Two forms of the DNA polymerase of bacteriophage T7.

Abstract:

The DNA polymerase induced by bacteriophage T7 can be isolated in two different forms. The distinguishing properties are: 1) the specific activities of the associated 3' to 5' single- and double-stranded DNA exonuclease activities, 2) the ability to catalyze DNA synthesis and strand displacement at nicks, and 3) the degree of stimulation of DNA synthesis on nicked, duplex DNAs by the gene 4 protein of phage T7. Form I is obtained when purification is carried out in the absence of EDTA while Form II is obtained if all purification steps are carried out in the presence of 0.1 mM EDTA. Form I has low levels of both exonuclease activities, less than 5% of those of Form II. Form I can initiate DNA synthesis at nicks leading to strand displacement, a consequence of which is its ability to be stimulated manyfold by the helicase activity of gene 4 protein on nicked, duplex templates. On the other hand, Form II cannot initiate synthesis at nicks even in the presence of gene 4 protein. In keeping with its higher exonuclease activities, Form II of T7 DNA polymerase has higher turnover of nucleotides activity (5-fold higher than Form I) and exhibits greater fidelity of nucleotide incorporation, as indicated by the rate of incorporation of 2-aminopurine deoxynucleoside monophosphate. Both forms of T7 DNA polymerase exhibit higher fidelity of nucleotide incorporation than bacteriophage T4 DNA polymerase. In the absence of EDTA or in the presence of FeSO4 or CaCl2, Form II irreversibly converts to Form I. The physical difference between the two forms is not known. No difference in molecular weight can be detected between the corresponding subunits of each form of T7 DNA polymerase as measured by gel electrophoresis in the presence of sodium dodecyl sulfate.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Other Enzymatic Activities, Fidelity, Accessory Proteins/Complexes, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 (purified without EDTA) Engler MJ1983 Nick Extension Yes, via strand displacement
T7 (purified without EDTA) Engler MJ1983 Molecular Weight 9.6E+04 Dalton
T7 (purified without EDTA) Engler MJ1983 3-5' Exonuclease (proofreading) Yes
T7 (purified without EDTA) Engler MJ1983 Cloned or native Cloned in E. coli
T7 (purified without EDTA) Engler MJ1983 Tagged No
T7 (purified without EDTA) Engler MJ1983 Nucleotide Substitution Rate 0.045 errors/bp
T7 (purified without EDTA) Engler MJ1983 Full length or truncated Full length
T7 (purified without EDTA) Engler MJ1983 Extension from RNA primer Yes
T7 (purified without EDTA) Engler MJ1983 Specific Activity 5800 units/mg
T7 (purified without EDTA) Engler MJ1983 Strand Displacement Yes
T7 (purified without EDTA) Engler MJ1983 Other accessory protein(s) E. coli thioredoxin
T7 Engler MJ1983 Molecular Weight 9.6E+04 Dalton
T7 Engler MJ1983 Cloned or native Cloned in E. coli
T7 Engler MJ1983 5-3' Exonuclease Yes
T7 Engler MJ1983 Tagged No
T7 Engler MJ1983 Nucleotide Substitution Rate 0.028 errors/bp
T7 Engler MJ1983 Nucleotide Substitution Rate 0.045 errors/bp
T7 Engler MJ1983 Full length or truncated Full length
T7 Engler MJ1983 Extension from RNA primer Yes
T7 Engler MJ1983 Specific Activity 5800 units/mg
T7 Engler MJ1983 Specific Activity 1.03E+04 units/mg
T7 Engler MJ1983 Nick Extension No
T7 Engler MJ1983 Other accessory protein(s) E. coli thioredoxin

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