PMID:23012374

Citation	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
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 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.
Links	PubMed PMC3501039 Online version:10.1074/jbc.M112.409235
Keywords	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
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT7:DPOL		GO:0003887: DNA-directed DNA polymerase activity	ECO:0000314:		F		Fig 3	complete CACAO 11449
9CAUD:A0A024AZS4		GO:0097619: PTEX complex	ECO:0000315:		C		Mutagenesis of T7 gene 5 and thioredoxin.	complete CACAO 11451

References

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