Spermidine-Deoxyribonucleic acid interaction in vitro and in Escherichia coli

Abstract

The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 105 M-I in 1 mM sodium cacodylate, pH 7.5, to 3 x 102 M-' in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered.The function of the naturally occurring polyamines continues to be the elusive subject of efforts involving a wide range of experimental systems. One approach to this problem has been to attempt to isolate native complexes between polyamines and various cellular constituents. A wide range of polyamine-macromolecular complexes has been identified (see reviews by Stevens [37], Cohen [7], and Tabor and Tabor [41].) However, where adequate controls had been performed, artifactual associations were readily demonstrated. Thus the polyamine content of Escherichia coli ribosomes was highly dependent on the method of isolation (40), exogenous nucleic acids greatly reduced the amount of [3H]spermine bound to endogenous nucleic acids of an E. coli lysate (20), and spermidine in E. coli that appeared bound to a membrane fraction completely equilibrated with [14C]spermidine added at the time of lysis (27). Major alterations of the polyamine levels in E. coli and phage T4 (1, 2) were brought about by modifications in the composition of the growth medium. It appears that the chemical activity of the polyamines is particularly sensitive to seemingly small alterations in environment, and therefore the possibility of polyamine exchange in any biological system needs rigorous assessment.An alternative approach toward elucidating polyamine function has been to exploit in vitro binding studies involving various polyamines and their suspected reactants. Interactions of putrescine, spermidine, and spermine with nucleic acids have been demonstrated in vitro (12,36,39) and have led to numerous proposals concerning in vivo associations (3,8,20,29,36). However, inorganic ions compete with polyamines for binding to nucleic acids (4, 17, 29). There is less association of polyamines with nucleic acids as the ionic strength increases (17,21,36,39), but the extent of this salt-induced dissociation has not been quantitated. Analyses of the inorganic-salt concentration within...

Aggregation of DNA by analogs of spermidine: enzymatic and structural studies

“…In this paper, we have shown that the effectiveness of these spermidine analogs in aggregating supercoiled DNA correlates with their ability to support cell growth, opening the possibility that compaction of DNA could be a physiologically relevant role of spermldine in prokaryotic and eukaryotic cells. The compaction of DNA by polyamines in vitro, albeit an attractive model, is clearly an oversimplification because of the high ionic strength found in living cells (45). However, intracellular compartmentation of polyamines, together with macromolecular crowding and the presence of DNA-binding proteins, could aid in this process.…”

Ligand-Induced DNA Condensation: Choosing the Model

“…However, this picture is incomplete without the curves of ligand binding coupled to DNA aggregation-resolubilization. The polyamine binding to DNA was measured previously at small ligand concentrations (28,29) and at the intermediate concentrations in the regime of DNA condensation (30,31). The determination of polyamine binding curves in the whole interval of ligand concentrations including the onset of DNA aggregation and the beginning of resolubilization is carried out in the present study.…”

Spermine induced reversible collapse of deoxyribonucleic acid-bridged nanoparticle-based assemblies

“…The negative control is not affected by spermine concentrations as high as 10 mM, demonstrating that any collapse or change in conformation of the DNA attached to single 80 AuNPs does not contribute to the observed shifts in the plasmon peak. For the polyamine spermidine, counter-ions can compete for binding sites on the DNA [42], inhibiting DNA collapse. To investigate if a similar competition process occurs here, spermine was added to the buffer without sodium chloride.…”