Single-molecule studies of the effect of template tension on T7 DNA polymerase activity.


T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3'-->5' exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a 'right hand' shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an 'open fingers' state in which the active site samples nucleotides, and a 'closed' state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of approximately 34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.




Exonuclease Activity, Source / Purification, Nucleotide Incorporation, Kinetic Parameters


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Polymerase Reference Property Result Context
T7 Wuite GJ2000 3-5' Exonuclease (proofreading) Yes
T7 Wuite GJ2000 Full length or truncated Full length
T7 Wuite GJ2000 Processivity 420bp
T7 Wuite GJ2000 Vmax 100 /second Reaction: Nucleotide incorporation; Substrate: dNTPs

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