ATR associates with the regulatory protein ATRIP that has been proposed to localize ATR to sites of DNA damage through an interaction with single-stranded DNA (ssDNA) coated with replication protein A (RPA). We tested this hypothesis and found that ATRIP is required for ATR accumulation at intranuclear foci induced by DNA damage. A domain at the N terminus of ATRIP is necessary and sufficient for interaction with RPA-ssDNA. Deletion of the ssDNA-RPA interaction domain of ATRIP greatly diminished accumulation of ATRIP into foci. However, the ATRIP-RPA-ssDNA interaction is not sufficient for ATRIP recognition of DNA damage. A splice variant of ATRIP that cannot bind to ATR revealed that ATR association is also essential for proper ATRIP localization. Furthermore, the ATRIP-RPA-ssDNA interaction is not absolutely essential for ATR activation because ATR phosphorylates Chk1 in cells expressing only a mutant of ATRIP that does not bind to RPA-ssDNA. These data suggest that binding to RPA-ssDNA is not the essential function of ATRIP in ATR-dependent checkpoint signaling and ATR has an important function in properly localizing the ATR-ATRIP complex.

The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP.ATR (ATM and Rad3-related) kinase is a protein kinase that coordinates cellular responses to genotoxic stress. ATR activation occurs primarily in S phase due to replication stress induced by DNA-damaging agents or replication inhibitors. More specifically, ATR activation is stimulated when the replication machinery encounters a DNA lesion and becomes uncoupled (the helicase continues to unwind DNA while the polymerase becomes stalled at the site of DNA damage) (9).The critical factor that promotes ATR activation is believed to be the accumulation of RPA (replication protein A)-coated single-stranded DNA (ssDNA) (11,33,43). At least two separate checkpoint complexes accumulate in distinct foci that colocalize with RPA. Rad17, a PCNA-like clamp loader protein, is recruited to RPA-ssDNA and loads the Rad9-Rad1-Hus1 checkpoint clamp at the junction of double-stranded and single-stranded DNA (4,14,53). Independently, ATR is recruited by ATR-interacting protein (ATRIP), which binds the RPA-ssDNA that accumulates at DNA lesions (3,15,37,52).ATRIP is required for ATR function, and mutation of either ATR or ATRIP causes the same phenotypes (3, 12). The strict requirement for ATRIP is conserved in Schizosaccharomyces pombe (Rad3 and Rad26), Saccharomyces cerevisiae (Mec1 and Ddc2/Lcd1/Pie1), and Xenopus laevis (xATR and xATRIP) (13,38,41,51). An N-terminal domain of ATRIP binds RPAssDNA and is necessary for stable ATR-ATRIP localization to damage-induced nuclear foci (3, 25).The ATR signaling pathway is currently viewed as an important target for the development of cancer therapies (10,22,24,32,34). However, the mechanism by which ATR is activated remains unclear. Localization to sites of DNA damage or replication stress has been suggested to be essential and perhaps sufficient to promote ATR signaling. However, mutations in ATRIP that disrupt the stable RPA-A...

