ATP half-sites in RadA and RAD51 recombinases bind nucleotides.

Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.We would like to thank Dr Timothy Sharpe for help with MALS analysis of the monomeric RadA protein and Dr Tara Pukala for the mass spectrometric analysis of the same protein. We would like to thank X-ray crystallographic and Biophysics facilities at the Department of Biochemistry for access to their instrumentation. We thank Diamond Light Source for access to beamline I04 (proposal MX315), European Synchrotron Radiation Facility for access to beamline ID23-1 (proposal MX-705 17 and MX-857) and Swiss Light Source for access to beamline PXIII that contributed to the results presented here. This work was funded by Translational Award from the Wellcome Trust (080083/Z/06/Z).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Wiley