DNA polymerase β:: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (k(pol)) and the apparent equilibrium dissociation constant of dNTP (K-d) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates, For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the K-d of dNTP. Although k(pol) was little affected, the catalytic efficiency k(pol)/K-d increased significantly owing to the decreases in K-d. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the K-d of dNTP appreciably but caused the k(pol) (and thus k(pol)/K-d) for incorrect dNTP incorporation to increase, As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates, In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filing synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed, However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected, A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.