The Rad9A checkpoint protein is required for nuclear ...

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The Rad9A checkpoint protein is required for nuclear localization of the claspin adaptor protein Megan L. Sierant, Nicole E. Archer & Scott K. Davey To cite this article: Megan L. Sierant, Nicole E. Archer & Scott K. Davey (2010) The Rad9A checkpoint protein is required for nuclear localization of the claspin adaptor protein, Cell Cycle, 9:3, 548-556, DOI: 10.4161/cc.9.3.10553 To link to this article: http://dx.doi.org/10.4161/cc.9.3.10553

Published online: 01 Feb 2010.

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Cell Cycle 9:3, 548-556; February 1, 2010; © 2010 Landes Bioscience

The Rad9A checkpoint protein is required for nuclear localization of the claspin adaptor protein Megan L. Sierant,1,2 Nicole E. Archer1 and Scott K. Davey1-3,* Department of Cancer Biology and Genetics, Cancer Research Institute; and 2Department of Biochemistry; 3Department of Pathology and Molecular Medicine; Queen’s University; Kingston, ON Canada

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Key words: Rad9A, claspin, cell cycle checkpoint control, nuclear localization, DNA repair, 911 complex

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Abbreviations: 911, Rad9A, Rad1, Hus1; ATR, atm and rad3-related; IB, immunoblot; IF, immunofluorescence; IP, immunoprecipitation; IR, γ-irradiation; mES, murine embryonic stem cell; RA, all-trans retinoic acid; TopBP1, topoisomerase IIβ binding protein 1

The interaction between the 911 complex, via Rad9A, and Claspin is required for activation of the Chk1-mediated checkpoint response, along with ATR, TopBP1, and the 911 clamp loader complex Rad17/RFC. Despite the importance of the Rad9A-Claspin interaction in the cell cycle, this interaction has yet to be characterized. In this work we show this interaction persists in a variety of different conditions. During the course of this study we also determined the nuclear localization of Rad9A affected the localization of the Claspin protein, leading us to the conclusion that Rad9A is able to affect Claspin cellular localization. This was verified experimentally using a Rad9A-null cell line and reconstitution of WT Rad9A. We also show that in mES cells the Rad9A paralog, Rad9B, is also capable of affecting Claspin localization. Together, these data suggest that Rad9 plays a role in locating Claspin to sites of DNA damage, facilitating its role during the Chk1-mediated checkpoint response. Since disruption of both Rad9A and Claspin has been shown to abolish Chk1 activation, we postulate that Rad9A-mediated Claspin localization is a vital step during checkpoint activation.

Introduction Activation of Chk1 is a key event in the atm and rad3-related (ATR)-mediated branch of the checkpoint response pathway, which is required for maintaining genomic stability and protection from endogenous and exogenous DNA damage. ATR,1-3 Claspin,2,4,5 TopBP1,6 the 911 complex6-10 and the 911 clamploader Rad17/RFC complex11 have been reported to be required for successful Chk1 activation during the DNA damage response (DDR) at the G2/M boundary. This research has led to a model of Chk1 activation that requires the presence of particular checkpoint proteins in order for ATR-mediated phosphorylation of Chk1 to occur. Previous work has demonstrated the 911 complex is required for localization of TopBP1 to sites of DNA damage.6,12,13 Evidence also shows that Rad17 and ATR are required localize the 911 complex and that this is required for ATR activation.14,15 Claspin is currently thought to act downstream of TopBP1 during checkpoint activation although identification of any recruitment or loading proteins is lacking.16 The presence of these proteins is required for ATR to phosphorylate, and thereby activate, the Chk1 kinase.1-11 Combined these data point to a model in which these proteins assemble into a Chk1-activation complex responsible for checkpoint initiation.

