Intramolecular Binding of the Rad9 C-terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 is closely linked with its DNA Binding [Cell Biology]

June 18th, 2015 by Takeishi, Y., Iwaya-Omi, R., Ohashi, E., Tsurimoto, T.

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA-damage. The 120 amino-acid (aa) stretch of the Rad9 C-terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent works showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9∆C-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA-binding activity that manifests in the absence of C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino-acid sequence in C-tail necessary for CRS binding to a 15 aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing variant C-tail deficient for CRS binding, and demonstrated that the mutant form restored the DNA binding as efficiently as 9∆C-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15 aa stretch, demonstrating that TopBP1 and CRS share the same binding region in C-tail. Indeed, we observed their competitive binding to C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA-damage signaling suggests that the competitive interactions of TopBP1 and CRS with C-tail will be crucial for the activation mechanism.