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 10-subunit complex that coordinates simultaneous replication on the leading and lagging strands. The 1160-residue pol III alpha subunit copies DNA accurately, making one error per 105 nucleotide incorporations. The goal of this research is to determine the residues that contribute to this high fidelity and activity of the polymerase subunit. Homology modeling and the computational methods of Theoretical Microscopic Anomalous Titration Curve Shapes (THEMATICS) and Partial Order Optimum Likelihood (POOL) have been used to predict functionally important amino acid residues. Site-directed mutagenesis and primer extension assays have been used to validate these predictions. This and previous work show that the top 11 residues predicted by POOL, which includes the three catalytic aspartates and two distal residues, are important for function. Fluorescence binding assays, thermal denaturation assays and steady-state kinetic assays are being performed to understand better the contribution of these residues to the function of the polymerase. Work supported by NSF-MCB-0843603, NSF-MCB-0845033, NSF-MCB-1158176 and Research Corporation for Science Advancement.