Members of the 911 complex, Rad9A, Hus1 and Rad1, interact to form a toroidal heterotrimer capable of encircling the DNA strand.17-22 Of the three members of the 911 complex, Rad9A is arguably the key component due to the presence of a nuclear localization sequence (NLS), a consensus region for interacting with the large subunit of the RPA complex, and an interacting motif for the apoptotic control proteins Bcl-2/Bcl-X L .10,23,24 All of these motifs have yet to be identified in either Hus1 or Rad1. In addition, there are extensive post-translational modifications of the Rad9A C-terminal tail, an extra 120 amino acids sharing no homology to either Hus1, Rad1 or PCNA.9,20,25 The Rad9A post-translational modifications have been shown to have no effect on the interactions with the other members of the 911 complex or with the clamp-loader Rad17/RFC.20,25,26 However, they have been shown to be required for the interaction with the topoisomerase IIβ binding protein 1 (TopBP1), specifically the S387 constitutive phosphorylation site.12 Ablation of this site disrupts the interaction between the 911 complex and TopBP1, and inhibits the formation of TopBP1 foci following checkpoint activation, suggesting that the 911 complex is required for proper recruitment of TopBP1.12 Based on data such as this, the current hypothesized role of the 911 complex is as a sensor/transducer responsible for locating to the sites of DNA damage and

*Correspondence to: Scott K. Davey; Email: [email protected] Submitted: 09/14/09; Accepted: 11/06/09 Previously published online: www.landesbioscience.com/journals/cc/article/10553 548

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interaction was examined under a variety of conditions. These included in the absence and presence of DNA DSBs produced via exposure to IR, different phases of the cell cycle, overexpression of different Rad9A truncation mutants, and overexpression of the Rad9A S272A point mutant. The Claspin protein has previously been shown to peak during the S/G2 phases of the cell cycle.4 In order to determine if the Rad9A-Claspin interaction was cell cycle-dependent, HeLa cells were synchronized to various points of the cell cycle and immunoprecipitation (IP) against Rad9A performed. The co-IP reaction, when probed by α-Claspin immunoblot (IB), revealed bands at all time points examined (Fig. 1A). The inverse reaction, in which α-Claspin co-IP reactions were followed by IB against Rad9A, was performed and revealed complimentary data (Fig. 1B). We observed slight variations in the bands of the secFigure 1. Rad9A and Claspin interact during all phases of the cell cycle in HeLa cells. ondary IBs in both panels A and B and densitomAsynchronously growing HeLa cells were synchronized via double thymidine block and etry values were obtained to determine if these released for the indicated number of hours before harvest. (A) α-Rad9 IP followed by α-Claspin IB produces reactive bands in all lanes above the 175 kDa marker. (B) variations were the result of cell cycle-dependent α-Claspin IP followed by α-Rad9A IB produces bands between the 63 kDa and 48 kDa changes in the Rad9A-Claspin interaction or if markers in all lanes, characteristic of Rad9A. they were the result of small differences during loading. When the densitometry values obtained facilitating the localization and recruitment of other key down- from panel (A) were compared to the values obtained in panel stream transducer/effector proteins. (B), no trends were observed. This led to the conclusion that the The Claspin adaptor protein was first identified in Xenopus as slight differences in band signal strength were most likely the a protein required for successful activation of the Chk1-mediated result of small differences in loading and not of any significant checkpoint response.27 Based on this, paralogs were discov- cell cycle-dependent changes in the interaction between Rad9A ered in mammals and yeast that shared similar functions with and Claspin. xClaspin.4,27,28 Furthermore, work on Claspin revealed that it The interaction between TopBP1 and Rad9A requires prior behaves in analogous manner to Rad9A during TopBP1 recruit- phosphorylation at the S387 residue.12 To determine if elements ment: Claspin is currently hypothesized to act as a loading dock present in the C-terminal tail of Rad9A affected Claspin binding, for the proteins required for Chk1 activation, namely the 911 various myc-tagged C-terminal truncation mutants, in which the complex, Rad17/RFC, ATR/ATRIP and TopBP1.1-11 Claspin has last 17 (∆17), 36(∆36) or 59(∆59) amino acids have been deleted, also been show to regular fork stability during DNA replication were transfected into HeLa cells along with a full-length Rad9A in a manner that is separate from its ability to regulate Chk1 construct (WT). IPs against the myc epitope tag were followed by activation, suggesting that this protein is an important member IB against Claspin and, again, showed that Claspin was present of the genome surveillance machinery.29 in all reactions indicating that the last 59 amino acids, including While the interaction between Rad9A and Claspin has pre- the S387 residue, of Rad9A are not required for this interaction viously been shown, no work on the mechanism or functional (Fig. 2A). Densitometry revealed minimal differences in band significance has been reported.4 Given the importance of the signal between the lanes. Rad9A-Claspin interaction for Chk1 activation and given that One of the key residues in the Rad9A tail is the S272 damRad9A has previously been shown to be required for the localiza- age-dependent phosphorylation site, phosphorylated in response tion of other members of the Chk1-activating complex, TopBP1 to IR-induced DNA damage.26 We investigated whether the specifically, we explore the possibility that the Rad9A subunit of the damage-dependent phosphorylation site at S272 was involved in 911 complex controls the localization of the Claspin protein. We Claspin binding. This particular phosphorylation is intact in the utilized biochemical and molecular biological techniques to exam- truncation mutant analyzed in Figure 2A, unlike the S387 phosine the interaction between Rad9A and Claspin experimentally. phorylation site, and so it seemed reasonable to assume this site was involved in the Rad9A-Claspin interaction. Figure 2B reveals Results that expression of Rad9A-S272A did not affect the interaction. In addition, we also examined whether the presence of DNA damPreviously, it was shown via biochemical techniques that Claspin age, via exposure to IR, affected the interaction between Rad9A interacts with Rad9A of the 911 complex.4 To confirm and elab- and Claspin. Our results show that, in both the presence and orate on their observations, the nature of the Rad9A-Claspin absence of DNA damage, there was no effect on Claspin binding

