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Cancer Research 67, 6100, July 1, 2007. doi: 10.1158/0008-5472.CAN-07-0369
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Phosphorylation of ATR-Interacting Protein on Ser239 Mediates an Interaction with Breast-Ovarian Cancer Susceptibility 1 and Checkpoint Function

Monica Venere1,2, Andrew Snyder1, Omar Zgheib3,4 and Thanos D. Halazonetis1,4

1 The Wistar Institute; Programs in 2 Cell and Molecular Biology and 3 Biochemistry, Biomedical Graduate Studies, University of Pennsylvania, Philadelphia, Pennsylvania; and 4 Departments of Molecular Biology and Biochemistry, University of Geneva, Geneva, Switzerland

Requests for reprints: Thanos D. Halazonetis, Department of Molecular Biology, University of Geneva, CH-1211, Geneva 4, Switzerland. Phone: 41-22-379-61-12; Fax: 41-22-379-68-68; E-mail: thanos.halazonetis{at}molbio.unige.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signaling of DNA damage and replication stress involves a multitude of proteins, including the kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), and proteins with BRCA1 COOH-terminal (BRCT) domains. The BRCT domain–containing proteins facilitate the phosphorylation of ATM/ATR substrates and can be coimmunoprecipitated with ATM or ATR. However, their mode of interaction with the ATM/ATR kinases remains elusive. Here, we show that breast-ovarian cancer susceptibility 1 (BRCA1) interacts directly with ATR-interacting protein (ATRIP), an obligate partner of ATR. The interaction involves the BRCT domains of BRCA1 and Ser239 of ATRIP, a residue that is phosphorylated in both irradiated and nonirradiated cells. Consistent with a role of BRCA1 in ATR signaling, substitution of Ser239 of ATRIP with Ala leads to a G2-M checkpoint defect. We propose that a direct physical interaction between BRCA1 and ATRIP is required for the checkpoint function of ATR. [Cancer Res 2007;67(13):6100–5]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monitoring DNA replication is critical for maintaining genomic stability (1–3). DNA replication forks may stall as a result of DNA damage, low concentrations of nucleotides, or in the process of replicating specific DNA sequences (sequences that are "hard to replicate"). In eukaryotes, the DNA replication checkpoint prevents collapse of stalled forks (a term referring to dissociation of the replication machinery from DNA) and inhibits entry into mitosis, thus ensuring completion of DNA replication (2–6). The key transducing kinase of the DNA replication checkpoint is the ataxia-telangiectasia mutated (ATM) and Rad3-related (ATR) kinase (2, 3). ATR phosphorylates several substrates, including the kinase Chk1, which inhibits the Cdc25C phosphatase and, thereby, inhibits mitotic entry (7–10).

Evidence from yeast to human cells indicates that ATR localizes to sites of stalled DNA replication forks (11, 12). This localization is mediated by the ATR-interacting protein (ATRIP), an obligate partner of ATR (13–17); however, the mechanism is being debated. In Saccharomyces cerevisiae, Lcd1/Ddc2, the yeast orthologue of ATRIP, may be recruited to stalled forks by binding to DNA via evolutionarily conserved basic residues (15). Another, nonmutually exclusive, model suggests that ATRIP binds to replication protein A (RPA), which coats the ssDNA present at sites of stalled DNA replication forks (16, 17).

In addition to ATR, ATRIP, and Chk1, several other proteins are required for the integrity of the DNA replication checkpoint, such as breast-ovarian cancer susceptibility 1 (BRCA1) and topoisomerase II binding protein 1 (TopBP1; refs. 18–22). BRCA1 and TopBP1 colocalize with ATRIP/ATR and RPA at stalled DNA replication forks (11, 17, 20–23) and both contain BRCA1 COOH-terminal (BRCT) domains, which were recently shown to be phosphopeptide-binding domains (24, 25). The physiologic ligands of the BRCT domains of TopBP1 remain elusive, but the BRCT domains of BRCA1 are known to interact with BRCA1-associated COOH-terminal helicase 1 (BACH1) and with CtBP-interacting protein (CtIP; refs. 24–26). Three-dimensional structures of BRCA1 with phosphopeptides derived from BACH1 and CtIP have been determined by X-ray crystallography and explain at the atomic level the specificity of BRCA1 for its ligands (27–30).

