A THUMB SUBDOMAIN MUTANT OF THE LARGE FRAGMENT OF ESCHERICHIA-COLI DNA-POLYMERASE-I WITH REDUCED DNA-BINDING AFFINITY, PROCESSIVITY, AND FRAMESHIFT FIDELITY

In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been sugg ested to interact with the duplex template-primer (Beese, L.S., Derbys hire, V. and Steitz, T.A. (1993) Science 260, 352-355). The present st udy investigates the properties of Klenow polymerase containing a 24-a mino acid deletion (residues 590-613) that removes a portion of the ti p of the thumb. The mutant polymerase has relatively normal dNTP bindi ng and catalytic rate. However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced, Although the mutant p olymerase has relatively normal base substitution fidelity, it has str ongly reduced frameshift fidelity, being especially error-prone for si ngle nucleotide addition errors in homopolymeric runs. The addition er ror rateincreases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage. The se observations suggest a role for the tip of the thumb of Klenow poly merase in determining DNA binding, processivity and frameshift fidelit y, perhaps by tracking the minor groove of the duplex DNA. The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory su bunit that affects these same properties.