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 transient 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 many proteins involved in various aspects of DNA repair, recombination and checkpoint control [2][3]. We produced yeast strains expressing Rad9 in which the BRCT domain had been deleted or which harboured point mutations in the highly conserved aromatic residue of each BRCT motif. Rates of survival and checkpoint delay of the mutants after ultraviolet (UV) irradiation were essentially equivalent to those of rad9Delta (null) cells, demonstrating that the BRCT domain is required for Rad9 function. Rad9 hyperphosphorylation, which occurs after DNA damage [4][5][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 interacted with the hyperphosphorylated forms of Rad9. This interaction was suppressed by mutations of the BRCT motifs that caused null phenotypes in vivo, suggesting that Rad9 oligomerization is required for Rad9 function after DNA damage.
Soulier
J
J
Imperial Cancer Research Fund, Clare Hall Laboratories, CDC Laboratory, South Mimms, Hertfordshire, EN6 3LD, UK.
Lowndes
N F
NF
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D005982:Glutathione Transferase / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; C069930:rad9 protein; D005982:Glutathione Transferase