Melting of the 5' nuclease domain of Pol 1 overlaps with the Klenow fragment. The 5' nuclease domain of Taq denatures as a separate peak, 10 degrees C before the Klentaq domain. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T(m) of 37 and 100 degrees C respectively at pH 9.5. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis.

Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy.

Klenow fragment error vs dna pol i