Abstract
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.
Highlights
Homologous recombination, the process of error-free repair of DNA double-strand breaks and stalled replication, has been well conserved through evolution from bacteria to humans [1,2,3,4]
We demonstrate that a monomerised form of human RAD51 (RAD51–BRC4 fusion) and a truncated monomeric form of P. furiosus RadA (RadA-ct) retain their ability to bind ATP and other nucleotides in the low micromolar range, and we present the crystal structures of monomeric ATPase domain of RadA (RadA-ct) bound to ATP, ADP, AMPPNP and GTP
In order to study the binding of ATP and other nucleotides to nonoligomeric RadA, we engineered the protein by removing the N-terminal domain and the linker that contains the FxxA oligomerisation sequence (Fig. 1A)
Summary
In order to study the binding of ATP and other nucleotides to nonoligomeric RadA, we engineered the protein by removing the N-terminal domain and the linker that contains the FxxA oligomerisation sequence (Fig. 1A). Of RadA-ct in the absence of nucleotides; this revealed the presence of a phosphate ion within the ATP-binding site We used these crystals to exchange the bound phosphates with various nucleotides by soaking the crystals in a stepwise fashion in mother liquor containing increasing concentrations of the nucleotide ligand and decreasing phosphate concentration. Using this procedure, the phosphate ion was successfully displaced from the ATP-binding site by nucleotide ligands, enabling us to solve the structures of RadA-ct bound to ATP, ADP, AMPPNP and GTP. In contrast to our monomeric RadA structures, in nucleotide-bound filament structures, this loop is shifted towards the nucleoside, with a corresponding shift in the adenine moiety
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