EFFECTS OF MUTATIONS ON THE PARTITIONING OF DNA SUBSTRATES BETWEEN THE POLYMERASE AND 3'-5'-EXONUCLEASE SITES OF DNA-POLYMERASE-I (KLENOW FRAGMENT)

Site-directed mutagenesis and time-resolved fluorescence spectroscopy were used to evaluate the contributions of individual amino acid side chains to the binding of DNA primer-templates to the 3'-5' exonuclease site of the large proteolytic fragment (Klenow fragment) of DNA polym erase I. Mutations were introduced into side chains that have been sho wn crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease site. The wild-type residues were repla ced by alanine in each case. To assess the effects of the mutations on DNA binding, time-resolved fluorescence anisotropy measurements were performed on dansyl-labeled primer-templates bound to the mutant enzym es. In contrast to techniques that simply monitor the overall binding of proteins to DNA, the time-resolved fluorescence anisotropy techniqu e was used to determine the fractional occupancies of the polymerase a nd 3'-5' exonuclease active sites of Klenow fragment. Equilibrium cons tants describing the partitioning of DNA between the two active sites were obtained for nine different mutant enzymes bound to both matched and mismatched DNA sequences. Mutations of Leu361 and Phe473 caused th e largest effects, significantly destabilizing the binding of mismatch ed DNA substrates to the 3'-5' exonuclease site relative to DNA bound at the polymerase site, consistent with structural data showing that t he side chains of these residues are involved in intimate hydrophobic interactions with the 3' terminal and penultimate bases of the primer strand [Beese, L., and Steitz, T. A, (1991) EMBO J. 10, 25-33]. Mutati ons of the His660 and Glu357 side chains also resulted in significant effects on the binding of mismatched DNA to the 3'-5' exonuclease site . Surprisingly, mutation of Tyr497 increased the partitioning of misma tched DNA into the 3'-5' exonuclease site, suggesting that the tyrosin e side chain in the wild-type enzyme destabilizes substrate binding, d espite crystallographic data showing that Tyr497 is H-bonded to the DN A substrate, The effects of mutating the amino acid side chains that s erve as ligands to two divalent metal ions bound at the 3'-5' exonucle ase site, designated A and B, indicated that metal A also helps to bin d DNA to the 3'-5' exonuclease site. These results demonstrate that th e time-resolved fluorescence anisotropy technique can be used to quant ify the energetic contributions associated with each of the crystallog raphically defined DNA-protein contacts at the 3'-5' exonuclease site.