Synopsis Preservation of genome integrity via the DNA damage response is critical to prevent disease. Ataxia-telangiectasia mutated and Rad3-related (ATR) is essential for life and functions as a master regulator of the DNA damage response, especially during DNA replication. ATR controls and coordinates DNA replication origin firing, replication fork stability, cell cycle checkpoints, and DNA repair. Since its identification 15 years ago, a model of ATR activation and signaling has emerged that involves localization to sites of DNA damage and activation through protein-protein interactions. Recent research has added an increasingly detailed understanding of the canonical ATR pathway, and an appreciation that the canonical model does not fully capture the complexity of ATR regulation. Here we review the ATR signaling process, focusing on mechanistic findings garnered from the identification of new ATR interacting proteins and substrates. We discuss how to incorporate these new insights into a model of ATR regulation, and point out the significant gaps in our understanding of this essential genome maintenance pathway.

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter–β-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.

The Saccharomyces cerevisiae Mec1-Ddc2 checkpoint kinase complex (the ortholog to human ATR-ATRIP) is an essential regulator of genomic integrity. The S. cerevisiae BRCT repeat protein Dpb11 functions in the initiation of both DNA replication and cell cycle checkpoints. Here, we report a genetic and physical interaction between Dpb11 and Mec1-Ddc2. A C-terminal domain of Dpb11 is sufficient to associate with Mec1-Ddc2 and strongly stimulates the kinase activity of Mec1 in a Ddc2-dependent manner. Furthermore, Mec1 phosphorylates Dpb11 and thereby amplifies the stimulating effect of Dpb11 on Mec1-Ddc2 kinase activity. Thus, Dpb11 is a functional ortholog of human TopBP1, and the Mec1/ATR activation mechanism is conserved from yeast to humans.

The DNA damage response kinases ataxia telangiectasia-mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ataxia telangiectasia-mutated and Rad3-related (ATR) signal through multiple pathways to promote genome maintenance. These related kinases share similar methods of regulation, including recruitment to specific nucleic acid structures and association with protein activators. ATM and DNA-PK also are regulated via phosphorylation, which provides a convenient biomarker for their activity. Whether phosphorylation regulates ATR is unknown. Here we identify ATR Thr-1989 as a DNA damageregulated phosphorylation site. Selective inhibition of ATR prevents Thr-1989 phosphorylation, and phosphorylation requires ATR activation. Cells engineered to express only a non-phosphorylatable T1989A mutant exhibit a modest ATR functional defect. Our results suggest that, like ATM and DNA-PK, phosphorylation regulates ATR, and phospho-peptide specific antibodies to Thr-1989 provide a proximal marker of ATR activation.The DNA damage response is an evolutionarily conserved signal transduction network that coordinates cell cycle transitions, DNA replication, DNA repair, and apoptosis to guard against genomic instability. At the apex of the DNA damage response are three related kinases belonging to the PI3K-related protein kinase (PIKKs) 2 family, including ataxia telangiectasia-mutated (ATM), ATM and RAD3-related (ATR), and DNA-dependent protein kinase (DNA-PK). The PIKKs are large proteins that share domain architecture and regulatory mechanisms. A large portion of the PIKK proteins consists of an array of antiparallel helices called Huntingtin, elongation factor 3, protein phosphatase 2A, and PI3K TOR1 (HEAT) repeats (1). The C-terminal kinase domain of the PIKKs is flanked by the FRAP, ATM, TRRAP (FAT) domain (2); PIKK regulatory domain (PRD) (3); and FAT C terminus domain (2).ATR is essential for viability in replicating human cells, and disruption of ATR in mice results in embryonic lethality prior to embryonic day 7.5 (4 -6). ATR regulates replication fork stability, restart of collapsed forks, and late-origin firing during S-phase. ATR also activates the G 2 checkpoint to prevent entry into mitosis in the presence of damaged DNA (7,8). Clinically, hypomorphic mutations in ATR cause Seckel syndrome (9), and heterozygous ATR mutations are associated with poor prognosis of tumors with microsatellite instability (10, 11).The ATR activation process involves recruitment of ATR and its obligate partner ATR-interacting protein (ATRIP) (4) to a DNA lesion or stalled replication fork. Single-stranded DNA coated with replication protein A often mediates this recruitment (12) because ATRIP interacts directly with the 70-kDa subunit of replication protein A (13). Independently, the RAD9-RAD1-HUS1 (9-1-1) complex is loaded onto sites of damage by the clamp loader RAD17 (14 -17). This loading is specific to a 5Ј recessed junction, perhaps because of the interaction of RAD9 with the 70N domain of replication protein A (18). The 9-1-1 com...

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.