The ATM and ATR kinases signal cell cycle checkpoint responses to DNA damage. Inactive ATM is an oligomer that is disrupted to form active monomers in response to ionizing radiation. We examined whether ATR is activated by a similar mechanism. We found that the ATRIP subunit of the ATR kinase and ATR itself exist as homooligomers in cells. We did not detect regulation of ATR or ATRIP oligomerization after DNA damage. The predicted coiled-coil domain of ATRIP is essential for ATRIP oligomerization, stable ATR binding, and accumulation of ATRIP at DNA lesions. Additionally, the ATRIP coiled-coil is also required for ATRIP to support ATR-dependent checkpoint signaling to Chk1. Replacing the ATRIP coiled-coil domain with a heterologous dimerization domain restored stable binding to ATR and localization to damage-induced intranuclear foci. Thus, the ATR-ATRIP complex exists in higher order oligomeric states within cells and ATRIP oligomerization is essential for its function.DNA damage causes the activation of signaling pathways that promote cell cycle arrest, DNA repair, and apoptosis. Ataxia-Telangiectasia-mutated (ATM) 1 and ATM and Rad3-related (ATR), the two kinases at the apex of the checkpoint response, are members of a family of atypical kinases that preferentially phosphorylate serine or threonine residues followed by glutamine (1-3). ATM initiates the immediate cell cycle checkpoint response to DNA double strand breaks whereas ATR is the predominant initiator of the checkpoint in response to lesions that stall replication forks (2). ATM deficiency causes the human disease ataxia telangiectasia. Cells and animals lacking ATR are not viable (4, 5), but a hypomorphic allele of ATR was recently associated with rare cases of seckel syndrome (6, 7).ATR and ATM share significant sequence homology and many substrates but are activated by different stimuli. ATM is held inactive in undamaged cells as an oligomer, with the kinase domain of one molecule bound intermolecularly to the FAT domain of another molecule (8). Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 and dimer dissociation, leading to the activation of ATM kinase signaling. Association with the Mre11-Rad50-Nbs1 complex can also facilitate monomerization and activation of ATM (9). The ATR activation mechanism is less well understood and may involve recruitment to sites of DNA lesions and interactions with specific DNA structures (10, 11).ATR exists in a stable complex with an associated protein ATRIP (ATR-interacting protein) (4). Similarly, in Schizosaccharomyces pombe and Saccharomyces cerevisiae, the ATR homologs Rad3 and Mec1 are found closely associated with ATRIP homologs Rad26 and Ddc2 respectively (12-15). Mutations in either Rad3 or Rad26 yield almost identical phenotypes, as do mutations in either Mec1 or Ddc2. Depletion of ATRIP by RNA inhibition also causes similar phenotypes the loss of ATR in mammalian cells (4). Furthermore, depletion of either ATRIP or ATR from cells causes a decrease in the intracellul...

Shwachman-Diamond syndrome (SDS; OMIM 260400) results from loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene. It is a multi-system disorder with clinical features of exocrine pancreatic dysfunction, skeletal abnormalities, bone marrow failure and predisposition to leukemic transformation. Although the cellular functions of SBDS are still unclear, its yeast ortholog has been implicated in ribosome biogenesis. Using affinity capture and mass spectrometry, we have developed an SBDS-interactome and report SBDS binding partners with diverse molecular functions, notably components of the large ribosomal subunit and proteins involved in DNA metabolism. Reciprocal co-immunoprecipitation confirmed the interaction of SBDS with the large ribosomal subunit protein RPL4 and with DNA-PK and RPA70, two proteins with critical roles in DNA repair. Function for SBDS in response to cellular stresses was implicated by demonstrating that SBDS-depleted HEK293 cells are hypersensitive to multiple types of DNA damage as well as chemically induced endoplasmic reticulum stress. Furthermore, using multiple routes to impair translation and mimic the effect of SBDS-depletion, we show that SBDS-dependent hypersensitivity of HEK293 cells to UV irradiation can be distinguished from a role of SBDS in translation. These results indicate functions of SBDS beyond ribosome biogenesis and may provide insight into the poorly understood cancer predisposition of SDS patients.

The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of COXs and LOs to oxygenate arachidonyl-derived lipoamino acids and vanilloids was investigated. COX-1 and COX-2 were able to minimally metabolize many of these species. However, the lipoamino acids were efficiently oxygenated by 12S-and 15S-LOs. The kinetics and products of oxygenation by LOs were characterized. Whereas 15S-LOs retained positional specificity of oxygenation with these novel substrates, platelet-type 12S-LO acted as a 12/15-LO. Fatty acid oxygenases may play an important role in the metabolic inactivation of lipoaminoacids or vanilloids or may convert them to bioactive derivatives.