DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition.

The values of Kd and Gibbs energy (delta G degrees) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+ (NH3)2OH](pT)7 and d[(Tp2)C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357-369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19-20-nucleotide fragments of the template appear to interact with the protein molecule.