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RecA Protein self-assembly

  • Brenner S
  • Zlotnick A
  • Stafford W
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Abstract

We have investigated the self-association of RecA protein from Escherichia coli by equilibrium ultracentrifugation. Monomeric RecA (Mr = 37,842) was observed in reversible equilibrium with trimers, hexamers and dodecamers in the presence of 1·5 m-KCl, 5 mm-Hepes, 1 mm-EDTA, 2 mm-ATP (pH 7·0) at 1°C. The equilibrium was strongly temperature-dependent, with polymerization being favored as the temperature was raised from 1°C to 21°C, and was reversible with respect to temperature. The values of both the standard enthalpy and entropy of self-association were positive, indicating that it is an entropy-driven process under these conditions. In the absence of KCl, in 50 mm-citrate, 5 mm-ATP, 5% (v/v) glycerol (pH 6·0) at 4°C, only small amounts of RecA monomer could be detected, while in 10 mm-Tris-acetate, 10% glycerol (pH 7·5) at 4°C, the smallest species present in significant concentration appeared to be the trimer. The majority of the species observed had molecular weights between 228,000 and 456,000, suggesting dominant stoichiometries of six to 12 monomers per oligomer. At pH 6·0, in the absence of ATP, much larger oligomers containing at least 24 monomers also appeared to be present. The data are consistent with an equilibrium mixture of monomers, trimers, hexamers, dodecamers, 24-mers and higher oligomers, with the distribution of oligomers being dependent on solution conditions. Thermodynamic analysis indicates that these oligomeric species are in reversible equilibrium with each other. It is not certain whether trimers assemble directly into hexamers, or whether disassembly into monomers is a prerequisite for the formation of higher oligomers. The possible role of higher-order RecA oligomers in the formation of RecA nucleoprotein filaments is discussed.

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Brenner, S. L., Zlotnick, A., & Stafford, W. F. (1990). RecA Protein self-assembly. Journal of Molecular Biology, 216(4), 949–964. https://doi.org/10.1016/s0022-2836(99)80013-8

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