Structure and dynamics of a DNA. Polymerase complex by time-resolved fluorescence spectroscopy

David P. Millar, Dwayne J. Allen, Steven J. Benkovic

Research output: Chapter in Book/Report/Conference proceedingConference contribution

4 Citations (Scopus)

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 Klenow 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 Klenow 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 Klenow fragment was consistent with this being the active site for 3'→5' exonuclease activity.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
EditorsJoseph F. Lakowicz
PublisherPubl by Int Soc for Optical Engineering
Pages392-403
Number of pages12
ISBN (Print)0819402451
StatePublished - Dec 1 1990
EventTime-Resolved Laser Spectroscopy in Biochemistry II - Los Angeles, CA, USA
Duration: Jan 15 1990Jan 17 1990

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
Volume1204 pt 1
ISSN (Print)0277-786X

Other

OtherTime-Resolved Laser Spectroscopy in Biochemistry II
CityLos Angeles, CA, USA
Period1/15/901/17/90

Fingerprint

Fluorescence Spectroscopy
DNA Polymerase I
primers
Fluorescence spectroscopy
Fragment
DNA
deoxyribonucleic acid
fluorescence
fragments
spectroscopy
Probe
strands
Site-directed mutagenesis
Excited States
mutagenesis
Elongation
Exonucleases
Mutagenesis
DNA Primers
probes

All Science Journal Classification (ASJC) codes

  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics
  • Computer Science Applications
  • Applied Mathematics
  • Electrical and Electronic Engineering

Cite this

Millar, D. P., Allen, D. J., & Benkovic, S. J. (1990). Structure and dynamics of a DNA. Polymerase complex by time-resolved fluorescence spectroscopy. In J. F. Lakowicz (Ed.), Proceedings of SPIE - The International Society for Optical Engineering (pp. 392-403). (Proceedings of SPIE - The International Society for Optical Engineering; Vol. 1204 pt 1). Publ by Int Soc for Optical Engineering.
Millar, David P. ; Allen, Dwayne J. ; Benkovic, Steven J. / Structure and dynamics of a DNA. Polymerase complex by time-resolved fluorescence spectroscopy. Proceedings of SPIE - The International Society for Optical Engineering. editor / Joseph F. Lakowicz. Publ by Int Soc for Optical Engineering, 1990. pp. 392-403 (Proceedings of SPIE - The International Society for Optical Engineering).
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Millar, DP, Allen, DJ & Benkovic, SJ 1990, Structure and dynamics of a DNA. Polymerase complex by time-resolved fluorescence spectroscopy. in JF Lakowicz (ed.), Proceedings of SPIE - The International Society for Optical Engineering. Proceedings of SPIE - The International Society for Optical Engineering, vol. 1204 pt 1, Publ by Int Soc for Optical Engineering, pp. 392-403, Time-Resolved Laser Spectroscopy in Biochemistry II, Los Angeles, CA, USA, 1/15/90.

Structure and dynamics of a DNA. Polymerase complex by time-resolved fluorescence spectroscopy. / Millar, David P.; Allen, Dwayne J.; Benkovic, Steven J.

Proceedings of SPIE - The International Society for Optical Engineering. ed. / Joseph F. Lakowicz. Publ by Int Soc for Optical Engineering, 1990. p. 392-403 (Proceedings of SPIE - The International Society for Optical Engineering; Vol. 1204 pt 1).

Research output: Chapter in Book/Report/Conference proceedingConference contribution

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N2 - 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 Klenow 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 Klenow 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 Klenow fragment was consistent with this being the active site for 3'→5' exonuclease activity.

AB - 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 Klenow 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 Klenow 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 Klenow fragment was consistent with this being the active site for 3'→5' exonuclease activity.

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Millar DP, Allen DJ, Benkovic SJ. Structure and dynamics of a DNA. Polymerase complex by time-resolved fluorescence spectroscopy. In Lakowicz JF, editor, Proceedings of SPIE - The International Society for Optical Engineering. Publ by Int Soc for Optical Engineering. 1990. p. 392-403. (Proceedings of SPIE - The International Society for Optical Engineering).