Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNAdamage responsive checkpoint complex

DNA damage activates cell cycle checkpoint signaling pathways that coordina te cell cycle arrest and DNA repair. Three of the proteins involved in chec kpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immu noprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study w e demonstrated that in human cells all the hRad9 and hHus1 and approximatel y one-half of the cellular pool of hRad1 interacted as a stable, biochemica lly discrete complex, with an apparent molecular mass of 160 kDa. This comp lex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical c hromatographic behavior as the endogenous complex. Interaction studies usin g differentially tagged proteins demonstrated that the proteins did not sel f-multimerize. Rather, each protein had a binding site for the other two pa rtners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting w ith the C terminus of hRad9's predicted PCNA-like region. Collectively, the se analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.