Fig. 6.

(A) Crystal structure of T7 DNAP with the N-terminal 3′-5′ exonuclease domain (residues 1–187) colored in pink, the C-terminal polymerization domain (residues 188–698) colored in green, and thioredoxin colored in orange. Catalytic active site residues from the polymerization and exonuclease domains are shown as orange spheres, whereas Asp⁹⁰ is represented as yellow spheres (PDB ID code 1TK8) (9). (B) Polymerization and exonuclease activities of wild-type and mutant T7 DNAPs. The assay was performed as described in Materials and Methods using the 5′-labeled primer/template molecule sp1/sp1c+18 (15mer/33 mer) depicted on top of the figure, in the presence (+) or absence (−) of dNTPs and thioredoxin. Polymerization or 3′-5′ exonucleolysis is detected as an increase or decrease, respectively, in the size (15mer) of the 5′-labeled primer. After incubation for 10 min at 30 °C, samples were analyzed by 7 M urea/20% polyacrylamide gel electrophoresis and autoradiography. Asterisk indicates the 5′-³²P-labeled end of the primer strand.