Structure and dynamics of a DNA: polymerase complex by time-resolved fluorescence spectroscopy

David P. Millar; Stephen J. Benkovic

doi:10.1117/12.17704

1 May 1990 Structure and dynamics of a DNA: polymerase complex by time-resolved fluorescence spectroscopy

David P. Millar, Stephen J. Benkovic

Proceedings Volume 1204, Time-Resolved Laser Spectroscopy in Biochemistry II; (1990) https://doi.org/10.1117/12.17704
Event: OE/LASE '90, 1990, Los Angeles, CA, United States

Abstract

The interaction of a fluorescent DNA primer:template with the Klenow fragment of DNA polymerase I has been studied in solution using time-resolved fluorescence spectroscopy. The excited-state decay behavior and internal reorientation dynamics of a dansyl sulfonamide probe connected by a propyl chain to a modified uridine base in the primer strand were very sensitive to the local probe environment and exhibited characteristic changes upon binding of Kienow fragment to the DNA and elongation of the primer strand. Between 5 and 7 bases of duplex DNA upstream of the 3' primer terminus were protected from the solvent by the Kienow fragment and the strength of DNA:protein contacts varied within this region, being strongest at the 3' primer terminus. About 5% of the substrates were bound in a second spatially distinct site on the enzyme. Site-directed mutagenesis of the Kienow fragment was consistent with this being the active site for 3'->5' exonuclease activity.

Citation Download Citation

David P. Millar and Stephen J. Benkovic "Structure and dynamics of a DNA: polymerase complex by time-resolved fluorescence spectroscopy", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); https://doi.org/10.1117/12.17704

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