Equilibrium Analysis of Ethidium Binding to DNA Containing Base Mismatches and Branches

Luis I. Hernández, Min Zhong, Scott H. Courtney, Luis A. Marky, Neville R. Kallenbach

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

In the processes of DNA replication, recombination, and repair, duplex DNA can transiently form branched structures, such as Holliday junctions, as well as base pair mismatches and bulges. These states have altered ligand and protein binding properties from normal double helical DNA. A variety of ligands have been reported to interact more tightly at branches and bulges than to normal duplex sites. The stoichiometry, structural basis, and thermodynamics of this effect have not been determined. We have investigated the binding of the intercalator, ethidium bromide, to several DNA constructs including base mismatches, bulges, and three- and four-arm branched structures, using chemical footprinting, titration calorimetry, and fluorescence lifetime measurements. Two classes of binding sites are detected in three- and four-arm junctions in our high ionic strength conditions: one class is characterized by a small number of ligands (2-4 per DNA), with high binding affinity (K > 105), and the second by a larger number of sites (10-12 per DNA) with lower affinity (K ∼ 104). By use of appropriate control experiments, the former appear to be associated with sites at or near the branch point or mismatch, while the latter are consistent with binding to the normal duplex DNA region(s) of the molecule. Titration calorimetry indicates an enthalpy of −10 to −13 kcal/mol for binding of ethidium to a mismatch or three- and four-arm branch point. The tight binding class is associated with a fluorescence lifetime of 12-16 ns, distinct from that of free ethidium (ca. 2 ns) and the longer lifetime observed for ethidium intercalated in duplex DNA (22–26 ns).

Original languageEnglish (US)
Pages (from-to)13140-13146
Number of pages7
JournalBiochemistry
Volume33
Issue number44
DOIs
StatePublished - Nov 1 1994

Fingerprint

Ethidium
DNA
Calorimetry
Ligands
Titration
Base Pair Mismatch
Fluorescence
Cruciform DNA
Intercalating Agents
Recombinational DNA Repair
DNA Replication
Thermodynamics
Protein Binding
DNA Repair
Osmolar Concentration
Ionic strength
Stoichiometry
Enthalpy
Binding Sites
Repair

ASJC Scopus subject areas

  • Biochemistry

Cite this

Hernández, L. I., Zhong, M., Courtney, S. H., Marky, L. A., & Kallenbach, N. R. (1994). Equilibrium Analysis of Ethidium Binding to DNA Containing Base Mismatches and Branches. Biochemistry, 33(44), 13140-13146. https://doi.org/10.1021/bi00248a025

Equilibrium Analysis of Ethidium Binding to DNA Containing Base Mismatches and Branches. / Hernández, Luis I.; Zhong, Min; Courtney, Scott H.; Marky, Luis A.; Kallenbach, Neville R.

In: Biochemistry, Vol. 33, No. 44, 01.11.1994, p. 13140-13146.

Research output: Contribution to journalArticle

Hernández, LI, Zhong, M, Courtney, SH, Marky, LA & Kallenbach, NR 1994, 'Equilibrium Analysis of Ethidium Binding to DNA Containing Base Mismatches and Branches', Biochemistry, vol. 33, no. 44, pp. 13140-13146. https://doi.org/10.1021/bi00248a025
Hernández, Luis I. ; Zhong, Min ; Courtney, Scott H. ; Marky, Luis A. ; Kallenbach, Neville R. / Equilibrium Analysis of Ethidium Binding to DNA Containing Base Mismatches and Branches. In: Biochemistry. 1994 ; Vol. 33, No. 44. pp. 13140-13146.
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