Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity

The
process of DNA replication is carried out with high efficiency
and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10
different subunits that coordinates simultaneous replication on the
leading and lagging strands. The 1160-residue Pol III alpha subunit
is responsible for the polymerase activity and copies DNA accurately,
making one error per 105 nucleotide incorporations. The
goal of this research is to determine the residues that contribute
to the activity of the polymerase subunit. Homology modeling and the
computational methods of THEMATICS and POOL were used to predict functionally
important amino acid residues through their computed chemical properties.
Site-directed mutagenesis and biochemical assays were used to validate
these predictions. Primer extension, steady-state single-nucleotide
incorporation kinetics, and thermal denaturation assays were performed
to understand the contribution of these residues to the function of
the polymerase. This work shows that the top 15 residues predicted
by POOL, a set that includes the three previously known catalytic
aspartate residues, seven remote residues, plus five previously unexplored
first-layer residues, are important for function. Six previously unidentified
residues, R362, D405, K553, Y686, E688, and H760, are each essential
to Pol III activity; three additional residues, Y340, R390, and K758,
play important roles in activity.