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Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12610-5. Epub 2007 Jul 18.

Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase.

Author information

1
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.

Abstract

We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.

PMID:
17640918
PMCID:
PMC1937514
DOI:
10.1073/pnas.0700920104
[Indexed for MEDLINE]
Free PMC Article

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