DNA bending by bZIP charge variants: A unified study using electrophoretic phasing and fluorescence resonance energy transfer

Philip R. Hardwidge, Jiong Wu, Sarah L. Williams, Kay M. Parkhurst, Lawrence J. Parkhurst, L. James Maher

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

The role of asymmetric charge neutralization as a primary determinant of protein-induced DNA helical bending remains controversial. Electrophoretic phasing experiments have been conducted previously for peptides derived from the yeast basic leucine zipper (bZIP) transcription factor GCN4 bound to AP-1 sites in duplex DNA. Mutations altering the electrostatic character of amino acids close to the DNA backbone result in phase-dependent gel mobility changes, interpreted as evidence of DNA bending. However, alternate interpretations are possible. The effect of electrostatic interactions on DNA conformation has now been investigated further, using purified peptides having indistinguishable AP-1 DNA affinity. Two independent techniques have been employed: electrophoretic phasing and fluorescence resonance energy transfer (FRET). The phasing results imply DNA bending by bZIP charge variants, consistent with earlier findings. FRET studies yield the mean 5′ end to 3′ end distance of AP-1 DNA when free or bound to neutral or charged bZIP peptides. These distances were reduced in the charged variant complexes relative to those in the free duplex and the wild-type complex. Bending of the DNA helical axis is shown by molecular modeling to be the simplest interpretation of these results. The electrophoretic phasing and FRET results thus offer two mutually supportive lines of evidence for induced bending of the DNA helical axis due to asymmetric changes in charge density caused by the electrostatic character of the amino acids residing near the DNA backbone.

Original languageEnglish (US)
Pages (from-to)7732-7742
Number of pages11
JournalBiochemistry
Volume41
Issue number24
DOIs
StatePublished - Jun 18 2002

ASJC Scopus subject areas

  • Biochemistry

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