NBS1 binds directly to TOPBP1 via disparate interactions between the NBS1 BRCT1 domain and the TOPBP1 BRCT1 and BRCT2 domains

The TOPBP1 and NBS1 proteins are key components of DNA repair and DNA-based signaling systems. TOPBP1 is a multi-BRCT domain containing protein that plays important roles in checkpoint signaling, DNA replication, and DNA repair. Likewise, NBS1, which is a component of the MRE11-RAD50-NBS1 (MRN) complex, functions in both checkpoint signaling and DNA repair. NBS1 also contains BRCT domains, and previous works have shown that TOPBP1 and NBS1 interact with one another. This interaction occurs efficiently under basal conditions and efficiency increases during a DNA damage response (DDR). In this work we examine the basal interaction between TOPBP1 and NBS1, and we show that the two proteins bind to one another in a non-canonical manner. We report that NBS1 uses its BRCT1 domain to directly interact with a central region of TOPBP1’s BRCT1, and that it also binds to a C-terminal region within TOPBP1’s BRCT2 domain. Thus, NBS1 can interact with TOPBP1 via two different modes of binding. For both of TOPBP1’s BRCT1 and BRCT2 domains we delineate short peptide sequences, less than 30 amino acids, that are sufficient for binding to NBS1 and can confer binding upon transfer to a heterologous protein, such as maltose binding protein (MBP). We also show that NBS1 requires an intact phosphate-binding pocket (PBP) within its BRCT1 domain to interact with TOPBP1, however the presence of phosphate is not required for binding. These data expand our understanding of how BRCT-containing proteins bind to one another by revealing a non-canonical mode of binding and by showing that PBPs can contribute to binding in a phosphate-independent manner.