Here, we identify Ser239 as a novel phosphorylation site in ATRIP and show that it is recognized specifically by the BRCT domains of BRCA1. Substitution of Ser239 with Ala compromises the interaction of ATRIP with BRCA1 and the DNA replication checkpoint. Thus, our studies provide a molecular handle to begin to understand the checkpoint function of BRCA1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant plasmids. A plasmid expressing human ATRIP in mammalian cells was constructed by cloning a PCR fragment of human atrip into the NheI and NotI restriction sites of the pIRESN2 bicistronic vector (Clontech Laboratories). Ser239 of ATRIP was substituted with Ala in the context of the pIRESN2-ATRIP plasmid by site-directed mutagenesis (Quik-Change mutagenesis kit, Stratagene). Small interfering RNA (siRNA)–resistant pIRESN2-ATRIP wild-type and A239 plasmids were generated also by site-directed mutagenesis using the oligonucleotide 5'-GATCATAAGGTCCATCGTTTGTTAGATGGCATGTC-3', which introduces silent nucleotide substitutions in the sequence of atrip that is targeted by the siRNA. Fragments of human BRCA1 or TopBP1 were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins by cloning corresponding PCR fragments into the NcoI and NotI restriction sites of the pGEX4T1 vector (Pharmacia).

Antibodies. Primary antibodies recognizing the following proteins were used for immunoblotting, immunoprecipitation, and/or immunofluorescence: p53 binding protein 1 (53BP1) and ATRIP (Wistar Hybridoma Facility), ATRIP phosphorylated at Ser68 or at Ser239 (PhosphoSolutions), BRCA1 (Calbiochem), RPA (Lab Vision Corp. and GeneTex), TopBP1 (Abcam, Inc.), actin (Calbiochem), GST (Calbiochem), and HA.11 epitope (Covance). Secondary antibodies conjugated to alkaline phosphatase recognizing mouse IgG (heavy and light chains), rabbit IgG (Fc; Promega), or mouse {kappa} light chains (Southern Biotechnology) were used for immunoblotting. For immunofluorescence, the secondary antibodies were conjugated to AlexaFluor 488 or 568 (Molecular Probes).

Plasmid and siRNA transfections. U2OS cells were obtained from the American Type Culture Collection. To generate stable pools, 1 x 105 U2OS cells/100 mm dish were transfected by calcium phosphate precipitation with 5 µg of pIRESN2 plasmid encoding siRNA-resistant hemagglutinin-tagged wild-type ATRIP or the A239 point mutant. Stable transfectants were selected by adding 0.5 mg/mL G418 (Invitrogen) in the tissue culture medium. After ~2 weeks, surviving clones were pooled and maintained in medium containing 0.25 mg/mL G418. For siRNA transfections, cells seeded in 60-mm plates were incubated with mixtures of Oligofectamine (Invitrogen) and 400 to 800 pmol control (luciferase) or one of the following siRNAs (Dharmacon): 800 pmol atrip (GGUCCACAGAUUAUUAGAUdTdT) or 400 pmol brca1 (1:1 ratio of CUGUGAGAACUCUGAGGACdTdT and CUUAGGUGAAGCAGCAUCUdTdT). Cells were analyzed 72 h after siRNA transfection.

Genotoxic stress. Genotoxic stress was induced by exposing the cells to the indicated dose of ionizing radiation (IR; J.L. Shepherd Mark 1 Model 30, 137 Cesium Irradiator; J.L. Shepherd and Associates) or by incubating them with 1 mmol/L hydroxyurea for 6 or 24 h.

Immunofluorescence. Cells destined for immunofluorescence analysis were cultured on 12-mm autoclaved glass coverslips. For processing, the coverslips were transferred into 24-well plates and washed quickly in 1x PBS. The cells were then fixed with 100% methanol for 10 min at –20°C, washed twice in 1x PBS, and permeabilized with 0.2% Triton X for 10 min at 4°C. The cells were washed again in 1x PBS and incubated with primary antibodies diluted in 1x PBS (for the hemagglutinin.11 antibody, 0.1% Triton X was added) for 1 h at room temperature or overnight at 4°C for the phosphospecific antibodies. The cells were then washed in 1x PBS for 10 min and incubated with secondary antibodies for 30 min at room temperature in the dark. After washing, the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 1 min at room temperature in the dark, washed twice in 1x PBS for 10 min, and then mounted with Fluoromount G (Southern Biotechnology). Images were acquired with an ORCA ER digital camera (Hamamatsu) and processed using ImageVision software (Silicon Graphics, Inc.).

