Origins of Sequence Selectivity in Homologous Genetic Recombination: Insights from Rapid Kinetic Probing of RecA-mediated DNA Strand Exchange

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

Research output: Contribution to journalArticle

Abstract

Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog. We observed that the reaction system was sensitive to heterology between the DNA substrates; however, such a "heterology effect" was not manifest when functional groups were added to or removed from the edges of the base-pairs facing the minor groove of the substrate duplex. Hence, RecA-mediated recombination must occur without the involvement of a triple helix, even as a transient intermediate in the process. The fastest detectable reaction phase was accelerated when the structure or stability of the substrate duplex was perturbed by internal mismatches or the replacement of G·C by I·C base-pairs. These findings indicate that the sequence specificity in recombination is achieved by Watson-Crick pairing in the context of base-pair dynamics inherent to the extended DNA structure bound by RecA during strand exchange.

Original languageEnglish (US)
Pages (from-to)343-359
Number of pages17
JournalJournal of Molecular Biology
Volume360
Issue number2
DOIs
StatePublished - Jul 7 2006
Externally publishedYes

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Keywords

  • base flipping
  • homologous recombination
  • pre-steady-state kinetics
  • stopped-flow fluorescence
  • transient intermediates

ASJC Scopus subject areas

  • Virology

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