Horace B. Gray

Extracellular nucleases of Alteromonas espejiana BAL 31.IV. The single strand-specific deoxyriboendonuclease activity as a probe for regions of altered secondary structure in negatively and positively supercoiled closed circular DNA

The dependence of the initial rate of introduction of the first single-chain scission (initial nicking rate) into covalently closed circular phage PM2 DNA by the single strand-specific nuclease from Alteromonas espejiana BAL 31 upon the superhelix density (sigma) of the DNA has been examined. The initial nicking rate decreases with decreasing numbers of negative superhelical turns (decreasing values of -sigma), which behavior is characteristic of other single strand-specific nucleases as reported earlier. In contrast to earlier work, the initial nicking rates of closed circular DNAs by the action of the Alteromonas nuclease have been shown to be readily measurable at values of -sigma as low as 0.02. However, even at the elevated concentrations of enzyme and extended digestion periods required to cause nicking at an appreciable rate at near-zero values of sigma, closed circular DNA containing very few superhelical turns (form IO DNA) is not cleaved at a detectable rate. When this DNA is rendered positively supercoiled by ethidium bromide (EtdBr), it is not affected by the nuclease until very high positive values of sigma are attained, at which low rates of cleavage can be detected at elevated enzyme concentrations. The effects of EtdBr on the enzyme activity have been tested and are entirely insufficient to allow the interpretation of zero nicking rates as the result of inhibition of the nuclease activity by the dye. Positively supercoiled DNA is concluded not to contain regions having significant single-stranded character until values of sigma are reached which are very much higher than the values of -sigma for which negatively supercoiled DNAs behave as if they contain unpaired or weakly paired bases.

Catenanes of closed circular intracellular PM2 phage DNA.

Abstract The closed circular intracellular DNA isolated from Pseudomonas BAL 31 20 min after infection with PM2 phage contains 5% catenated forms. The existence of catenated dimers is demonstrated by isolation of this species as a middle band in a propidium iodide—CsCl gradient, by electron microscopy and by sedimentation studies. The sedimentation patterns of the doubly closed dimer and the singly closed dimer in both neutral and alkaline media have been investigated in detail. The sedimentation pattern of the doubly nicked species has been examined in a neutral medium. Observed sedimentation coefficients are in sufficiently good agreement with those calculated from an existing empirical relationship to extend the useful range of applicability of this relationship to higher superhelix densities and lower molecular weights.

Type I DNA topoisomerases from mammalian cell nuclei interlock strands and promote renaturation of denatured closed circular PM2 DNA

Type I DNA topoisomerases from mouse ascites cell nuclei and from rat liver cell nuclei act on denatured viral closed circular PM2 DNA to produce molecules with a highly contracted structure as well as fully duplex non-supercoiled covalently closed circular molecules. Highly contracted DNA molecules contain a novel type of topological linkage in which a strand in one region of the double-stranded molecule passes between the strands in another region of the circular molecule one or more times. Since it is also found that the action of the topoisomerase promotes renaturation of complementary strands in denatured closed circular DNA, it is suggested that formation of contracted DNA structures proceeds through renatured, duplex intermediates with highly negative superhelix densities that contain small single-stranded regions.