Helix-random coil transitions in DNA homopolymer pairs

The thermal stability (Tm) of each homopolymer pair has been determined over a wide range of ionic strength and pHm is determined by the base pair stability and by the pKa′ of the base titrated during the transition; thus there is a relation between pHm–pKa′ and Tm.

Abstract

Conditions have been found where simple helix-random coil transitions occur for each of the DNA homopolymer pairs dGdC,† dIdC and dIdBC¯ and it is shown that these correspond to the melting of dG : dC, dI : dC and dI : dBC¯ base pairs. The complementary strands of the homopolymer pairs can be separated and identified by density-gradient centrifugation in Cs2SO4 at alkaline pH. Although this technique demonstrates complete separation for homopolymer pairs, incomplete separation results when dAT : dABU¯ copolymer hybrids are studied in the same way. The isolated homopolymer strands have been used to re-form dIdC and dIdBC¯; mixing experiments show maximum interaction for a purine: pyrimidine mole ratio of 1, as expected for dI : dC and dI : dBC¯ base pairs. The changes in absorbancy and in viscosity which accompany the thermal transition are qualitatively like those of natural DNA's. However, there are some interesting quantitative differences. The width of the melting zone is independent of salt concentration over at least a 20-fold range. This contrasts with the behavior of natural DNA's whose melting curves broaden in low salt concentrations, and also contrasts with the dAT copolymer which shows a sharp transition in low-salt but a broad transition in high salt concentrations. The thermal stability (Tm) of each homopolymer pair has been determined over a wide range of ionic strength. After melting, the homopolymer pairs re-form on cooling below Tm. The kinetics are second-order for most of the reaction and the initial rate increases with Tm–T but decreases as the salt concentration is reduced, much like the results of Ross & Sturtevant (1960) for rA : rU. In contrast, re-formation of dAT : dAT is rapid even at low ionic strength, presumably because the double helix re-forms from a single strand. The alkaline transition for each homopolymer pair has also been investigated. At constant ionic strength and temperature, pHm is determined by the base pair stability and by the pKa′ of the base titrated during the transition; thus there is a relation between pHm–pKa′ and Tm. On cooling through the transition zone, the viscosity rises as the homopolymer pair re-forms. However, the viscosity continues to increase gradually on further cooling down to 0°C; this probably is due to end-to-end aggregation of helical units.