Search

The ATR kinase controls cell cycle transitions and the DNA damage response. ATR activity is regulated through two ATR-activating proteins, ETAA1 and TOPBP1. To examine how each activator contributes to ATR signaling, we used quantitative mass spectrometry to identify changes in protein phosphorylation in ETAA1- or TOPBP1-deficient cells. We identified 724, 285, and 118 phosphosites to be regulated by TOPBP1, ETAA1, or both ATR activators, respectively. Gene ontology analysis of TOPBP1- and ETAA1-dependent phosphoproteins revealed TOPBP1 to be a primary ATR activator for replication stress, while ETAA1 regulates mitotic ATR signaling. Inactivation of ATR or ETAA1, but not TOPBP1, results in decreased Aurora B kinase activity during mitosis. Additionally, ATR activation by ETAA1 is required for proper chromosome alignment during metaphase and for a fully functional spindle assembly checkpoint response. Thus, we conclude that ETAA1 and TOPBP1 regulate distinct aspects of ATR signaling with ETAA1 having a dominant function in mitotic cells. [ABSTRACT FROM AUTHOR]

Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR) causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway. [ABSTRACT FROM AUTHOR]

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11- deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR. [ABSTRACT FROM AUTHOR]

Presents a study indicating that multiple ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related substrates including CHK1 and -2 are hyperphosphorylated in cells treated with caffeine and genotoxic agents such as hydroxyurea or ionizing radiation. Role of ATM-ATR in the regulation of cell cycle checkpoints by phosphorylating multiple substrates; Use of caffeine to study ATM and ATR signaling; Ability of caffeine to prevent phosphorylation of ATM and ATR substrates.

Although the herpes simplex virus type 1 (HSV-1) genome might be expected to induce a DNA damage response, the ATR kinase is not activated in infected cells. We previously proposed that spatial uncoupling of ATR from its interaction partner, ATRIP, could be the basis for inactivation of the ATR kinase in infected cells; however, we now show that ATR and ATRIP are in fact both recruited to HSV-1 replication compartments and can be coimmunoprecipitated from infected-cell lysates. ATRIP and replication protein A (RPA) are recruited to the earliest detectable prereplicative sites, stage II microfoci. In a normal cellular DNA damage response, ATR/ATRIP are recruited to stretches of RPA-coated single-stranded DNA in an RPA- and kinase-dependent manner, resulting in the phosphorylation of RPA by ATR in damage foci. In contrast, in HSV-1-infected cells, RPA is not phosphorylated, and endogenous phosphorylated RPA is excluded from stage II microfoci; in addition, the recruitment of ATR/ATRIP is independent of RPA and the kinase activity of ATR. Furthermore, we show that ATR/ATRIP play a beneficial role in viral gene expression and virus production. Although ICP0 has been shown to be important for partial inactivation of other cellular DNA repair pathways, we show that ICP0 is not responsible for the inactivation of ATR signaling and, furthermore, that neither ATR nor ATRIP is a target of ICP0 degradation. Thus, ATR and ATRIP may function outside the context of the canonical ATR damage signaling pathway during HSV-1 infection to participate in the viral life cycle. [ABSTRACT FROM AUTHOR]

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 damage-regulated 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. [ABSTRACT FROM AUTHOR]

The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity. [ABSTRACT FROM AUTHOR]