RAD51 Protein ATP Cap Regulates Nucleoprotein Filament Stability

• Published in: The Journal of biological chemistry Vol. 287; no. 12; pp. 8724 - 8736
• Main Authors: Amunugama, Ravindra, He, Yujiong, Willcox, Smaranda, Forties, Robert A., Shim, Kang-Sup, Bundschuh, Ralf, Luo, Yu, Griffith, Jack, Fishel, Richard
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.03.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Crystallography, X-Ray; DNA and Chromosomes; DNA Recombination; DNA Repair; Enzyme Turnover; Humans; Methanococcus - chemistry; Methanococcus - enzymology; Methanococcus - genetics; Molecular Sequence Data; Nucleoproteins - chemistry; Nucleoproteins - genetics; Nucleoproteins - metabolism; Protein Stability; Protein Turnover; Protein-DNA Interaction; Rad51 Recombinase - chemistry; Rad51 Recombinase - genetics; Rad51 Recombinase - metabolism; Sequence Alignment; Protein-DNA Interaction; Protein Turnover; DNA Recombination; DNA Repair; Enzyme Turnover
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.239426

RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP γ-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity. RAD51 and RecA homologs form a nucleoprotein filament (NPF) that includes an ATP cap, which sandwiches the adenosine nucleotide. The HsRAD51(D316K) ATP cap substitution dramatically enhances recombinase and NPF stability. The HsRAD51(Asp-316) residue forms a salt bridge with ATP that allows more rapid protein turnover. HsRAD51(D316K) provides a useful reagent for the study of recombinase function in physiological conditions.