Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins

Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 06/2007; 27(9):3367-77. DOI: 10.1128/MCB.02238-06
Source: PubMed


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.

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    • "Kinase assays were performed as previously described [5], [26]. Briefly, ATR-ATRIP complexes were isolated from HEK293T cells transfected with FLAG-ATR and HA-ATRIP expression vectors using anti-HA beads (Sigma). "
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    ABSTRACT: 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.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "–RPA interaction is primarily responsible for inhibiting Dmc1-driven stand exchange We generated Rad22 mutant proteins defective in their interactions with RPA. Several proteins interact with RPA via acidic amino acid clusters (Ball et al. 2007); two such clusters located in the middle region of Rad22 (D240–E241 and E250–D251) are conserved among Schizosaccharomyces species (Fig. 4D). To determine which cluster mediated RPA interaction with Rad22, each cluster was mutated separately and used in GST pulldown assays with RPA. "
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    ABSTRACT: Both ubiquitously expressed Rad51 and meiosis-specific Dmc1 are required for crossover production during meiotic recombination. The budding yeast Rad52 and its fission yeast ortholog, Rad22, are "mediators;" i.e., they help load Rad51 onto ssDNA coated with replication protein A (RPA). Here we show that the Swi5-Sfr1 complex from fission yeast is both a mediator that loads Dmc1 onto ssDNA and a direct "activator" of DNA strand exchange by Dmc1. In stark contrast, Rad22 inhibits Dmc1 action by competing for its binding to RPA-coated ssDNA. Thus, Rad22 plays dual roles in regulating meiotic recombination: activating Rad51 and inhibiting Dmc1.
    Full-text · Article · Nov 2013 · Genes & development
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    • "Interestingly, previous reports have indicated that CHK1 phosphorylation can occur without ATRIP focus formation, in cells harboring an ATRIP N-terminal deletion (45,46) or an LG mutant (47), which is incapable of binding to RPA or is deficient in dimerization (37,48). Further, stimulation of ATR activity by TOPBP1 can occur in the absence of RPA binding to ATRIP (49). Thus, an association of a residual amount of ATRIP-ATR kinase with TOPBP1 in chromatin might be sufficient to phosphorylate CHK1. "
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    ABSTRACT: When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.
    Full-text · Article · May 2013 · Nucleic Acids Research
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