Cell extracts, immunoblotting, and immunoprecipitation. Cells were lysed for 45 min in cell lysis buffer [50 mmol/L Tris (pH 8.0), 120 mmol/L NaCl, 0.5% NP40, 1 mmol/L DTT, 8 µg/mL Pefabloc SC, 2 µg/mL pepstatin, 15 mmol/L NaF, 0.1 mmol/L NaVO4, 0.1 µmol/L staurosporine, 2 µg/mL aprotinin, 2 µg/mL leupeptin, 4 mmol/L caffeine, and 0.4 µmol/L wortmannin]. Then, 0.1 unit/µL DNase I, RNase-free, and 5 mmol/L MgCl2 were added and allowed to incubate at 16°C for 1 h. The lysates were cleared by centrifugation and the protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories). For immunoblotting, the samples were resolved by SDS-PAGE and transferred to 0.2 µm polyvinylidene difluoride membranes (Bio-Rad Laboratories). For immunoprecipitation, cell extracts (200–1,000 µg) were precleared by incubation with 40 µL Protein G Sepharose beads (Pharmacia) that had been washed in immunoprecipitation buffer [50 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 5 mmol/L MgCl2, 1 mmol/L EDTA, and 0.1% Tween 20] or hemagglutinin-immunoprecipitation buffer [50 mmol/L BTP (pH 6.5), 150 mmol/L NaCl, 5 mmol/L MgCl2, and 0.1% NP40]. In separate reactions, primary antibodies were conjugated for 1 h at 4°C to 40 µL Protein G beads, after which the beads were washed once in immunoprecipitation buffer or hemagglutinin-immunoprecipitation buffer. Then, the precleared extracts were incubated with the antibody-conjugated beads in immunoprecipitation buffer or hemagglutinin-immunoprecipitation buffer at 4°C for 1 h or overnight. The beads were then washed thrice, after which the immunoprecipitated proteins were released from the beads by boiling in 2x SDS loading buffer, resolved by SDS-PAGE, and immunoblotted.

GST pull down assay. GST-BRCA1 or GST-TopBP1 fusion proteins or GST alone were bound to glutathione Sepharose beads (Pharmacia). After washing, the beads were incubated with 500 µg cell extract at 4°C for 1 h in cell lysis buffer. After three washes with cell lysis buffer, proteins bound to the beads were resolved by SDS-PAGE and were immunoblotted.

Cell cycle checkpoint assay. Parental U2OS cells or stably transfected U2OS cells expressing hemagglutinin-tagged wild-type or Ala239 mutant ATRIP were seeded on 60-mm culture dishes. A day later, the cells were transfected with control or atrip-specific siRNA. After 48 h, the cells were irradiated (9 Gy), and 4 h later 1 µmol/L nocodazole was added. After an additional 20 h, the cells were harvested by trypsinization, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X in PBS, washed, and spun onto slides using a Shandon Cytospin cytocentrifuge (Thermo Scientific). The cells were stained by immunofluorescence for expression of hemagglutinin-tagged ATRIP and counterstained with DAPI. The cells were scored under the microscope as either mitotic (condensed chromatin) or interphase. At least 100 cells per condition were scored. For the stably transfected cells, only hemagglutinin-positive cells were counted and the experiment was done in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intracellular localization of ATRIP. To study ATRIP, we generated a monoclonal antibody against a protein fragment corresponding to residues 1 to 288 of the full-length protein and a polyclonal antibody against ATRIP phosphorylated on Ser68, the latter representing a modification of ATRIP induced by agents that block DNA replication (31). We validated these antibodies by examining the intracellular localization of ATRIP in cells exposed to IR. In nonirradiated cells, there were between 0 and 5 ATRIP foci per cell. The number of these foci did not increase at early time points after irradiation (5–60 min), but at later time points some cells exhibited a significant number of ATRIP foci (Fig. 1A ). The fraction of these cells increased with time (from 1 to 8 h), probably reflecting a larger fraction of cells in S phase. At all time points examined, the ATRIP foci also stained positive for ATRIP phosphorylated on Ser68 and for RPA, BRCA1, and TopBP1 (Fig. 1B and data not shown), as previously described (11, 17, 20, 22, 31).


