A SINGLE RESIDUE IN DNA-POLYMERASES OF THE ESCHERICHIA-COLI DNA-POLYMERASE-I FAMILY IS CRITICAL FOR DISTINGUISHING BETWEEN DEOXYRIBONUCLEOTIDES AND DIDEOXYRIBONUCLEOTIDES

Bacteriophage T7 DNA polymerase efficiently incorporates a chain-termi nating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for t his difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chai n is critical for selectivity. Replacing tyrosine-526 of T7 DNA polyme rase with phenylalanine increases discrimination against the four dide oxynucleotides by >2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discri mination against the four dideoxynucleotides 250- to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced p roperties for use in DNA sequence analysis.