Structural characterization and kinetic evaluation of RecA-mediated strand exchange

[摘要] The RecA protein of Escherichia coli plays essential roles in homologous recombination. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for thein vivo process. During this process, a key intermediate comprised of three DNA strands simultaneously bound to a RecA filament (RecA·tsDNA complex) forms. To date, the questions about the high-resolution structure of this intermediate and how the it forms have not been addressed.We present a systematic characterization of the helical geometry of the three DNA strands of the RecA·tsDNA complex using fluorescence resonance energy transfer (FRET). Measurements of the helical parameters for the RecA·tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex.We then monitored the formation of the RecA·tsDNA complex in real time using a fluorescent base analog. Time-dependent changes of polarized emission from the fluorophore demonstrated this process involves at least three phases. The first phase is strongly dependent on substrate concentration and reaction temperature. Kinetic simulation revealed a sequential four-steps mechanism as the best description for the reaction. The association rate constant approaches the magnitude of 107 M-1s -1. Thermodynamic analysis indicates the reaction is entropy favorable and a large activation energy is necessary for the association step. A comparison of the energy diagrams between a homologous, partially homologous and a completely heterologous dsDNA substrates suggests that the RecA protein discriminates homology both kinetically and thermodynamically. Based on these data, a structural and mechanistic model for RecAmediated strand exchange was constructed.

[发布机构] Rice University

[效力级别] biology