Thioredoxin, the processivity factor, sequesters an exposed cysteine in the thumb domain of bacteriophage T7 DNA polymerase. Academic Article uri icon

abstract

  • Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). Gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. Gp5 contains ten cysteines. Under non-reducing condition exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin-binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA350/dT25. On this primer-template gp5/trx in which C313 or C275 are replaced with serine have 50% and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. Gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle X-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.

publication date

  • September 25, 2012