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-, 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.
Wu
Xiaoming
X
Department of Biochemistry and Molecular Biology, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA.
Shell
Steven M
SM
Zou
Yue
Y
D018797:Cell Cycle Proteins / Q000235:genetics*; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000502:physiology*; D005092:Exonucleases / Q000235:genetics*; D005786:Gene Expression Regulation; D018816:Genes, cdc; D006367:HeLa Cells; D006801:Humans; D034741:RNA, Small Interfering; D051716:Replication Protein A; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; D029702:Schizosaccharomyces pombe Proteins; C106332:hus1 protein, S pombe; C069930:rad9 protein; D005092:Exonucleases; C113574:Rad1 protein, human