding angle and consequently superhelix densities are probably between two and three times the previously assumed values.

Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.

Synthesis and DNA cleaving properties of hybrid molecules containing propargylic sulfones and minor groove binding lexitropsins

Abstract A series of hybrids incorporating propargylic sulfones and minor groove bindin goligopeptide carriers 2-11 were synthesized and their abilities to cleave DNAd have been demonstrated.

Design of DNA-cleaving molecules which incorporate a simplified metal-complexing moiety of bleomycin and lexitropsin carriers

Abstract The syntheses of 1a-c, functional models for bleomycin, which are composed of a simplified metal-complexing moiety of bleomycin and poly-N-methylpyrrole peptides are described. Their functional cleavage of DNA in the presence of reductants is demonstrated.

Interconversion of replication and recombination structures : implications for terminal repeats and concatemers

Replication and recombination structures can be interconverted by branch-migration. Using this simple concept a novel mechanism is proposed for generating concatemers through an initial single-strand DNA invasion into a duplex. Only DNAs with terminal repeats can form concatemers, and Herpes Simplex Virus DNA replication is considered in detail. The model is more parsimonious than other models such as Watsons for concatemer formation.

Thymine-guanine base pairing during transcription of polydeoxypyrimidines in vitro.

Abstract RNA polymerase from Escherichia coli catalyses poly(rG) synthesis in the presence of defined, repeating DNAs having only thymine and cytosine residues in one strand. Poly(rG) is the only ribohomopolymer formed. The reaction is strongly inhibited by 1 μ m -dATP or 1 μ m -ATP, and to a lesser extent by dAMP, CTP or UTP. With ATP and GTP present, the ribopolymer which is complementary to the polydeoxypyrimidine strand in the strict Watson-Crick sense is synthesized, as demonstrated earlier (Nishimura, Jacob & Khorana, 1964; Nishimura, Jones & Khorana, 1965; Morgan, 1970). Mn2+ is a better divalent cation than Mg2+ for poly(rG) synthesis, and the reaction is sensitive to actinomycin D or rifampicin. At 12 °C with d(T-C-C)n as template, a 1 1 complex is formed between the single-stranded DNA template and the newly synthesized poly(rG). This complex can be isolated in Cs2SO4 density gradients. These experiments indicate that thymine · guanine base pairs can form when the thymine residues are part of polydeoxypyrimidines also containing cytosine.

An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence

Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of lambda phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their proteinase inhibitors. The assay is based on ethidium bromide fluorescence, according to the following principles: (i) Ethidium bromide increases its fluorescence by 25-fold when it intercalates between base pairs of double-stranded DNA. (ii) Histones prevent this large increase in fluorescence by binding with high affinity to DNA thus blocking ethidium bromide intercalation. (iii) A proteinase that digests histones will make more DNA available for ethidium bromide intercalation, thereby producing an increase of fluorescence. Proteinase activity can easily be determined, in the presence of a DNA/histone complex, from the rate of ethidium fluorescence increase. In contrast, activity of a proteinase inhibitor is quantitated by the inhibition of fluorescence gain in the presence of a known amount of proteinase. This assay is rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow quantitation of nanogram amounts of various broad-specificity proteinases and their inhibitors. We show some possible applications of the assay (i) in testing column fractions during protein purifications, (ii) quantitation of alpha 1-antitrypsin in human serum, and (iii) detection of proteinase activity in cell extracts.

Conformational isomerization of the Holliday junction associated with a cruciform during branch migration in supercoiled plasmid DNA.

The variable positions of a branch-migrating cruciform junction in supercoiled plasmid DNA were mapped following cleavage of the DNA with bacteriophage T7 endonuclease I. T7 endonuclease I specifically cleaved, and thereby resolved, the Holliday junction existing at the base of the cruciform in the circular bacterial plasmid pSA1B.56A. Cruciform extrusion of cloned sequences in pSA1B.56A (containing a 322 base-pair inverted repeat insert composed of poxvirus telomeric sequences) topologically relaxed the plasmid substrate in vitro. Thus, numerous crossover positions were identified within the region of cloned sequences, reflecting the range of superhelical densities in the native plasmid preparation. Endonuclease I-sensitive crossover positions, mapped to both strands of the viral insert following the T7 endonuclease I digestion of either plasmid preparations or individual topoisomers, were regularly separated by approximately ten nucleotides. The appearance of sensitive crossovers every ten nucleotides corresponds to a change in linking difference (delta Lk) of +/- 2 in the circular core domain of the plasmid during branch point migration. In contrast, individual topoisomers of a plasmid preparation differ in linking number in increments of +/- 1. Thus, the observed linearization of each individual topoisomer following enzyme treatment, as a result of resolution of the crossovers associated with each topoisomer, showed that branch point migration to sensitive crossover positions must have occurred facilely. T7 endonuclease I randomly resolved across either axis of the cruciform, though some discrimination (related to the sequence specificity of the enzyme) was observed. The ten-nucleotide spacing between sensitive crossover positions is accounted for by an isomerization of the cruciform junction on branch point migration. An hypothesis is that this isomerization was imposed upon the cruciform junction by the change in helix twist (delta Tw) in the two branches that compose the topologically closed, circular domain of the plasmid. T7 endonuclease I may discriminate between the various isomeric forms and cleave a sensitive conformation that appears with every turn of branch migration which leads to the extrusion, or absorption, of two turns of helix from the circular core.

The isolation of a high molecular weight terminal deoxynucleotidyl transferase from calf thymus

Summary A new terminal deoxynucleotidyl transferase (TDT) 1 has been isolated from calf thymus of higher molecular weight than that originally isolated by Bollum (1962) . The enzymes are probably metabolically related as one or the other is found depending on the purification procedure. However a direct conversion from one to the other has not been achieved. Although most of the enzymatic properties are very similar in contrast the new TDT also has the ability to use a template. This suggests that TDTs may be proteolytic degradation products of template-requiring DNA polymerases. Thus the finding of TDTs in a variety of rapidly metabolizing cells could be due to the uncovering of proteolytic activity rather than the synthesis of a new class of template-independent polymerases.