An exposed cysteine of T7 gene 5 are critical for interaction with its processive factor, thioredoxin

DNA polymerase (gp5) of bacteriophage T7 is a non‐processive polymerase encoded by gene 5 of the phage. Escherichia coli encoded thioredoxin (trx) binds tightly to the thumb subdomain of gp5 (gp5/trx) and increases processivity to approximately 800 nucleotides per binding event. Gene 5 protein has ten cysteine residues, of which only Cys275 and Cys313 are exposed on the protein surface; both residues are located on a flexible loop of the thioredoxin binding domain. In the present study, we replaced either Cys275 or Cys313 with a serine to examine the role of these two exposed cysteines on the function of T7 DNA polymerase. Replacement of Cys275 with a serine (gp5‐C275S/trx) has little effect on DNA polymerase activity as compared with wild‐type gp5/trx. However, substitution of a serine for Cys313 decreases DNA polymerase activity 3‐ to 4‐fold on linear and circular primed DNA, respectively. In particular, substitution of either cysteine decreases activity of the altered gp5 10‐fold on linear DNA templates in the absence of thioredoxin. Biological and physical interaction studies indicate that gp5‐C313S fails to form a stable complex with thioredoxin. Probing the envelope structure of gp5‐C313S using small angle X‐ray scattering analysis reveals a distinct conformation from wild‐type gp5. The data suggest that Cys313 is important for a conformational interaction with thioredoxin and is likely a part of the thioredoxin binding domain surface. The thioredoxin binding domain not only interacts with trx but also with the primer‐template.