Interaction and colocalization of Rad9/Rad1/Hus1 checkpoint complex with replication protein A in human cells

Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-kDa, 32-kDa, and 14-kDa (RPA70, RPA32, RPA14, respectively). It is a protein essential for most cellular DNA metabolic pathways. Checkpoint proteins Rad9, Rad1 and Hus1 form a clamp-like complex which plays a central role in the DNA damage-induced checkpoint response. In this report, we presented the evidence that Rad9-Rad1-Hus1 complex directly interacted with RPA in human cells, and this interaction was mediated by the binding of Rad9 protein to both RPA70 and RPA32 subunits. In addition, the cellular interaction of Rad9-Rad1-Hus1 with RPA or hyperphosphorylated RPA was stimulated by UV irradiation or camptothecin treatment in a dose dependent manner. Such treatments also resulted in the co-localization of the nuclear foci formed with the two complexes. Consistently, knockdown of the RPA expression in cells by the small interference RNA (siRNA) blocked the DNA damage-dependent chromatin association of Rad9-Rad1-Hus1, and also inhibited the Rad9-Rad1-Hus1 complex formation. Taken together, our results suggest that Rad9-Rad1-Hus1 and RPA complexes collaboratively function in DNA damage responses, and that the RPA may serve as a regulator for the activity of Rad9-Rad1-Hus1 complex in the cellular checkpoint network.