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Figure 2. Mutations in the Rad9A C-terminal tail do not affect Rad9A-Claspin interactions regardless of the presence of DNA damage. Asynchronously growing HeLa cells were transfected with different Rad9A mutant constructs and IP performed against either myc or endogenous Rad9A, as indicated. (A) Expression of either full-length or different truncation mutants of Rad9A results in an α-Claspin reactive band above the 175 kDa marker after IB. (B) Cells were transfected with different point mutations or wild-type Rad9A and treated as indicated with 10 Gy IR followed by a 30 min recovery period. IPs were against either endogenous Rad9A (end) or the myc tag of different Rad9A constructs (WT and S272A). All samples show α-Claspin reactive bands above the 175 kDa marker.

(Fig. 2B). Combined, the data presented in both Figures 1 and 2 suggest the Rad9A-Claspin interaction is constitutive in at least a subset of the total Rad9A and Claspin in a cell and does not depend on phosphorylation at either the S272 or S387 residues. Previous work on these truncation mutants revealed aberrant Rad9A expression patterns, it was found that deletion of either the 36 or 59 C-terminal amino acids resulted in primarily cytoplasmic Rad9A due to deletion of the NLS sequence.12,23 During the course of the experiments involving the ∆59 mutant, we utilized confocal microscopy to determine if the expression patterns of the mutants were as previously described.12 Given the persistence of the Rad9A-Claspin interaction, we found that overexpression of the ∆59 mutant in HeLa cells, compared to WT (Fig. 3A), resulted in a shift in Claspin localization, following the mutant Rad9A protein into the cytoplasm (Fig. 3B). Exposure to irradiation did not alter this effect (Fig. 4) nor did expression in the hTERT-RPE1 cell line, a non-transformed immortalized epithelial background (Fig. S1). Also displayed in Figure 3, we repeated this experiment in a third cell line, a WT mES background and observed the same result. From this we hypothesized that Rad9A may be responsible for the nuclear localization of Claspin.

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To test this hypothesis we performed a series of experiments on the mES cell lines, which revealed that simply staining for Claspin localization in both the WT and Rad9A-null backgrounds showed no change in Claspin localization (Fig. 5). IBs on whole cell extracts from both WT and the Rad9A-deficient mES cells revealed αRad9-reactive bands in all samples consistent with the size of the Rad9B protein (Osorio W, unpublished results). We postulated that the undifferentiated mES cells may be expressing the Rad9A paralog, Rad9B, which had previously been described to be confined to testicular tissue and transformed cells.30,31 Quantitative PCR techniques were utilized and it was found that both the WT and Rad9A-null mES cells expressed detectable levels of Rad9B mRNA (Fig. 6C). The lack of difference between the panels in Figure 4 supports the findings by Hopkins et al. who found that Rad9B was able to interact with all three members of the 911 complex.31 Differentiation of these mES cells, via exposure to RA, caused both a decrease in the levels of Rad9B mRNA detected (Fig. 6C) as well as a change in Claspin localization in Rad9A-null cells (Fig. 6B) compared to WT cells (Fig. 6A). Overexpression of a WT human Rad9A cDNA construct in

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Figure 3. Overexpression of non-nuclear Rad9A protein changes the subcellular localization of Claspin in undamaged, asynchronous cells. Asynchronous HeLa and WT mES cells were grown on coverslips, coated with fibronectin in the case of the mES cells, transfected with either WT or ∆59, as indicated, myc-tagged Rad9A constructs and processed for indirect IF against Rad9A (green), Claspin (red) and myc-tag (blue), while nuclei were visualized via DAPI (white). The white bar represents 10 nm.


