The BRCT domain of the S-cerevisiae checkpoint protein Rad9 mediates a Rad9-Rad9 interaction after DNA damage

The Saccharomyces cerevisiae checkpoint protein Rad9 is required for transi ent cell-cycle arrest and transcriptional induction of DNA-repair genes in response to DNA damage [1]. It contains a carboxyterminal tandem repeat of the BRCT (BRCA1 carboxyl terminus) motif, a motif that is also found in man y proteins involved in various aspects of DNA repair, recombination and che ckpoint control [2,3]. We produced yeast strains expressing Rad9 in which t he BRCT domain had been deleted or which harboured point mutations in the h ighly conserved aromatic residue of each BRCT motif, Rates of survival and checkpoint delay of the mutants after ultraviolet (UV) irradiation were ess entially equivalent to those of rad9 Delta (null) cells, demonstrating that the BRCT domain is required for Rad9 function. Rad9 hyperphosphorylation, which occurs after DNA damage [4-6], was absent in the BRCT mutants, as was Rad9 dependent phosphorylation of the Rad53 protein. A two hybrid approach identified a specific interaction between the Rad9 BRCT domain and itself. Biochemical analysis in vitro and in vivo confirmed this interaction and, furthermore, demonstrated that the Rad9 BRCT domain preferentially interact ed with the hyperphosphorylated forms of Rad9. This interaction was suppres sed by mutations of the BRCT motifs that caused null phenotypes in vivo, su ggesting that Rad9 oligomerization is required for Rad9 function after DNA damage. (C) Elsevier Science Ltd ISSN 0960-9822.