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Tran, NQ, Lee, SJ, Akabayov, B, Johnson, DE and Richardson, CC (2012) Thioredoxin, the processivity factor, sequesters an exposed cysteine in the thumb domain of bacteriophage T7 DNA polymerase. J. Biol. Chem. 287:39732-41


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 10 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 dA(350)/dT(25). On this primer-template gp5/trx in which Cys-313 or Cys-275 is 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.


PubMed PMC3501039 Online version:10.1074/jbc.M112.409235


Bacteriophage T7/enzymology; DNA-Directed DNA Polymerase/genetics; DNA-Directed DNA Polymerase/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli/virology; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Thioredoxins/genetics; Thioredoxins/metabolism; Viral Proteins/genetics; Viral Proteins/metabolism



Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status


GO:0003887: DNA-directed DNA polymerase activity



Fig 3

CACAO 11449


GO:0097619: PTEX complex



Mutagenesis of T7 gene 5 and thioredoxin.

CACAO 11451


See also


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