T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Abstract:

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.

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 S1979 3-5' Exonuclease (proofreading) Yes
T7 Adler S1979 Cloned or native Native organism
T7 Adler S1979 3-5' Exo Specific Activity 2580 units/mg
T7 Adler S1979 Tagged No
T7 Adler S1979 Full length or truncated Full length
T7 Adler S1979 Specific Activity 3370 units/mg
T7 Adler S1979 Other accessory protein(s) E. coli thioredoxin

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