Ion induced coil to helix transition of dialyzed poly d(A-T) from Cancer borealis.

Abstract

Naturally occurring poly d(A-T) from testes of Cancer borealis was prepared starting with a fibrous DNA-RNA complex. The complex was chromatographed on a methylated albuminkieselguhr column, fractions with a high poly d(A-T) content were collected, treated with a large amount of pancreatic ribonuclease and rechromatographed on methylated albumin-kieselguhr. The purest preparations obtained had 0.74 mole % of cytosine and 0.71 mole % of guanine. Controlled dialysis of the poly d(A-T) against distilled water resulted in a hyperchromic shift closely resembling thermal melting. Graded addition of bivalent metal (preferably Mg++) to the dialyzed polymer at room temperature reversed the hyperchromic shift. The term “ionic freezing” is suggested for the transition from coil to helix induced by the addition of metal. The midpoint of this transition, designated If, is as characteristic for a preparation of DNA as Tm. In the Mg++ to P ratio range of 0 to 0.5 both values are directly proportional, and the change in If of 10 μM Mg++ corresponds to the change in Tm of 5°. In this Mg++ to P ratio range, the metal is bound stoichiometrically. Divalent cations differ in their efficiency of reforming helix and are arranged in the following series starting with the least effective (highest Tm at 50 μM): Zn>Mg>Ba>Ca>Mn>Co. The ion-induced coil to helix transition is pH dependent. The ion-induced transition may also be accomplished in 8 M urea provided the temperature is lowered to – 0.5°. From several considerations it seems probable that the bases participate in metal binding. Partial separation of the strands of poly d(A-T) after dialysis and their reunification by the added Mg++ was confirmed by electron microscopy. Biological implications of this finding were discussed in terms of the possible role of Mg in the initiation of strand separation.