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differentiating Rad9A-null cells restored the number of cells expressing nuclear Claspin when compared to untransfected Rad9A-deficient cells (Fig. 6E). No change in Claspin localization was observed in either untransfected or transfected cells in the WT background (Fig. 6E), which was as expected. Differentiation of the mES cells was confirmed by decreasing levels of Oct4 expression, a common marker for pluripotency, as detected by IB on whole cell extracts (Fig. 6D). From these data, we conclude that the Rad9A protein regulates the nuclear localization of Claspin. Rad9B is able also able to affect Claspin localization in cells expressing this paralog.


Discussion Claspin and Rad9A have been shown to interact and this interaction is vital for the activation of the Chk1 DNA damage response kinase.4,7,8,14,32 Our investigations into the Rad9A-Claspin relationship revealed that the association persists in a variety of conditions studied and can be considered constitutive in a fraction of the Rad9A and Claspin present in a cell. We have also discovered that both Rad9A and Rad9B can affect the nuclear localization of the Claspin adaptor protein resulting exclusion of Claspin from the nucleus upon disruption of the Rad9A NLS. Reconstitution of WT human Rad9A protein in a mouse Rad9A- and Rad9Bdeficient background reverses this shift from predominantly cytoplasmic in untransfected cells to predominantly nuclear in transfected cells, recreating the effect of endogenous Rad9A/B on Claspin localization. Defining the conditions of the Rad9A-Claspin interaction is important for a complete understanding of the events occurring during the Chk1-mediated DNA damage response. Cytoplasmic sequestration, via deletion of the Rad9A NLS, effectively depletes both Rad9A and Claspin in the nucleus and, according to reports, prevents activation of the Chk1 effector kinase.2,4-10 This effectively renders the Rad9A-null mES cells Claspin-null as well and explains the degree of lethality encountered during the course of working with these cells. In this report we confirmed the interaction between two key members of the Chk1 activation pathway and suggest that disruption of the Rad9A-Claspin interaction would be one way of leading to the previously reported Chk1 activation defects resulting from disruption of either protein2,4-9 Expression of a fusion protein of the Rad9A NLS with Claspin would give the same result as depleting the 911 complex in the nucleus, which has already been shown to result in decreased activation of Chk1 in response to DNA damage,7-9 by removing the PCNA domain of the Rad9A protein from the cell, required for 911 complex formation, and possibly by preventing the nuclear localization of both Hus1 and Rad1. Both the 911 complex and Claspin are ring-shaped molecules, one possibility concerning the structure of this interaction is that these rings stack along the damaged DNA strand, which would then act as locator signals to other members of the checkpoint and DNA repair machinery.17,22,33-36 Future work on this interaction could focus on determining if Claspin is able to interact with the other members of the 911 complex, Hus1 and Rad1, and would reveal whether this interaction is based on the PCNA-

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Figure 4. Overexpression of non-nuclear Rad9A protein changes the subcellular localization of Claspin in damaged, asynchronous HeLa cells. Asynchronous cells were grown on coverslips, transfected with either WT or ∆59, as indicated, myc-tagged Rad9A constructs, exposed to 10 Gy IR, and all to recover for 30 min then processed for IF as previously described. Transfected cells were identified by strong signals in the myc channel. Nuclei were stained with DAPI (white), while antibodies were used to detect Rad9A (green), Claspin (red) and myc (blue). The white bar represents 10 nm.

