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• W2054813812 abstract "ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA. ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA. In eukaryotic cells, various checkpoint control mechanisms help to safeguard the integrity of the genomic DNA. These signaling cascades participate in the detection of abnormal DNA replication intermediates and DNA damage. Thereupon, these regulatory systems trigger a cell cycle delay until the defects are rectified (1.Melo J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Crossref PubMed Scopus (399) Google Scholar, 2.Nyberg K.A. Michelson R.J. Putnam C.W. Weinert T.A. Annu. Rev. Genet. 2002; 36: 617-656Crossref PubMed Scopus (650) Google Scholar, 3.Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Key components in these pathways are members of the phosphoinositide kinase-related family of protein kinases, including ATM 2The abbreviations used are: ATM, ataxia-telangiectasia mutated; ATR, ATM- and Rad3-related; RPA, replication protein A. and ATR (4.Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar, 5.Shiloh Y. Kastan M.B. Adv. Cancer Res. 2001; 83: 209-254Crossref PubMed Scopus (262) Google Scholar). There are many similarities between ATM and ATR, including sequence homology and common substrates. However, activation of these kinases depends upon different types of genomic signals. ATM responds primarily to double-stranded DNA breaks induced by ionizing radiation and other agents. Conversely, ATR responds to problems that arise during the course of DNA replication (6.Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 7.Hekmat-Nejad M. You Z. Yee M. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 8.Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (199) Google Scholar, 9.Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (870) Google Scholar). In turn, ATR promotes the activation of Chk1, a downstream kinase that inhibits cell cycle progression. Importantly, several members of the phosphoinositide kinase-related family of protein kinases function in cooperation with partner proteins that help recruit these kinases to DNA (10.Falck J. Coates J. Jackson S.P. Nature. 2005; 434: 605-611Crossref PubMed Scopus (998) Google Scholar). The partner for human ATR, which is called ATRIP (ATR-interacting protein), is functionally conserved in eukaryotic cells (11.Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (754) Google Scholar). For example, Mec1 and Rad3, the budding and fission yeast homologs of ATR, form stable complexes with Ddc2 and Rad26, respectively (12.Rouse J. Jackson S.P. EMBO J. 2000; 19: 5801-5812Crossref PubMed Scopus (118) Google Scholar, 13.Wakayama T. Kondo T. Ando S. Matsumoto K. Sugimoto K. Mol. Cell. Biol. 2001; 21: 755-764Crossref PubMed Scopus (109) Google Scholar, 14.Paciotti V. Clerici M. Lucchini G. Longhese M.P. Genes Dev. 2000; 14: 2046-2059PubMed Google Scholar, 15.Edwards R.J. Bentley N.J. Carr A.M. Nat. Cell Biol. 1999; 1: 393-398Crossref PubMed Scopus (172) Google Scholar). In Aspergillus nidulans, the uvsB and uvsD genes encode ATR and ATRIP homologs (16.De Souza C.P. Ye X.S. Osmani S.A. Mol. Biol. Cell. 1999; 10: 3661-3674Crossref PubMed Scopus (47) Google Scholar). ATRIP and its relatives appear to be critical for the function of ATR. Genetic studies in yeast (12.Rouse J. Jackson S.P. EMBO J. 2000; 19: 5801-5812Crossref PubMed Scopus (118) Google Scholar, 13.Wakayama T. Kondo T. Ando S. Matsumoto K. Sugimoto K. Mol. Cell. Biol. 2001; 21: 755-764Crossref PubMed Scopus (109) Google Scholar, 14.Paciotti V. Clerici M. Lucchini G. Longhese M.P. Genes Dev. 2000; 14: 2046-2059PubMed Google Scholar, 15.Edwards R.J. Bentley N.J. Carr A.M. Nat. Cell Biol. 1999; 1: 393-398Crossref PubMed Scopus (172) Google Scholar, 17.Uchiyama M. Galli I. Griffiths D.J. Wang T.S. Mol. Cell. Biol. 1997; 17: 3103-3115Crossref PubMed Scopus (26) Google Scholar), ablation of ATRIP by treatment of human cells with small interfering RNA (11.Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (754) Google Scholar), and immunodepletion of ATRIP from Xenopus egg extracts (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) have all shown that absence of ATRIP functionally resembles the lack of ATR. Despite these insights, important aspects regarding the function of ATRIP remain unresolved. For example, recruitment of ATRIP to single-stranded DNA can clearly occur in an RPA-dependent manner (19.Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2056) Google Scholar). However, there appear to be RPA-independent means for association of ATRIP with DNA as well (20.Bomgarden R.D. Yean D. Yee M.C. Cimprich K.A. J. Biol. Chem. 2004; 279: 13346-13353Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 21.Ünsal-Kaçmaz K. Sancar A. Mol. Cell. Biol. 2004; 24: 1292-1300Crossref PubMed Scopus (82) Google Scholar). Furthermore, recent studies have indicated that recruitment of ATRIP to RPA-containing, DNA damage-induced foci in mammalian cells is not essential for the ATR-dependent activation of Chk1 (22.Ball H.L. Myers J.S. Cortez D. Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (187) Google Scholar). Another important issue involves the native quaternary structure of the ATR-ATRIP complex in cells, about which there is not a clear consensus, and its potential regulation during checkpoint responses (20.Bomgarden R.D. Yean D. Yee M.C. Cimprich K.A. J. Biol. Chem. 2004; 279: 13346-13353Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 21.Ünsal-Kaçmaz K. Sancar A. Mol. Cell. Biol. 2004; 24: 1292-1300Crossref PubMed Scopus (82) Google Scholar, 23.Wright J.A. Keegan K.S. Herendeen D.R. Bentley N.J. Carr A.M. Hoekstra M.F. Concannon P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7445-7450Crossref PubMed Scopus (198) Google Scholar, 24.Ball H.L. Cortez D. J. Biol. Chem. 2005; 280: 31390-31396Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In view of the fact that activation of ATM involves a change in its oligomerization (25.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2702) Google Scholar), this issue is highly pertinent. Finally, it is not known whether the binding of ATRIP to ATR is directly necessary for ATR to phosphorylate its targets appropriately. Extracts from Xenopus egg have proven to be a valuable tool for functional analysis of checkpoint regulatory pathways (6.Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 7.Hekmat-Nejad M. You Z. Yee M. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 26.Dasso M. Newport J.W. Cell. 1990; 61: 811-823Abstract Full Text PDF PubMed Scopus (253) Google Scholar, 27.Kumagai A. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 199-213Crossref PubMed Scopus (111) Google Scholar, 28.Kumagai A. Guo Z. Emami K.H. Wang S.X. Dunphy W.G. J. Cell Biol. 1998; 142: 1559-1569Crossref PubMed Scopus (212) Google Scholar, 29.Michael W.M. Ott R. Fanning E. Newport J. Science. 2000; 289: 2133-2137Crossref PubMed Scopus (160) Google Scholar). Our laboratory has previously identified and characterized a Xenopus homolog of ATRIP called Xatrip (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Xatrip forms a tight complex with Xenopus ATR (Xatr). Immunodepletion of Xatrip from egg extracts strongly compromises the checkpoint-dependent activation of Xenopus Chk1 (Xchk1). In this study, we have performed a systematic analysis to identify the various functional domains of Xatrip involved in association with DNA, interaction with Xenopus ATR (Xatr), and potential oligomerization of the Xatr-Xatrip complex. In parallel, we have examined how these domains contribute to the ability of Xatr-Xatrip to phosphorylate Xchk1 in response to checkpoint-inducing DNA templates. The results indicate that binding of Xatrip to Xatr is required for Xatr-Xatrip to associate with DNA in egg extracts, to adopt an oligomeric state, and to phosphorylate Xchk1. However, using model DNA templates that induce a checkpoint response, we have been able to show directly that mutants of Xatrip that have lost the ability to recruit Xatr stably to these templates are fully competent in supporting the Xatr-dependent phosphorylation of Xchk1, consistent with recent studies of human ATRIP (22.Ball H.L. Myers J.S. Cortez D. Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (187) Google Scholar). These observations suggest that proper physical interaction of Xatrip with Xatr is directly and inextricably linked with the ability of Xatr to phosphorylate its targets effectively. Egg Extracts and Oligonucleotides—Xenopus egg extracts were prepared as described (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The use of single-stranded (dA)70 and annealed, double-stranded (dA)70-(dT)70 to study checkpoint responses in egg extracts was reported previously (30.Kumagai A. Dunphy W.G. Mol. Cell. 2000; 6: 839-849Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The random sequence single-stranded and double-stranded 70-mer oligonucleotides used in this study were described previously (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Production of Recombinant Proteins—For small scale protein production in Sf9 insect cells, sequences from wild-type and mutant Xatrip proteins were cloned into a pIEx-1 vector (Novagen) with a FLAG epitope that was engineered into the 3′-end of the coding region by standard procedures. The constructs were transfected into Sf9 insect cells with the Cellfectin reagent (Invitrogen) according to the instructions of the manufacturer. For larger scale expression, Xatrip sequences were cloned into pFastBacHTa vectors with a His6 tag at the N-terminal end and either a FLAG or HA tag at the C-terminal or N-terminal ends, respectively. Purification with nickel-agarose beads was performed as described (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Recombinant human RPA was purified from Escherichia coli CodonPlus RIL cells as described (31.Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). Antibodies—Antibodies against Xatrip, Xatr, and Xenopus RPA70 were described previously (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 32.Lee J. Kumagai A. Dunphy W.G. Mol. Cell. 2003; 11: 329-340Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Anti-human RPA70 and anti-FLAG antibodies were purchased from U.S. Biological and Sigma, respectively. Immunodepletion from Egg Extracts—For immunodepletion of Xatr and Xatrip, antibodies coated on protein A-magnetic beads (Dynal, Inc.) were incubated with extracts on ice for 45 min. The beads were removed with a magnet, and the procedure was repeated to ensure complete removal of the proteins. Binding of Recombinant Xatrip to Oligonucleotides—Either cell lysates containing recombinant proteins or purified proteins were incubated with various biotinylated oligonucleotides coated on streptavidin-conjugated magnetic beads (Dynal) in buffer A (10 mm HEPES-KOH (pH 7.5), 80 mm NaCl, 20 mm β-glycerol phosphate, 2.5 mm EGTA, and 0.1% Nonidet P-40) containing 10 mm MgCl2, 100 μg/ml bovine serum albumin, and 10 mm dithiothreitol (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The beads were isolated with a magnet and processed for immunoblotting as described previously (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In order to test binding activity in egg extracts, either insect cell lysates or purified recombinant proteins were incubated with extracts at room temperature for 15 min. Next, the oligonucleotide-coated beads were added to the extracts, and the incubation was continued for 90 min. The beads were treated as described above after being collected by centrifugation through a sucrose cushion. In Vitro RPA Binding Assay—Anti-FLAG antibodies were coupled to GammaBind Plus Sepharose (Amersham Biosciences) and incubated with Sf9 cell lysates containing various Xatrip recombinant proteins at 4 °C for 1 h. Beads were washed four times with buffer A containing 1 mm phenylmethylsulfonyl fluoride and 1 mm dithiothreitol and mixed with RPA (50–100 nm) in buffer A in the absence or presence of 20 μg/ml (dA)70. After incubation at room temperature for 30 min, the beads were washed four times with buffer A. Bound proteins were eluted with SDS gel sample buffer and processed for immunoblotting. Gel Filtration Chromatography—A Superdex-200 10/300 GL column was equilibrated at 4 °C in a buffer containing 20 mm HEPES-KOH (pH 7.5), 80 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride. Egg extract (50 μl) was loaded onto the column, and the column was developed with the same buffer. Aliquots of fractions were subjected to SDS-PAGE, and elution profiles of the proteins of interest were