LARGE-SCALE OPENING OF A-MOLECULES IS SUPPRESSED BY SALT(T RICH REGIONS WITHIN SUPERCOILED DNA)

Large-scale cooperative helix opening has been previously observed in A+T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circ les. We have found that localised large-scale stable melting in superc oiled DNA is strongly suppressed by modest elevation of salt concentra tion, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A+T rich region surrounding the inverted repeat (the C- type pathway, first observed in the extrusion of the ColE1 inverted re peat) is elevated by addition of salt. The temperature required for ex trusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A+T rich regions in supercoiled DNA exhibit hyperreactivity towards o smium tetroxide as the temperature is raised; this reactivity is stron gly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of tempera ture. Above 30 mM NaCl the reactivity of the ColE1 sequences is comple tely supressed at normal temperatures. 3. Stable helix opening transit ions in A+T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become pr ogressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitio ns shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistic al mechanical simulation of the data indicate that the cooperativity f ree energy for the transition is unaltered by addition of salt, but th at the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentration s of salt strongly suppress the large-scale helix opening of A+T rich regions, even at high levels of negative supercoiling. While the openi ng at low salt concentrations may reveal a propensity for such transit ions, spontaneous opening is very unlikely under physiological conditi ons of salt, temperature and superhelicity, and we conclude that prote ins will therefore be required to facilitate opening transitions in ce llular DNA.