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Figure 5. Claspin localization is not affected by Rad9A deletion in mES cells. Asynchronous mES were grown on fibronectin-coated coverslips and stained for indirect IF against Claspin (red), nuclei were visualized via DAPI (white) in both WT and Rad9A-null cells, as indicated. The white bar represents 10 nm.

like ring-shaped domains or based on other elements present in the Rad9A C-terminal that were not examined here. Other future experiments could include examination of the mechanism behind Claspin loading as this has not been addressed in the current literature. Initial experiments with the Rad9A-null mES cells revealed no change in Claspin localization when compared to WT mES cells (Fig. 4). Subsequent experimentation with these cells revealed the presence of Rad9B mRNA that decreased after treatment with RA, a potent initiator of differentiation (Fig. 6C). We postulate the undifferentiated mES cells are able to survive due to partial rescue by substitution of Rad9B in the 911 complex but as levels decrease during differentiation the true phenotype of Rad9 ablation is revealed. The Rad9A-null cells have been shown to be sensitive to genotoxic agents so the rescue by Rad9B is not a full rescue.37 Currently, there is nothing in the literature addressing the mechanism of Claspin loading, the only previous report on Claspin nuclear translocation demonstrates that Claspin nuclear localization is dependent upon the two circadian proteins, TIM and its partner TIPIN.38 The work described in this paper does not address the role of these two proteins in Claspin localization. The possibility that Tim/TIPIN and Rad9A interact with different pools of the Claspin protein cannot be excluded and is the subject of future experiments. Due to mounting evidence


supporting the roles of circadian proteins during the DDR,38,39 the possibility of an interaction between the 911 complex and Tim/TIPIN is a logical next step. In this work, we have provided additional information about the mechanism, and possible purpose for this interaction, between two important members of the DDR. Understanding the details of this interaction is important for defining the function of Rad9A and the 911 complex during the DDR. This is important, particularly in light of new evidence supporting the role of Rad9A as an oncogene, upregulated in both non-small cell lung cancer and breast cancer.40-43 Materials and Methods Cell culture conditions and manipulation techniques. HeLa cells (CCL-2), obtained from the ATCC repository (Manassas, VA), were cultured in Dulbecco’s Modified Eagle media (DMEM-Invitrogen, Burlington, Canada) supplemented with 10% fetal calf serum (Sigma Aldrich, Oakville, Canada) and grown in a humidified environment at 37°C and 5% CO2. Murine embryonic stem cells (mES), both wild-type and containing a genomic deletion of part of the rad9A gene were a gift from Dr. Howard Lieberman, and are previously described in Hopkins et al.37 mES were maintained in gelatinized culture dishes in KnockOut DMEM (Invitrogen) supplemented with

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Figure 6. Levels of Rad9B mRNA decrease after exposure to retinoic acid as detected by quantitative PCR. WT (A) and Rad9A-null (B) mES cells were exposed to RA for the indicated time and harvested for mRNA extraction. The mRNA was used a template for cDNA and then qPCR utilizing a probes against Rad9B and GAPDH, was performed. The qPCR data was processed as previously described and the normalized values plotted vs. the RA exposure time (C). Differentiaion was confirmed via IB against Oct4 on whole cell extracts (D). Approximately 200 cells from slides used in (A and B) were scored for the number of cells showing cytoplasmic and nuclear claspin signal for both WT and Rad9A-null mES cells as well as cells transfected with WT-Rad9A and exposed to RA for 72 hrs (E).

the following: 15% FBS (ES qualified-Invitrogen), 0.1 mM β-mercaptoethanol (Bioshop, Burlington, Canada), 0.1 mM nonessential amino acids (Invitrogen), 2 mM L-glutamine (Bioshop), 50 ug/mL penicillin/streptomycin (Sigma), and 103 units/mL ESGRO (Millipore, Etobicoke, Canada). In order to differentiate the mES cells, media was supplemented with 10 µM all-trans retinoic acid (RA-Bioshop). mES cells were either harvested for

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mRNA or replated on coverslips (described below), as the experiment required. Transfections were performed with the FuGENE 6 transfection reagent (Roche Diagnostics, Mississauga, Canada) according to the manufacturer’s instructions. Unless otherwise indicated, cells were cultured for 48 hrs post-transfection and treated as indicated. The ratio of reagent (µL) to DNA (µg) for all reactions was