determined by immunoblotting analysis. The standard proteins used for size estimation were thyroglobulin (669 kDa), ferritin (440 kDa), and catalase (232 kDa). Phosphorylation of 35S-Xchk1 in Egg Extracts—35S-Labeled Xchk1 proteins were synthesized in reticulocyte lysates as described (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Egg extracts were incubated with 35S-Xchk1 (one-tenth volume of a reaction), 50 μg/ml of (dA)70-(dT)70,3 μm tautomycin, 100 μg/ml cycloheximide for 90 min at room temperature. Aliquots of the reactions were removed for SDS-PAGE and phosphorimaging. Xatrip Can Associate with DNA in Vitro through both RPA-dependent and RPA-independent Mechanisms—To characterize the interaction of Xatrip with DNA, we immobilized various biotinylated oligonucleotide templates on streptavidin-coated beads and incubated the beads with recombinant His6-Xatrip-FLAG in the presence or absence of RPA. The beads were recovered, washed extensively, and analyzed for the binding of Xatrip by immunoblotting with anti-FLAG antibodies (Fig. 1A). In the presence of RPA, Xatrip showed a high affinity for all tested oligonucleotides, including a random sequence single-stranded 70-mer, a random sequence double-stranded 70-mer, single-stranded (dA)70, and double-stranded (dA)70-(dT)70. Interestingly, however, all of the templates except for (dA)70 did show detectable, albeit considerably lower, binding in the absence of RPA. To pursue this issue, we compared the binding of Xatrip to the homopolymers (dA)70, (dT)70, (dG)70, and (dC)70 in the presence and absence of RPA. As shown in Fig. 1B, Xatrip displayed a strict dependence on RPA for binding to (dA)70. By contrast, Xatrip bound quite well to (dT)70, (dG)70, and (dC)70 in both the presence and absence of RPA. These results suggest that Xatrip has an intrinsic single-stranded DNA binding activity with a preference for stretches of dT, dG, and dC. These observations could help to explain apparently discordant findings in the literature on the RPA dependence of human ATRIP for binding to DNA (19.Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2056) Google Scholar, 20.Bomgarden R.D. Yean D. Yee M.C. Cimprich K.A. J. Biol. Chem. 2004; 279: 13346-13353Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 21.Ünsal-Kaçmaz K. Sancar A. Mol. Cell. Biol. 2004; 24: 1292-1300Crossref PubMed Scopus (82) Google Scholar) (see “Discussion”). The N-terminal Region of Xatrip Is Required for in Vitro Binding to DNA—To determine which part of Xatrip is involved in association with DNA, we first constructed fragments encompassing the N-terminal half (residues 1–405), C-terminal half (residues 406–801), and a central region (residues 244–598) of the protein and tested binding to (dA)70, (dT)70, and (dA)70-(dT)70. Consistent with the previous observations (22.Ball H.L. Myers J.S. Cortez D. Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (187) Google Scholar, 33.Itakura E. Takai K.K. Umeda K. Kimura M. Ohsumi M. Tamai K. Matsuura A. FEBS Lett. 2004; 577: 289-293Crossref PubMed Scopus (19) Google Scholar), the N-terminal 1–405 fragment bound to all three DNA templates as efficiently as wild-type Xatrip, but there was no binding of either the 244–598 or 406–801 fragments (Fig. 2, A–D). The 1–405 fragment displayed both RPA-dependent binding to (dA)70 and RPA-independent binding to (dT)70 (Fig. 2, C and D). We proceeded to map which regions of Xatrip were important for these two different modes of binding. For these experiments, we made serial deletions from the N-terminal and C-terminal ends of the 1–405 fragment. We tested binding of these fragments to (dA)70 in the presence of RPA and to (dT)70 in the absence of RPA, respectively. As shown in Fig. 2C, the N-terminal 80 amino acids of Xatrip were necessary for RPA-dependent binding to (dA)70. However, this region is not sufficient for RPA-dependent binding, because fragments smaller than N-terminal residues 1–185 could not bind to RPA-coated (dA)70. On the other hand, more extensive N-terminal deletion mutants (e.g. 121–405 and 140–405) displayed good binding to (dT)70 in the absence of RPA (Fig. 2D). Only RPA-dependent Binding of Xatrip to DNA Can Be Detected in Egg Extracts—We