Interaction of Rad51 with ATP and Mg2+ Induces a Conformational Change in Rad51

Abstract

The presumptive first step in the Rad51-promoted formation of joint molecules is binding of the protein to ssDNA in the presence of ATP and Mg²⁺. In this paper, we report that Rad51's ability to bind DNA is rapidly inactivated when incubated at 30−37 °C but is stabilized by the presence of ATP and Mg²⁺. Although unable to promote binding to DNA, ATP-γ-S also prevents inactivation of Rad51 at 37 °C. AMP-P-N-P lacks this property, while ADP protects partially but only at 5−10 times higher concentrations than ATP. These observations correlate with the dissociation constant of those nucleotides for Rad51 determined by equilibrium dialysis. Rad51 binds ATP and ATP-γ-S with a 1:1 stoichiometry and K_ds of 21 and 19 μM, respectively. The presence of DNA significantly increases the affinity of Rad51 for ATP, while DNA has a smaller effect on the affinity of ATP-γ-S. Competition binding studies show that ADP and AMP-P-N-P bind with a 5- and 55-fold lower affinity, respectively, than ATP. The CD spectrum of Rad51 with negative double minima at around 210 and 222 nm is characteristic of an α-helical protein. Upon binding ATP and Mg²⁺, the CD spectrum is altered in the regions 194−208 and 208−235 nm, changes that are indicative of a more structured state; this change does not occur with Rad51 that has been inactivated at 37 °C. We surmise that the active conformation is more resistant to inactivation at elevated temperature. Our data suggest that one of the roles of ATP and Mg²⁺ in Rad51-mediated strand exchange is to induce the proper protein structure for binding the two DNA substrates.