Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein.

Abstract:

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.

Polymerases:

T7

Topics:

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

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 Hori K1979 3-5' Exonuclease (proofreading) Yes
T7 Hori K1979 Cloned or native Native organism
T7 Hori K1979 3-5' Exo Specific Activity 0.13 umol/min/mg
T7 Hori K1979 Tagged No
T7 Hori K1979 Full length or truncated Full length
T7 Hori K1979 Specific Activity 0.427 umol/min/mg
T7 Hori K1979 KM 48nM Reaction: 3-5' Exonuclease; Substrate: DNA template
T7 Hori K1979 Isoelectric Point pH 5.9
T7 Hori K1979 Other accessory protein(s) E. coli thioredoxin

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