Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions

David P. Millar; Theodore E. Carver

doi:10.1117/12.182777

17 August 1994 Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions

David P. Millar, Theodore E. Carver

Proceedings Volume 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV; (1994) https://doi.org/10.1117/12.182777
Event: OE/LASE '94, 1994, Los Angeles, CA, United States

Abstract

Time-resolved fluorescence measurements of optical probes incorporated at specific sites in DNA provides a new approach to studies of DNA structure and DNA:protein interactions. This approach can be used to study complex multi-state behavior, such as the folding of DNA into alternative higher order structures or the transfer of DNA between multiple binding sites on a protein. In this study, fluorescence anisotropy decay of an internal dansyl probe attached to 17/27-mer oligonucleotides was used to monitor the distribution of DNA 3' termini bound at either the polymerase of 3' to 5' exonuclease sites of the Klenow fragment of DNA polymerase I. Partitioning of the primer terminus between the two active sites of the enzyme resulted in a heterogeneous probe environment, reflected in the associative behavior of the fluorescence anisotropy decay. Analysis of the anisotropy decay with a two state model of solvent-exposed and protein-associated dansyl probes was used to determine the fraction of DNA bound at each site. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3-fold increase in the equilibrium partitioning of DNA into the exonuclease site, while two or more consecutive G:G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250- fold greater than that of the corresponding matched DNA sequence. Internal single mismatches located up to four bases from the primer terminus produced larger effects than the same mismatch at the primer terminus. These results provide insight into the recognition mechanisms that enable DNA polymerases to proofread misincorporated bases during DNA replication.

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David P. Millar and Theodore E. Carver "Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); https://doi.org/10.1117/12.182777

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KEYWORDS

Polymers

Luminescence

Fluorescence anisotropy

Proteins

Time resolved spectroscopy

Anisotropy

Crystallography

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