Interaction of Rad51 with ATP and Mg2+ induces a conformational change in Rad51.
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 degrees C but is stabilized by 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 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-gamma-S with a 1:1 stoichiometry and Kds of 21 and 19 microM, respectively. The presence of DNA significantly increases the affinity of Rad51 for ATP, while DNA has a smaller effect on the affinity of ATP-gamma-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 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 indicative of a more structured state; this change does not occur with Rad51 that has been inactivated at 37 degrees 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.
Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine, School of Medicine, Stanford University, California 94305, USA.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051135:Rad51 Recombinase; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D008274:Magnesium