The highly conserved GXD sequence present in the Mycobacterium tuberculosis DNA Polymerase I corresponds to a hinge region in the finger subdomain connecting M and N helices of E.coli pol I. An examination of the crystal structures of pol I family polymerases reveals that the invariant aspartate of the hinge forms a salt bridge with the conserved arginine of the O-helix and an H–bond with Q708. In order to clarify the role of this region, we generated and characterized conserved and nonconserved mutant derivatives of this aspartate, the preceding glutamate and the Q in TB pol I. For comparison, D732A mutein of pol I was also included. The muteins representing conserved aspartate (D707 of TB pol I or D732 of pol I) showed a strong Km(dNTP) effect and minor alteration in Kd(DNA), with about 10-20 fold decrease in overall catalytic efficiency. The TB muteins, E706A and Q683A have less pronounced deviations from the wild-type enzyme. Further examination of D707A of TB pol I showed no alteration in the processivity or the dideoxynucleotide sensitivity patterns. However, both TB pol D707A and homologous E.coli D732A failed to form a stable E·DNA·dNTP ternary complex. These results suggest that the aspartate in the hinge region is catalytically important and is required for dNTP binding and in the formation of a prepolymerase ternary complex.