asked whether the two distinct in vitro DNA-binding modes of Xatrip (e.g. RPA-dependent and RPA-independent) could also be observed in Xenopus egg extracts. To address this question, we removed RPA from egg extracts by immunodepletion with anti-RPA antibodies (32.Lee J. Kumagai A. Dunphy W.G. Mol. Cell. 2003; 11: 329-340Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In parallel, we prepared mock-depleted extracts using control antibodies. Next, we incubated these extracts with streptavidin beads containing either (dA)70 or (dT)70 and subsequently reisolated the beads to examine binding of Xatrip by immunoblotting. In mock-depleted extracts, Xatrip bound to both (dA)70 and (dT)70 in comparable amounts, and the binding of RPA to these two templates was similar (Fig. 3A). By contrast, in RPA-depleted extracts, there was no detectable binding to either (dA)70 or (dT)70. One possible explanation for the difference between the in vitro DNA binding assays and binding to DNA in egg extracts is that competition from other DNA-binding proteins in egg extracts obscures RPA-independent binding of Xatrip to DNA (see “Discussion”). To pursue these observations further, we examined what regions of Xatrip are required for association with DNA in egg extracts. We observed that neither the ΔN80 nor the ΔN120 mutants could bind to any single-stranded or double-stranded template that we tested in egg extracts (Fig. 3B). In order to localize the DNA binding region more precisely, we introduced smaller deletions lacking 31 (ΔN31) and 53 (ΔN53) amino acids from the N-terminal end of Xatrip. As shown in Fig. 3C, both the ΔN31 and ΔN53 mutants showed significant binding to DNA, which suggests that the region between amino acids 54 and 80 is crucial for RPA-dependent binding of Xatrip to DNA in egg extracts. Interaction of Xatrip with RPA—Next, we asked if Xatrip could interact directly with RPA. To address this issue, we first performed immunoprecipitation experiments in egg extracts. As shown in Fig. 4A, we observed small amounts of Xatrip in anti-RPA immunoprecipitates, but we could not detect RPA in anti-Xatrip immunoprecipitates. Similarly, ATR-ATRIP is present in anti-RPA immunoprecipitates from human cells (21.Ünsal-Kaçmaz K. Sancar A. Mol. Cell. Biol. 2004; 24: 1292-1300Crossref PubMed Scopus (82) Google Scholar). Inclusion of (dA)70-(dT)70 in the egg extracts did not have an effect on coimmunoprecipitation of RPA and Xatrip. These experiments suggested that Xatrip might have a low affinity for RPA. Therefore, we incubated purified Xatrip and RPA together in order to increase the local concentration of the two components relative to one another. In these experiments, purified RPA bound very well to FLAG-agarose beads containing Xatrip but not to control beads lacking Xatrip (Fig. 4B). Binding was not affected by the addition of (dA)70. Using this assay, we examined what part of Xatrip is involved in binding to RPA. We observed that the ΔN31 and ΔN53 mutants, but not the ΔN80 mutant, could interact well with RPA (Fig. 4C). Therefore, the same region of Xatrip that is essential for RPA-dependent binding to DNA in egg extracts is also necessary for direct binding of RPA. We proceeded further by examining which part of RPA is involved in this interaction. For this purpose, we examined different mutant constructs including a trimeric form of RPA in which the RPA70 subunit lacks 168 N-terminal residues and two truncation mutants of the isolated RPA70 subunit (residues 1–441 and 169–447) (34.Lao Y. Lee C.G. Wold M.S. Biochemistry. 1999; 38: 3974-3984Crossref PubMed Scopus (96) Google Scholar). As expected, all three mutants bound well to DNA (Fig. 4, D and E). However, only the 1–441 construct could support the association of Xatrip with DNA. These results imply that the first 168 amino acids of RPA70 are required for interaction with Xatrip. It is well established that this domain of RPA70 is involved in protein-protein interactions (e.g. with DNA polymerase α-primase and SV40 T antigen) (35.Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1186) Google Scholar). These data are also consistent with studies on a mutant of budding yeast RPA70 that is compromised in checkpoint control and unable to mediate the RPA-dependent binding of Ddc2, the budding yeast homologue of ATRIP, to DNA (19.Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2056) Google Scholar, 36.Umezu K. Sugawara N. Chen C. Haber J.E. Kolodner R.D. Genetics. 1998; 148: 989-1005Crossref PubMed Google Scholar, 37.Kim H.S. Brill S.J. Mol. Cell. Biol. 2001; 21: 3725-3737Crossref PubMed Scopus (96) Google Scholar). This mutation (rpa-t11) maps to Lys45 in the N-terminal domain of the protein. The C-terminal End of Xatrip Is Necessary but Not Sufficient for Binding to Xatr—In order to determine which region of Xatrip binds to Xatr, we examined a series of N-terminal and C-terminal truncation mutants. For assaying these mutants, we took advantage of the fact that there is an excess of Xatr over Xatrip in Xenopus egg extracts so that about one-third of Xatr is free of Xatrip (18.Kumagai A. Kim S.-M. Dunphy W.G. J. Biol. Chem. 2004; 279: 49599-49608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). As shown in Fig. 5A, if we incubated full-length Xatrip-FLAG in egg extracts, we could readily coimmunoprecipitate endogenous Xatr with anti-FLAG antibodies. Using this assay, we found that N-terminal deletion mutants of Xatrip lacking up to 244 amino acids (ΔN244) could still associate with Xatr well (Fig. 5A). By contrast, a more severe deletion mutant lacking the N-terminal half of the protein (ΔN405) could not bind to Xatr anymore (Fig. 5B). On the other hand, for the C-terminal end of Xatrip, even mutants with deletions as small as 21 amino acids (ΔC780) were incapable of binding to Xatr (Fig. 5, A and C). These results are consistent with the recent report that there are small conserved C-terminal motifs within the human ATRIP, Nbs1, and Ku80 proteins that interact with ATR, ATM, and DNA-PK, respectively (10.Falck J. Coates J. Jackson S.P. Nature. 2005; 434: 605-611Crossref PubMed Scopus (998) Google Scholar). Nearly this entire motif, which is located at amino acids 779–786 in Xatrip, is missing from the ΔC780 Xatrip mutant (Fig. 5A). However, this motif appears not to be sufficient for high affinity binding to Xatr, because even the whole C-terminal half of Xatrip (the ΔN405 mutant) cannot associate stably with Xatr. Therefore, there may be additional sequences in Xatrip that are involved in binding to Xatr. In the case of human ATRIP, an N-terminal deletion mutant containing residues 108–790 but not one containing residues 218–790 can bind to ATR (11.Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (754) Google Scholar). In addition, a splice variant of human ATRIP lacking residues 658–684 (equivalent to residues 666–694 of Xatrip) is defective for interaction with ATR (22.Ball H.L. Myers J.S. Cortez D. Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (187) Google Scholar). Interaction of Xatr with Xatrip Is Required for Binding to DNA in Egg Extracts—Next, we assessed the relationship between interaction of Xatrip with Xatr and binding of these proteins to DNA. For this purpose, we assayed the ability of the ΔC718 Xatrip mutant (which cannot interact with Xatr) to bind to DNA in both a purified system and in Xenopus egg extracts. First, we incubated either full-length or ΔC718 Xatrip in a defined system with streptavidin beads containing (dA)70 in the absence and presence of RPA (Fig. 6, A and B). We observed that the ΔC718 protein bound as well as full-length Xatrip to DNA, with both proteins showing the expected dependence on RPA. We proceeded to incubate both Xatrip proteins in egg extracts containing beads coated with (dA)70. In this experiment, wild-type Xatrip, but not the ΔC718 mutant, is able to associate with the free pool of endogenous Xatr in the extracts. In contrast to the results with the purified system, there was no binding of the ΔC718 mutant to DNA in egg extracts, although wild-type Xatrip bound well under the same conditions (Fig. 6C). To characterize these observations further, we examined the ability of wild-type Xatrip to bind to DNA in extr" @default.
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• W2054813812 title "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" @default.
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