Calorimetric analysis of binding of two consecutive DNA strands to RecA protein illuminates mechanism for recognition of homology

Artikel i vetenskaplig tidskrift, 2007

RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change, (Delta H) with temperature (heat capacity change, Delta C-p). In the presence of the ATP analogue ATP gamma S, the Delta H for the binding of the first DNA strand depends upon temperature (large Delta C-p) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced Delta C-p, indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the Delta H of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the Delta H is larger than that for non-complementary DNA strand, but less than the Delta H of the annealing of the complementary DNA without RecA. This small Delta H could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The Delta H of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands. (c) 2006 Elsevier Ltd. All rights reserved.