A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Pol beta and TdT to investigate the region of Pol beta 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 termed TDT::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 POL beta. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of POL beta 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::Pol beta protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Pol beta are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Pol beta 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].