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.
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