Thermodynamics of the Helix‐Coil Transition of (dG‐dC) Oligomers

Abstract

The dissociation of short double-helical DNA with the alternating sequence (dG-dC),. (dG-dC), into the corresponding single strands was studied as a function of chain length, oligomer concentration and temperature in 1.0 M NaCl, pH 7.2 by hypochromicity and equilibrium centrifugation. The melting curves are analyzed according to an "all-or-none'' model and the following thermodynamic parameters were deduced.The change in free energy for formation of a dG . dC base pair adjacent to another one, AG:, ---2.2 kcal/mol at 37 "C; the corresponding change in enthaply, AH:, = -11.1 5 0.5 kcal/mol and in entropy, AS:, = -27.9 & 1 cal . K-l * mol base pair-l. The stability constant, s, is 104 f 5 at 25 "C. The formation of the first base pair from two single strands involves a nucleation parameter, j3, = 1.6 M-l a t 25 "C, which is similar to the corresponding values determined for different RNA oligomers. The enthaply change associated with the formation of the first base pair, -AH;,, = + 3 & 5 kcal/mol which is very close to zero.These thermodynamic parameters are compared with those for the dA . dT base pair. At physiological conditions, i t is estimated that the stability of one dG * dC pair is very close to that of two dA . dT pairs. The data imply that the opening of a unique sequence of DNA in a cell is a very improbable event.The thermodynamics of base-pair formation in DNA are of considerable biological interest. Processes like replication, recombination or recognition of specific base sequences very probably involve an opening of the double helix. Experimental techniques like the hybridization of nucleic acids also depend on the relative stability of base pairs.The higher stability of the dG * dC base pair as compared to the dA * dT pair has been known for a long time and can be deduced, for example, from the dependence of the melting temperature (T,,,) on the dG . dC content of DNA's from different organism, [ 11. Quantitative thermodynamic parameters for the two base pairs involved in the helix-coil transition of natural DNA, which is essentially a mixture of many different sequences, is rather difficult t o obtain. The situation is made somewhat easier by studying the melting of corresponding polymers with simple repeating sequences [2].On the other hand, information about the free energy and enthalpy of base pair formation and the nucleation parameters involved in the initiation of the double helix can be obtained in a rather direct way from melting experiments using short oligomers of defined length and sequence [3]. A considerable body of thermodynamic data on A . U and G * C base pairs has been accumulated during the last few years by studying synthetic and natural RNA oligomers [4-lo]. With respect to DNA the situation is less satisfying and most of the results are restricted to the dA dT base pais, obtained from the melting of oligomers of the type (dA),. (dT)m [11,12] or (dA-dT), [13,14]. I n order to establish the thermodynamic parameters of the dG . dC base pair in the double helix, experiments on t...

Predicting DNA duplex stability from the base sequence.

“…This calculation assumes that the free energy of a duplex results from the sum of its nearest-neighbor interactions. We have assigned a helix initiation free energy (Ag1) of 5 kcal for duplexes containing G-C base pairs and 6 kcal for duplexes composed exclusively of A'T base pairs (7)(8)(9)29). For a duplex formed from a self-complementary sequence, Agsym equals 0.4 kcal, whereas for a duplex formed from two complementary sequences, Agsym equals 0.…”

Thermodynamics of the B to Z transition in poly(m5dG-dC).

“…A cooperative length of 25 bp was reported for the trifluorethanol-induced B-Z transition in poly(dG-dC) (20). Previous investigators argued that the B-Z transition is entropically driven (7,8,13 [6][7][8] kcal/mol for the Z-B transition of (m5dC-dG)3 in solutions containing 20-40% methanol, and an entropy change of [21][22][23][24][25][26][27] cal/K-mol. These values are both much larger than ours and of opposite sign.…”

Reactions in vitro of the DNA polymerase‐primase from Xenopus laevis eggs

“…At a low enzyme/DNA ratio, R limited section of the template is copied, even after long incubation times, suggesting that barriers exist on the DNA template, which prevent the progression of the enzyme around the single-stranded D N A circle. At relatively high enzyme concentration several enzyme molecules are probably simultaneously engaged in DNA synthesis on one single-stranded An experiment with fd DNA, specifically primed by a hybridized Hue11 fragment, has shown that several of these stop sites coincide with stable [33] secondary structures in the DNA template. In the fd DNA region, about 400 nucleotides upstream from the 3' OH end of the hybridized HaeIIC fragment, we found only one stable loop-and-stem structure between nucleotides 4345 and 4371 [31].…”