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4:2 and 4 µg of DNA was used for a 6 cm dish. DNA constructs used in this study were constructed as previously described.12,26 Cell synchronization was performed as previously described.26 Irradiation was carried out using a Victoreen Electrometer (Atomic Energy of Canada, Mississauga, Canada) 137Cs γ-irradiator with an approximate dose rate of 0.5 Gy/min for a dose of 10 Gy. Unirradiated cells were transported and treated similarly as the irradiated samples but were not exposed to the γ-ray source. Confocal microscopy. For confocal microscopy experiments, cells were seeded at 20% confluence on glass coverslips and cultured for 48 hrs in either 6-well or 6 cm dishes. mES cells were plated on coverslips coated in fibronectin prior to processing and allowed sufficient time for adhesion. Cells were allowed to grow for 24 hrs prior to transfection. Coverslips were processed for immunofluorescence (IF) as previously described.12 Antibodies used in IF experiments were used in the following dilutions: α-Claspin 1/100 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), α-Rad9A 1/200 (RCH Antibodies, Kingston, Canada), α-cmyc 1/200 (Santa Cruz Biotechnology Inc.,). Alexa fluorochromeconjugated secondary antibodies (Invitrogen) were diluted 1/200. Coverslips were mounted in Antifade Gold supplemented with 4',6-diamidino-2-phenylindole (DAPI-Invitrogen) on clean glass slides. Images were acquired using a 100X oil-immersion objective mounted on a Leica TCS SP2 MP inverted confocal microscope (Leica, Richmond Hill, Canada). Images with signals from two or more different fluorochromes were captured sequentially to minimize spectral overlap. Raw TIFF images were processed and overlays of data from different channels were created in Adobe Photoshop (Adobe Systems Canada, Etobicoke, Canada). Molecular and biochemical analysis. Cellular extracts for immunoprecipitations (IP) and immunoblots (IB) were References 1.

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Kim SM, Kumagai A, Lee J, Dunphy WG. Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil domains of ATRIP. J Biol Chem 2005; 280:38355-64. Kumagai A, Kim SM, Dunphy WG. Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1. J Biol Chem 2004; 279:49599-608. Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol 2001; 21:412939. Chini CC, Chen J. Human claspin is required for replication checkpoint control. J Biol Chem 2003; 278:30057-62. Chini CC, Chen J. Claspin, a regulator of Chk1 in DNA replication stress pathway. DNA Repair (Amst) 2004; 3:1033-7. Delacroix S, Wagner JM, Kobayashi M, Yamamoto K, Karnitz LM. The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev 2007; 21:1472-7. Bao S, Lu T, Wang X, Zheng H, Wang LE, Wei Q, et al. Disruption of the Rad9/Rad1/Hus1 (9-1-1) complex leads to checkpoint signaling and replication defects. Oncogene 2004; 23:5586-93. Dang T, Bao S, Wang XF. Human Rad9 is required for the activation of S-phase checkpoint and the maintenance of chromosomal stability. Genes Cells 2005; 10:287-95.


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prepared as previously described.20 mRNA extracts for use in quantitative RT-PCR were prepared according to the manufacturer’s instructions using the RNEasy kit (Qiagen, Mississauga, Canada). Extracted RNA was analyzed via spectrometry using an Eppendorf BioPhotometer (Eppendorf Canada, Mississauga, ON, Canada) using the Factor 10, LP 1 mm Hellma TrayCell. Approximately 1 µg of RNA was used as a template for cDNA synthesis via the Promega Reverse Transcription system (Fisher Scientific, Ottawa, Canada) according to the manufacturer’s instructions. For the qRT-PCR reactions, Taqman probes (Applied Biosystems, Streetsville, Canada) against murine 18S RNA, Rad9A, Rad9B were used. One hundred nanograms of cDNA were used to quantify gene expression on an Eppendorf LightCycler and raw data was analyzed according to the 2-∆∆CT method described in Livak and Schmittgen.44 Acknowledgements

This work has been made possible by funding from the following agencies: the National Cancer Institute of Canada through funds from the Canadian Cancer Society, the Canadian Breast Cancer Fellowship (Ontario Chapter), and the Canadian Institute for Health Research to SD. We would like to thank Dr. Howard Lieberman for his generous gift of both the WT and Rad9A-null murine ES cells and to Kevin Hopkins for helping us with the culture techniques. We would also like to thank Matt Gordon and Jeff Mewburn for their assistance with the confocal microscopy and their technical expertise. Note

Supplementary materials can be found at: www.landesbioscience.com/supplement/SierantCC9-3-Sup.pdf

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Volume 9 Issue 3