title: Direct evaluation of a kinetic model for RecA-mediated DNA-strand exchange: the importance of nucleic acid dynamics and entropy during homologous genetic recombination.
abstract: The Escherichia coli RecA protein is the prototype of a class of proteins that play central roles 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 homologous recombination. We explored the mechanism of RecA-mediated DNA-strand exchange using oligonucleotide substrates with the intrinsic fluorophore 6-methylisoxanthopterin. Pre-steady-state spectrofluorometric analysis elucidated the earliest transient intermediates formed during recombination and delineated the mechanistic strategy by which RecA facilitates this process. The structural features of the first detectable intermediate and the energetic characteristics of its formation were consistent with interactions between a few bases of the single-stranded DNA and the minor groove of a locally melted or stretched duplex DNA. Further analysis revealed RecA to be an unusual enzyme in that entropic rather than enthalpic contributions dominate its catalytic function, and no unambiguously active role for the protein was detected in the earliest molecular events of recombination. The data best support the conclusion that the mechanistic strategy of RecA likely relies on intrinsic DNA dynamics.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Xiao
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lee
authors.forename: Scott F
authors.initials: SF
authors.lastname: Singleton
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D019277:Entropy; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D013696:Temperature
chemical_list: D004247:DNA; D011938:Rec A Recombinases