INTERACTION OF RAD51 WITH ATP AND MG2+ INDUCES A CONFORMATIONAL CHANGE IN RAD51

The presumptive first step in the Rad51-promoted formation of joint mo lecules 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 rap idly inactivated when incubated at 30-37 degrees C but is stabilized b y the presence of ATP and Mg2+. Although unable to promote binding to DNA, ATP-gamma-S also prevents inactivation of Rad51 at 37 degrees C. AMP-P-N-P lacks this property, while ADP protects partially but only a t 5-10 times higher concentrations than ATP. These observations correl ate with the dissociation constant of those nucleotides for Rad51 dete rmined by equilibrium dialysis. Rad51 binds ATP and ATP-gamma-S with a 1:1 stoichiometry and K(d)s Of 21 and 19 mu M, respectively. The pres ence of DNA significantly increases the affinity of Rad51 for ATP, whi le DNA has a smaller effect on the affinity of ATP-gamma-S. Competitio n binding studies show that ADP and AMP-P-N-P bind with a 5- and 55-fo ld lower affinity, respectively, than ATP. The CD spectrum of Rad51 wi th negative double minima at around 210 and 222 nm is characteristic o f an alpha-helical protein. Upon binding ATP and Mg2+, the CD spectrum is altered in the regions 194-208 and 208-235 nm, changes that are in dicative of a more structured state; this change does not occur with R ad51 that has been inactivated at 37 degrees C, We surmise that the ac tive conformation is more resistant to inactivation at elevated temper ature. Our data suggest that one of the roles of ATP and Mg2+ in Rad51 -mediated strand exchange is to induce the proper protein structure fo r binding the two DNA substrates.