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.
Highlights
Bacteriophage T7-induced DNA polymerase is composed of a 1:1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin
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 inthe native holoenzyme for modification by N-ethylmaleimide
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
Summary
Bacteriophage T7-induced DNA polymerase is composed of a 1:1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin. Subsequent analysis demonstrated occurrence of related proteins in a variety of organisms including other prokaryotes, yeast, plants, and mammals [10] Such studies led to proposed roles for the small protein in regulation of nitrogen metabolism (1I), control of C 0 2fixation reactionsinchloroplasts [12], participationinsulfateand phosphate group transfer reactions[13,14,15,16,17], and as a powerful reductant of disulfide bonds in proteins[18]. These activities have generally been attributed to the active center disulfide of thioredoxin, Cys-Gly-Pro-Cys, which can undergo reversible oxidation-reduction andwhich is invariant inE . These activities have generally been attributed to the active center disulfide of thioredoxin, Cys-Gly-Pro-Cys, which can undergo reversible oxidation-reduction andwhich is invariant inE . coli, yeast, and calf liver proteins [10]
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