RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.
The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.
van der Linden
Eddy
E
Department of Cell Biology and Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
Sanchez
Humberto
H
Kinoshita
Eri
E
Kanaar
Roland
R
Wyman
Claire
C
D017766:Acid Anhydride Hydrolases; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D045643:DNA Repair Enzymes / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D000076228:MRE11 Homologue Protein; D018625:Microscopy, Atomic Force; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D004247:DNA; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes