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Equilibrium analysis of ethidium binding to DNA containing base mismatches and branches.

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 stages 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 > 10(5)), and the second by a larger number of sites (10-12 per DNA) with lower affinity (K approximately 10(4)). 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).

journal_name

Biochemistry

journal_title

Biochemistry

authors

Hernández LI,Zhong M,Courtney SH,Marky LA,Kallenbach NR

doi

10.1021/bi00248a025

subject

Has Abstract

pub_date

1994-11-08 00:00:00

pages

13140-6

issue

44

eissn

0006-2960

issn

1520-4995

journal_volume

33

pub_type

杂志文章

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