Figure 1
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  		Figure 1. Validation of anti-ATRIP antibodies for immunofluorescence analysis and colocalization of ATRIP with BRCA1, TopBP1, and RPA, but not 53BP1, in nonirradiated and irradiated cells. A, ATRIP foci in nonirradiated and irradiated cells monitored by a monoclonal antibody that recognizes ATRIP and a polyclonal antibody that recognizes ATRIP phosphorylated on Ser68. Blue lines, nuclei of the cells. Rightmost panels, part of the nucleus (rectangle) of a cell that was examined 4 h after exposure to 9 Gy, shown under higher magnification. B, colocalization of ATRIP, BRCA1, TopBP1, and RPA, but not 53BP1, in irradiated cells. Only part of the nucleus is shown, similar to the rightmost panels in (A).

 
Costaining of cells for ATRIP and 53BP1 showed that these two proteins respond differently to irradiation. 53BP1 foci were evident 5 min after irradiation in practically all cells and their number decreased thereafter, whereas ATRIP foci formed later (Fig. 1B). Further, at all time points examined (up to 8 h after irradiation), the vast majority of ATRIP and 53BP1 foci did not colocalize, consistent with ATRIP and 53BP1 recognizing distinct lesions (Fig. 1B and data not shown). 53BP1 recognizes DNA DSBs, whereas ATRIP is likely to recognize DNA replication forks that have encountered IR-induced DNA damage lesions.

Phosphorylation of ATRIP on Ser239. ATRIP contains an evolutionarily conserved sequence starting at Ser239 that shares homology with the sequences of BACH1 and CtIP that are recognized by the BRCT domains of BRCA1. Indeed, all the residues of BACH1 and CtIP that are seen in the three-dimensional structures of the BRCA1-BACH1 and BRCA1-CtIP complexes to make sequence-specific contacts with BRCA1 are present in human ATRIP (Fig. 2A ; refs. 27–30). This raised the possibility that ATRIP would bind to BRCA1 with the interaction between these two proteins being mediated by an ATRIP fragment containing phospho-Ser239. To test this hypothesis, we first examined whether ATRIP is phosphorylated on Ser239. We generated a polyclonal antibody specific for phospho-Ser239 and confirmed its specificity by blotting extracts prepared from cells expressing hemagglutinin-tagged wild-type ATRIP or ATRIP containing a single substitution of Ser239 with Ala (Fig. 2B). Analysis of endogenous ATRIP using this phosphospecific antibody indicated that ATRIP was phosphorylated on Ser239 in both nonirradiated and irradiated cells and in cells exposed to hydroxyurea (Fig. 2C). The level of phosphorylation increased at late time points after exposure to IR or hydroxyurea. This may represent a cell cycle effect, as the related sequence in BACH1 is phosphorylated predominantly in the S and G2 phases of the cell cycle by cyclin-dependent kinases (25) and exposure of cells to IR or hydroxyurea leads to accumulation of cells in S and G2. Immunofluorescence analysis further indicated that the pool of ATRIP that localized at nuclear foci was also phosphorylated on Ser239 (Fig. 2D).


