title: RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling.
abstract: The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9- and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.
authors.affiliation: a From the Department of Biochemistry and Biophysics ; University of North Carolina School of Medicine ; Chapel Hill , NC USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lindsey-Boltz
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kemp
authors.forename: Christopher
authors.initials: C
authors.lastname: Capp
authors.forename: Aziz
authors.initials: A
authors.lastname: Sancar
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000528:radiation effects; D014466:Ultraviolet Rays
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C549605:HUS1 protein, human; D009687:Nuclear Proteins; C558820:RHNO1 protein, human; D034741:RNA, Small Interfering; C110513:TOPBP1 protein, human; C069930:rad9 protein; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; D005092:Exonucleases; C113574:Rad1 protein, human
keywords: 9-1-1, Rad9-Hus1-Rad1; ATR, Ataxia telangiectasia-mutated and Rad3-related; DNA damage checkpoint; DNA damage response; IP, immunoprecipitation; RHINO, Rad9, Hus1, Rad1 interacting nuclear orphan; RPA, Replication Protein A; TopBP1, Topoisomerase binding protein 1; UV, ultraviolet; checkpoint clamp; checkpoint kinase; chromatin; protein-protein interaction; ssDNA, single-stranded DNA; ultraviolet light