¹Department of Biochemistry and Molecular Biology, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA

Correspondence: Y Zou, E-mail: zouy@etsu.edu

Received 24 January 2005; Revised 16 February 2005; Accepted 17 February 2005; Published online 9 May 2005.

Abstract

Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-, 32-, 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 (9–1–1) 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 9–1–1 with RPA or hyperphosphorylated RPA was stimulated by UV irradiation or camptothecin treatment in a dose-dependent manner. Such treatments also resulted in the colocalization 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 9–1–1, and also inhibited the 9–1–1 complex formation. Taken together, our results suggest that 9–1–1 and RPA complexes collaboratively function in DNA damage responses, and that the RPA may serve as a regulator for the activity of 9–1–1 complex in the cellular checkpoint network.