The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis.
The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co-precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1-D SDS-PAGE of co-precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co-precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.
Rajesh
Changanamkandath
C
Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA.
Gruver
Aaron M
AM
Basrur
Venkatesha
V
Pittman
Douglas L
DL
D000818:Animals; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004268:DNA-Binding Proteins; C519208:RAD51C protein, mouse; C491807:Rad51d protein, mouse; C491866:Xrcc2 protein, mouse; D051135:Rad51 Recombinase