T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups.

Abstract:

Bacteriophage T7-induced DNA polymerase is composed of a 1:1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin. Preparation of active subunits in the absence of sulfhydryl reagents indicates the reduced form of thioredoxin is sufficient for formation of the active holoenzyme. The oxidized form of thioredoxin, thioredoxin modified at one active site sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified at both active site sulfhydryls by N-ethylmaleimide, are all inactive, being defective in complex formation with gene 5 protein. Thioredoxin sulfhydryl groups present in native T7 DNA polymerase do not appear to be involved in an intersubunit disulfide bond; one and probably both sulfhydryls are available in the native holoenzyme for modification by N-ethylmaleimide. Furthermore, DNA substrates alter the reactivity of thioredoxin cysteines within the holoenzyme with respect to this reagent. Substrates for the single strand exonuclease enhance the reactivity of thioredoxin sulfhydryl groups while those for the polymerase or double strand exonuclease functions afford protection. It, therefore, seems likely that thioredoxin sulfhydryl groups are present in the reduced state within the native polymerase.

Polymerases:

T7

Topics:

Historical Protein Properties (MW, pI, ...), Accessory Proteins/Complexes, Exonuclease Activity, Source / Purification

One line summary:

This paper concludes that thioredoxin cysteines are in the reduced form in the native T7 polymerase and suggests that thioredoxin sulfhydryl groups are located at or near the DNA binding site of the polymerase.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 Adler S1983 Molecular Weight 9.2E+04 Dalton
T7 Adler S1983 3-5' Exonuclease (proofreading) Yes
T7 Adler S1983 Cloned or native Native organism
T7 Adler S1983 3-5' Exo Specific Activity 9.1 umol/min/mg
T7 Adler S1983 Tagged No
T7 Adler S1983 Full length or truncated Full length
T7 Adler S1983 Specific Activity 10.7 units/mg
T7 Adler S1983 Other accessory protein(s) E. coli thioredoxin

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