A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Polβ and TdT to investigate the region of Polβ required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termedTDT POLB , in which the DNA region encoding amino acids (aa) 154-212 of TdT was replaced by the corresponding region encoding aa 1-60 of Polβ. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of Polβ represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT Polβ protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Polβ are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Polβ possesses ssDNA-binding activity [Kumar et al., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar et al., Biochemistry 29 (1990b) 7156-7159; Prasad et al., J. Biol. Chem. 268 (1993) 22746-22755].