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T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups
The Journal of Biological Chemistry
|June 10, 1983
Summary
The reduced form of Escherichia coli thioredoxin is sufficient for active T7 DNA polymerase holoenzyme formation. DNA substrates alter thioredoxin cysteine reactivity, suggesting reduced thioredoxin sulfhydryl groups in the native polymerase.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- Bacteriophage T7 DNA polymerase is a complex of gene 5 protein and E. coli thioredoxin.
- Thioredoxin's role in holoenzyme formation and activity is crucial but not fully understood.
Purpose of the Study:
- To investigate the role of thioredoxin sulfhydryl groups in T7 DNA polymerase activity.
- To determine the redox state of thioredoxin within the native holoenzyme.
Main Methods:
- Preparation of active T7 DNA polymerase subunits.
- Chemical modification of thioredoxin sulfhydryl groups using iodoacetate, methyl iodide, and N-ethylmaleimide.
- Assessing complex formation and enzyme activity after modification.
- Investigating the effect of DNA substrates on thioredoxin cysteine reactivity.
Main Results:
- The reduced form of E. coli thioredoxin is sufficient for active holoenzyme formation.
- Oxidized or modified thioredoxin is inactive and fails to complex with gene 5 protein.
- Native T7 DNA polymerase contains accessible thioredoxin sulfhydryl groups, not involved in intersubunit disulfide bonds.
- DNA substrates modulate the reactivity of thioredoxin cysteines towards N-ethylmaleimide.
Conclusions:
- Thioredoxin sulfhydryl groups are likely in a reduced state within the native T7 DNA polymerase.
- DNA binding alters the conformation of the holoenzyme, affecting thioredoxin reactivity.