Figure 2
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  		Figure 2. ATRIP is phosphorylated on Ser239 in nonirradiated and irradiated cells. A, sequence similarity of a human ATRIP (ATRIP_h) peptide to the phosphopeptides of human BACH1 (BACH1_h) and CtIP (CtIP_h) that interact with the BRCT domains of BRCA1. Corresponding sequences of ATRIP orthologues in lower species—S. cerevisiae Ddc2 (Ddc2_sc), S. pombe Rad26 (Rad26_sp), D. melanogaster Mus304 (Mus304_dm)—are also indicated. Green asterisks, residues of human BACH1 and CtIP that make sequence-specific contacts with the BRCT domains of BRCA1 (27–30). Blue asterisks, basic residues of S. cerevisiae Ddc2 that are required for checkpoint function and localization to DNA damage sites (15). B, specificity of an antibody raised against an ATRIP peptide containing phosphorylated Ser239. Extracts from parental U2OS cells (–) and U2OS cells expressing hemagglutinin (HA)-tagged wild-type (wt) ATRIP or ATRIP with Ser239 substituted with Ala (A239) were immunoprecipitated (IP) with an antibody that recognizes the hemagglutinin tag and then immunoblotted (IB) with the phospho-Ser239 (pS239)–specific antibody. A mock immunoprecipitation without antibody of extracts containing hemagglutinin-tagged wt ATRIP served as a control (no Ab lane). C, endogenous ATRIP is phosphorylated on Ser239. Extracts from untreated U2OS cells (–) and U2OS cells exposed to 9 Gy IR or 1 mmol/L hydroxyurea (HU) were immunoprecipitated with a monoclonal antibody against ATRIP and then immunoblotted with the same antibody or the pS239-specific antibody. Mock immunoprecipitations without antibody (no Ab) or without cell extract (no CE) served as controls. D, ATRIP at the nuclear foci is phosphorylated on Ser239. Immunofluorescence analysis of ATRIP in nonirradiated and irradiated cells. Only part of the nucleus is shown, similar to Fig. 1B.

 
Interaction of ATRIP with BRCA1. We next examined whether ATRIP interacts with BRCA1. In a GST pull down assay, the BRCT domains of BRCA1, but not GST protein alone, interacted with endogenous ATRIP extracted from untreated cells or cells exposed to IR or hydroxyurea (Fig. 3A ). Ectopically expressed hemagglutinin-tagged wild-type ATRIP also interacted with BRCA1 in the pull down assay, but an ATRIP protein with Ala at position 239 interacted very weakly, suggesting that the interaction between ATRIP and BRCA1 involves Ser239 of ATRIP (Fig. 3B). Finally, polypeptides encompassing all the BRCT domains of TopBP1 failed to interact with ATRIP, suggesting specificity for the BRCT domains of BRCA1 (Fig. 3C).


Figure 3
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  		Figure 3. Phospho-Ser239–dependent interaction of ATRIP with the BRCT domains of BRCA1. A, GST pull down (GSTpd) assay using GST protein or GST fused to the BRCT domains of human BRCA1 (residues 1,649–1,859) and extracts from untreated cells or cells exposed to IR or hydroxyurea. Endogenous ATRIP bound to BRCA1 was detected by immunoblotting with a monoclonal antibody against ATRIP or the phospho-Ser239–specific antibody. B, GST pull down assay using parental U2OS cells (–) or U2OS cells expressing hemagglutinin-tagged wild-type ATRIP or ATRIP with a Ser239 to Ala substitution. C, GST pull down assay of endogenous ATRIP from U2OS cells using GST proteins fused to fragments containing the indicated residues of human TopBP1 (these fragments encompass all the BRCT domains of human TopBP1) or to the BRCT domains of BRCA1.

 
Defective checkpoint in cells expressing ATRIP Ala239. Because both BRCA1 and ATRIP are required for maintenance of G2 arrest after irradiation (14, 25), we examined whether the interaction between these two proteins is also required for G2 arrest. We generated cells stably expressing hemagglutinin-tagged wild-type or mutant (Ala239) ATRIP and studied the integrity of the DNA replication checkpoint. To negate the effect of endogenous ATRIP, the cells were examined after being transfected with siRNA targeting the endogenous atrip gene, whereas the plasmids expressing the ectopic ATRIP proteins had silent nucleotide substitutions that rendered them resistant to the transfected siRNA (Fig. 4A ). Clones with similar levels of wild-type and A239 mutant ATRIP (Fig. 4B) were transfected with control or atrip-specific siRNA and examined for maintenance of G2 arrest 24 h after irradiation. When endogenous ATRIP was depleted by siRNA, the ectopically expressed wild-type ATRIP effectively inhibited mitotic entry, whereas the A239 mutant was defective (Fig. 4C). These results suggest that the interaction between BRCA1 and ATRIP is required for checkpoint function.


