A thermodynamic study on the formation and stability of DNA duplex at transcription site for DNA binding proteins...

Using isothermal titration calorimetry (ITC), we studied the thermodynamic parameters of the 15-mer duplex dsDNA [d(GAGATGACTCATCTC)].[d(GAGATGAGTCATCTC)] formation from its two complementary single strands (S1 and S2) over a range of temperatures. The two complementary single strands d(GAGATGACTCATCTC) (herein called S1) and d(GAGATGAGTCATCTC) (herein called S2) containing palindromic sequences may assume ordered structures at low temperatures, which made the duplex dsDNA formation rather complicated. The thermodynamic parameters for the duplex formation, such as the binding constants (Kb), the enthalpies (delta H0), the free energies (delta G0), the entropies (delta S0) are strongly temperature-dependent. The thermally-induced disruptions of the duplex and its two complementary single strands, S1 and S2, were measured using differential scanning calorimetry (DSC) and CD spectroscopy, the results demonstrate that the DNA duplex is very stable, and its component single strands have an ordered structure at low temperature. This 15-mer specific sequence DNA may act as recognition site for DNA binding proteins GCN4 and plays a key role in transcription regulation of gene expression. Our analyses of the thermodynamic data suggest that the duplex formation is a coupled process between conformational transitions in the two single strands and their binding to form duplex dsDNA.