Investigating the Nucleotide Selection Mechanism of Bacillus Stearothermophilus DNA Polymerase I

Abstract

The movement of the fingers subdomain of Bacillus stearothermophilus DNA polymerase I Large Fragment relative to a DNA substrate was monitored via Fӧrster Resonance Energy Transfer (FRET) during nucleotide (dNTP) selection. Changes in FRET were monitored in bulk solution and at the single molecule level in the presence of a variety of substrates and cofactors. The presence of the 3'‐hydroxyl at the primer terminus strongly influenced the ability of the polymerase to discriminate between correct and incorrect dNTPs. Using a deoxy‐terminated primer in Ca2+, the ground state dissociation constant (KdG) obtained for the correct dNTP was nearly 10‐fold lower than the KdG obtained for an incorrect dNTP. When a dideoxy‐terminated primer was used the difference in the KdGs was reduced to only 2‐fold. Using a deoxy‐terminated primer in Ca2+ with the D653A mutant, where the ability to bind the B‐metal ion is compromised, there was a complete loss of discrimination between correct and incorrect dNTPs. Using a dideoxy‐terminated primer in Mg2+, we observed a 10‐fold difference in the KdGs. Non‐hyrolyzable nucleotide analogs will be used to determine the difference in the KdGs using a deoxy‐terminated primer in Mg2+. When Mn2+ and a dideoxy‐terminated primer were used a 20‐fold difference in KdGs was observed favoring incorrect substrates over correct substrates. The changes in FRET efficiencies suggest the subdomain may adopt different substrate and cofactor dependent conformations. These results indicate that the 3'‐hydroxyl and the metal ions that coordinate the dNTP are integral to the fidelity of this polymerase and influence the way in which it interacts with its substrates. NIH Grant GM 063276−09