6D lanes 7 and 8). We conclude from our studies using the LacR-LacO tethering and enrichment approach that RHINO is a genuine regulator of ATR-Chk1 signaling in mammalian cells. Discussion The core molecular mechanism of ATR-Chk1 signaling is reasonably well-established and is based on studies using a wide range of model eukaryotic organisms and systems. vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV Rosavin 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 Rosavin fide regulator of ATR-Chk1 signaling in mammalian cells. Keywords: checkpoint clamp, checkpoint kinase, chromatin, DNA damage response, DNA damage checkpoint, protein-protein interaction, ultraviolet light Abbreviations 9-1-1Rad9-Hus1-Rad1UVultravioletRHINORad9, Hus1, Rad1 interacting nuclear orphanTopBP1Topoisomerase binding protein 1ATRAtaxia telangiectasia-mutated and Rad3-relatedRPAReplication Protein AIPimmunoprecipitationssDNAsingle-stranded DNA Introduction In response to DNA damage by endogenous or exogenous sources, eukaryotic cells activate DNA damage response signaling pathways that promote DNA repair, slow or arrest cell cycle progression, and maintain cellular and organismal viability.1 Genetic studies from a variety of model systems ranging from budding yeast to mouse models and human cells have demonstrated a key role for a heterotrimeric complex known as the 9-1-1 (Rad9-Hus1-Rad1) clamp in the cellular response to DNA damage and in preventing tumorigenesis.2-4 Structural analyses of the 9-1-1 complex demonstrated that 9-1-1 resembles PCNA,5-10 a homotrimeric sliding clamp protein that facilitates the activities of a multitude of DNA metabolic enzymes on DNA,11,12 including DNA synthesis by DNA polymerases. Though 9-1-1 is also capable of binding to many PNCA-interacting proteins,9,13-18 the best characterized function of the 9-1-1 clamp is in ATR-mediated DNA damage checkpoint signaling, where it is loaded onto primer-template junctions at sites of DNA damage and replication stress by an alternative clamp loader known as Rad17-Replication Factor C.19-21 A key feature of 9-1-1 that differentiates it from PCNA is the presence of an unstructured, highly phosphorylated extension on the C-terminus of the Rad9 subunit.22,23 This domain binds to a protein known as TopBP1, which serves as a direct stimulator of ATR kinase activity through DNA-independent and DNA-dependent mechanisms.24-26 Once active, ATR phosphorylates a number of proteins to maintain genomic stability, including the DNA damage checkpoint effector kinase Chk1.1,27 The role of the 9-1-1 clamp in activation of ATR-mediated DNA damage checkpoint signaling is therefore thought to involve the stabilization of TopBP1 at sites of damage so that it can activate ATR. Though biochemical studies using recombinant proteins of the yeast homologs of 9-1-1, TopBP1, and ATR support this general model28 and also a direct role for Rad9 in stimulating ATR kinase activity,28,29 experimental validation of the model using human proteins is currently lacking. Interestingly, a recent DNA damage response screen in human cells identified a novel factor termed RHINO (for Rad9, Hus1, Rad1 interacting nuclear orphan) that localized to sites of DNA damage, Rosavin mediated cell sensitivity and/or cell Rosavin cycle checkpoint response to ionizing radiation (IR) and other agents that induce double-strand breaks in DNA.30 Furthermore, mass spectrometric analysis of RHINO protein complexes following exposure of cells to IR identified both the 9-1-1 checkpoint clamp and the ATR activator TopBP1.30 These interactions were validated by co-immunoprecipitation approaches with ectopically expressed proteins in irradiated cells.30 The observation that the RHINO gene is only present in vertebrate genomes indicates the existence of a unique regulatory factor of the ATR-Chk1 pathway in higher eukaryotes. Here, we examined the interactions of RHINO with 9-1-1 and TopBP1 in vitro and in vivo and its role as a mediator of ATR DNA damage checkpoint signaling in mammalian cells. We Rabbit polyclonal to Complement C4 beta chain find that RHINO directly binds Rosavin to TopBP1 and forms a stable, heterotetrameric complex.