Construction and evaluation of a kinetic scheme for RecA-mediated DNA strand exchange

Jie Xiao, Andrew M. Lee, Scott F. Singleton

Research output: Contribution to journalArticlepeer-review

Abstract

The Escherichia coli RecA protein is the prototype of a class of proteins playing a central role in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding its recombinational activities. Toward a molecular-level understanding of RecA-mediated DNA strand exchange, we explored its mechanism using oligonucleotide substrates and the intrinsic fluorescence of 6-methylisoxanthopterin (6M1). Steady- and presteady-state spectrofluorometric data demonstrate that the reaction proceeds via a sequential four-step mechanism comprising a rapid, bimolecular association step followed by three slower unimolecular steps. Previous authors have proposed multistep mechanisms involving two or three steps. Careful analysis of the differences among the experimental systems revealed a previously undiscovered intermediate (N 1) whose formation may be crucial in the kinetic discrimination of homologous and heterologous sequences. This observation has important implications for probing the fastest events in DNA strand exchange using 6M1 to further elucidate the molecular mechanisms of recombination and recombinational repair.

Original languageEnglish (US)
Pages (from-to)473-496
Number of pages24
JournalBiopolymers
Volume81
Issue number6
DOIs
StatePublished - Apr 15 2006
Externally publishedYes

Keywords

  • Base flipping
  • Homologous recombination
  • Presteady-state kinetics
  • Stopped-flow fluorescence
  • Transient intermediates

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

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