E. coli DNA polymerase IV is one of two Y family polymerases involved in copying damaged DNA, which is a major mechanism of DNA damage tolerance. DinB is specialized for incorporating nucleotides across from adducts on deoxyguanine at the N2 position, with its cognate lesion bearing a furfuryl adduct. Biochemical characterization of enzymes is often focused on those amino acid residues that are in the active site and make direct contact with the substrate. In the case of DNA polymerases this usually includes negatively charged aspartate and glutamate residues that position the metal ions (2Mg2+) correctly to both activate and facilitate nucleophilic attack of the primer hydroxyl group onto the ?-phosphate group of the incoming deoxynucleotide triphosphate. Often overlooked are the second shell residues, which, although they are not in direct contact with the substrate, are in contact with the first shell residues, those amino acid residues that contact the substrate. We have identified second-shell residues in DinB using the active-site predictors THEMATICS and POOL. By carrying out site-directed mutagenesis and determining the change in activity of the resulting DinB variants, we find a wide range of effects of second-shell residues. Characterizing the roles of second shell residues will lead to a better understanding of enzyme function and could lead to the ability to manipulate enzyme activity and specificity.