Figure 4
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  		Figure 4. Ser239 of ATRIP is required for the integrity of the DNA replication checkpoint. A, depletion of endogenous ATRIP, but not ectopically expressed hemagglutinin-tagged ATRIP, in cells transfected with atrip-specific, but not control (ctl) siRNA. wt-st, U2OS cells stably expressing wild-type ATRIP. B, similar levels of wild-type and mutant (A239) hemagglutinin-tagged ATRIP in stably transfected U2OS cells. U2OS parental cells (–) express only endogenous ATRIP. Actin serves as a loading control. C, cell cycle checkpoint defect in U2OS cells stably expressing an A239 mutant of ATRIP. U2OS cells stably expressing wild-type or A239 mutant hemagglutinin-tagged ATRIP were transfected with control or atrip-specific siRNA, exposed to 0 or 9 Gy IR, and incubated with nocodazole to prevent exit from mitosis. The fraction of cells in mitosis 24 h after irradiation was scored visually under the microscope after immunofluorescence analysis to validate hemagglutinin-ATRIP expression and staining of the chromosomal DNA with DAPI. D, model showing that the interaction of ATRIP with BRCA1 is required for integrity of the DNA damage checkpoint.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several proteins with BRCT domains, such as BRCA1, TopBP1, 53BP1, NBS1, and MDC1, function in DNA damage and replication block checkpoint pathways (1–3). These proteins are thought to facilitate the function of the key checkpoint kinases, ATM and ATR/ATRIP, either by activating them or by facilitating the phosphorylation of their substrates (32, 33). Consistent with such a function, 53BP1 and NBS1 interact with ATM, whereas BRCA1 and TopBP1 interact with ATR/ATRIP (13, 33–35). The interaction between NBS1 and ATM involves the COOH-terminal 20 amino acids of NBS1 (13). The interaction of TopBP1 with ATR/ATRIP requires both ATR and ATRIP and involves a segment of TopBP1 between BRCT domains 6 and 7, whereas the domains mediating the interactions between endogenous ATM and 53BP1 and between endogenous ATR and BRCA1 proteins had not been previously mapped (33–35). Thus, the interaction of BRCA1 with ATRIP described here is, to our knowledge, the first interaction reported between ATM or ATR/ATRIP and a BRCT domain–containing protein that involves the BRCT domains of the latter protein. It remains to be determined whether other BRCT domain–containing proteins interact with ATM or ATR/ATRIP in a similar manner.

What is the physiologic significance of the BRCA1-ATRIP interaction? In mouse testis, during meiotic prophase I, localization of ATR to the unsynapsed chromosomes is dependent on BRCA1, suggesting that BRCA1 may recruit ATR/ATRIP to blocked DNA replication forks or sites of DNA damage (35). However, in our hands, depletion of BRCA1 by siRNA did not inhibit ATRIP focus formation in U2OS cells exposed to IR or hydroxyurea, and, further, the ATRIP A239 mutant was also not defective in forming foci in response to DNA damage (data not shown). These results argue that in interphase cells the interaction between BRCA1 and ATRIP does not recruit ATR/ATRIP to stalled DNA replication forks. However, we cannot rule out the possibility that BRCA1 has some role in ATR/ATRIP recruitment, because such recruitment may be mediated by multiple mechanisms, only one of which may involve BRCA1. Indeed, RPA can mediate recruitment of ATR/ATRIP to DNA damage sites (16, 17). Interestingly, the region of S. cerevisiae Lcd1/Ddc2 that shares homology to the sequence surrounding Ser239 of human ATRIP is required for targeting Lcd1/Ddc2 to DNA damage sites (15). A clear homologue of human BRCA1 is not present in either S. cerevisiae or S. pombe. Nevertheless, it remains possible that another BRCT domain–containing protein interacts with this region of Lcd1/Ddc2.

In conclusion, we report here a novel interaction between human ATRIP and BRCA1 mediated by phosphorylated Ser239 of ATRIP and the BRCT domains of BRCA1. This interaction is critical for the function of ATR/ATRIP in the DNA damage checkpoint and may therefore form the foundation for understanding the molecular mechanism by which BRCA1 exerts its checkpoint function.


   Acknowledgments
 
Grant support: Swiss National Foundation grant 3100A0-112434 and National Cancer Institute grant CA118827 (T.D. Halazonetis).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: M. Venere and A. Snyder contributed equally to this work.

Received 1/29/07. Revised 2/28/07. Accepted 3/23/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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