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The DNA damage checkpoint response is controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK), including ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). ATR forms a complex with its partner ATRIP. In budding yeast, ATR and ATRIP correspond to Mec1 and Ddc2, respectively. ATRIP/Ddc2 interacts with replication protein A-bound single-stranded DNA (RPA-ssDNA) and recruits ATR/Mec1 to sites of DNA damage. Mec1 is stimulated by the canonical activators including Ddc1, Dpb11 and Dna2. We have characterized the ddc2-S4 mutation and shown that Ddc2 not only recruits Mec1 to sites of DNA damage but also stimulates Mec1 kinase activity. However, the underlying mechanism of Ddc2-dependent Mec1 activation remains to be elucidated. Here we show that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo and through ssDNA recognition in vitro. The ddc2-S4 mutation diminishes damage-induced phosphorylation of the checkpoint mediators, Rad9 and Mrc1. Rad9 controls checkpoint throughout the cell-cycle whereas Mrc1 is specifically required for the S-phase checkpoint. Notably, S-phase checkpoint signaling is more defective in ddc2-S4 mutants than in cells where the Mec1 activators (Ddc1/Dpb11 and Dna2) are dysfunctional. To understand a role of Ddc2 in Mec1 activation, we reconstituted an in vitro assay using purified Mec1-Ddc2 complex, RPA and ssDNA. Whereas ssDNA stimulates kinase activity of the Mec1-Ddc2 complex, RPA does not. However, RPA can promote ssDNA-dependent Mec1 activation. Neither ssDNA nor RPA-ssDNA efficiently stimulates the Mec1-Ddc2 complex containing Ddc2-S4 mutant. Together, our data support a model in which Ddc2 promotes Mec1 activation at RPA-ssDNA tracts. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and F1-elated protein inase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that ype ne rotein hosphatase 1 (TOPP1) and its regulatory protein, nhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. [ABSTRACT FROM AUTHOR]