The culture medium of Pseudomonas BAL 31 contains endonuclease activities which are highly specific for single-stranged DNA and for the single-stranded or weakly hydrogen-bonded regions in supercoiled closed circular DNA. Exposure of nicked DNA to the culture medium results in cleavage of the strang opposite the sites of preexisting single-strand scissions. At least some of the linear duplex molecules derived by cleavage of supercoiled closed circular molecules contain short single-stranded ends. Single-strand scissions are not introduced into intact, linear duplex DNA or unsupercoiled covalently closed circular DNA. Under these same reaction conditions, 0X174 phage DNA is extensively degraded and PM2 form I DNA is quantitatively converted to PM2 form III linear duplexes. Prolonged exposure of this linear duplex DNA to the concentrated culture medium reveals the presence of a double-strand exonuclease activity that progressively reduces the average length of the linear duplex. These nuclease activities persist at ionic strengths up to 4 M and are not eliminated in the presence of 5% sodium dodecyl sulfate. Calcium and magnesium ion are both required for optimal activity. Although the absence of magnesium ion reduces the activities, the absence of calcium ion irreversibly eliminates all the activities.

synopsisThe sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sedimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments.When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in accord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7-8%) of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed Xbzbsc DNA circles. Possible explanations for some of the above results are discussed.A corresponding result is observed in the intrinsic viscosity vs. pH curve.

SynopsisCovalently closed intracellular DNA obtained from Pseudomonas BAL 31 20 min after infection with PM2 phage has been shown to be heterogeneous in superhelix density. Analytical band sedimentation, in the presence of low concentrations of ethidium bromide, has been carried out on fractions centripetal and centrifugal to the mode of a single band of closed circular DNA in a preparative propidium iodideCsC1 buoyant density gradient. Different average sedimentation rates, as well as different band shapes, have been observed for upper and lower fractions centrifuged at a dye concentration near the minimum in so versus ethidium bromide concentration titrations performed on DiVA from proximate intermediate fractions. Similar differences, although not as pronounced, have been obtained at a dye concentration corresponding to a point in the steep region of the titrations. Differential band sedimentation experiments performed on the same fractions have confirmed these results.Differential band sedimentation experiments on similarly fractionated mature PM2 I DNA (closed circular form) have shown slight differences in the relative sedimentation rates of upper and lower fractions at dye concentrations corresponding to the steep regions in the titrations. The same experiments, when performed on nicked circular DNA obtained from heating both the mature and intracellular fractions, showed no evidence of differences in sedimentation coefficients. Such results may indicate slight heterogeneity in the superhelix density of viral PM2 I DNA; however, the degree of this heterogeneity would be somewhat less than that of the intracellular DNA.The possibility that superhelix density heterogeneity may arise from displacement loops, which have been found at low levels in intracellular PM2 DNA, has been subjected to experimental tests. Unless such structures are originally present and removed by the isolation procedure, this possibility may be rejected. 953

No abstract