Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase.

Abstract:

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Accessory Proteins/Complexes, Nucleotide Incorporation, Exonuclease Activity, Nick Extension

One line summary:

This paper describes how T7 DNA polymerase purified without EDTA catalyzes strand displacement synthesis. It concludes that synthesis is terminated by a template-switching reaction that yields a duplex branch and proposes that the branch arises as a result of either primer relocation or rotational strand exchange.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 (purified without EDTA) Lechner RL1983 3-5' Exonuclease (proofreading) Yes
T7 (purified without EDTA) Lechner RL1983 Cloned or native Native organism
T7 (purified without EDTA) Lechner RL1983 Tagged No
T7 (purified without EDTA) Lechner RL1983 Full length or truncated Full length
T7 (purified without EDTA) Lechner RL1983 Specific Activity 5800 units/mg
T7 (purified without EDTA) Lechner RL1983 Other accessory protein(s) E. coli thioredoxin
T7 (purified without EDTA) Lechner RL1983 Nick Extension Yes, via strand displacement
T7 Lechner RL1983 3-5' Exonuclease (proofreading) Yes
T7 Lechner RL1983 Cloned or native Native organism
T7 Lechner RL1983 Tagged No
T7 Lechner RL1983 Full length or truncated Full length
T7 Lechner RL1983 Specific Activity 1.08E+04 units/mg
T7 Lechner RL1983 Specific Activity 5800 units/mg
T7 Lechner RL1983 Nick Extension No
T7 Lechner RL1983 Other accessory protein(s) E. coli thioredoxin

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