RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro

摘要

Mg²⁺ ion stimulates the DNA strand exchange reaction catalyzed by RecA, a key step in homologous recombination. To elucidate the molecular mechanisms underlying the role of Mg²⁺ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg²⁺ and sought to determine which step of the reaction is affected. Thermal stability, intrinsic fluorescence, and native mass spectrometric analyses of RecA revealed that RecA binds at least two Mg²⁺ ions with KD ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg²⁺ and similar to those of full-length RecA in the presence of saturating Mg²⁺. These observations, together with the results of a molecular dynamics simulation, support the idea that the acidic tail hampers the strand exchange reaction by interacting with other parts of RecA, and that binding of Mg²⁺ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg²⁺ stimulated joint molecule formation, whereas binding of the second stimulated progression of the reaction. Thus, RecA is actively involved in the strand exchange step as well as bringing the two DNAs close to each other.