1. TopBP1 Activates the ATR-ATRIP Complex
Akiko Kumagai, Joon Lee, Hae Yong Yoo, William G. Dunphy 
Cell 
Volume 124, Issue 5, Pages (March 2006)
DOI: /j.cell
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Recombinant XtopBP1 Induces Activation of the Xatr-Xatrip Complex
(A) Isolation of Xatr-Xatrip complexes. Control buffer (lane 1), His6-XatripΔN222-FLAG (lane 2), and full-length His6-Xatrip-FLAG (lane 3) were added to egg extracts containing anti-FLAG antibody beads. After incubation, the beads were reisolated, washed, and eluted with 3X-FLAG peptide. The eluates were immunoblotted with anti-Xatr (top) and anti-FLAG antibodies (bottom).
(B) Preparation of His6-XtopBP1. The protein was purified from Sf9 cells and stained with Coomassie blue.
(C) The kinase activity of isolated Xatr-Xatrip is greatly increased in the presence of His6-XtopBP1. Control eluate (lanes 1 and 2) and eluates containing either Xatr-XatripΔN222 (lanes 3 and 4) or Xatr-Xatrip complexes (lanes 5 and 6) from (A) were incubated in kinase buffer with [32P]ATP and PHAS-I in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of His6-XtopBP1. Reactions were subjected to SDS-PAGE. The gel was stained with Coomassie blue (top), and 32P incorporation into PHAS-I was detected by phosphorimaging (bottom).
(D) His6-XtopBP1 induces the kinase activity of Xatr in a dose-dependent manner. Xatr-XatripΔN222 (lanes 1–7) and control eluate (lane 8) were incubated either without XtopBP1 (lane 1) or with different amounts of XtopBP1 (0.5–30 μg/ml) (lanes 2–8) in the presence of [32P]ATP and PHAS-I. Reactions were processed for staining with Coomassie blue (top) and phosphorimaging (bottom).
(E) Quantitation of 32P incorporation into PHAS-I from (D).
Cell  , DOI: ( /j.cell )
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3. Figure 2
The XtopBP1-Induced Kinase Activity of the Xatr-XatripΔN222-FLAG Complex Is Due to Xatr
(A) Egg extracts were left untreated (lanes 1–4) or subjected to an immunodepletion procedure with either control (lane 5) or anti-Xatr antibodies (lane 6). Extracts were incubated with anti-FLAG beads in the absence (lanes 1 and 2) or presence (lanes 3–6) of XatripΔN222-FLAG. The beads were reisolated and eluted with 3X-FLAG peptide. Eluates were incubated with [32P]ATP and PHAS-I in the absence (lanes 1 and 3) or presence (lanes 2 and 4–6) of His6-XtopBP1. After SDS-PAGE, the top portion of the gel was immunoblotted with anti-Xatr and anti-FLAG antibodies (top two panels). The bottom portion was stained with Coomassie blue (second panel from bottom) and analyzed with a phosphorimager (bottom panel).
(B) Quantitation of the data in (A). Data are represented as mean ± standard deviation.
(C) Kinase activity of the activated form of Xatr is sensitive to caffeine. Xatr-XatripΔN222 was activated by His6-XtopBP1 and assayed for kinase activity toward PHAS-I in the absence (lane 1) or presence of 0.04, 0.2, 1, and 5 mM caffeine (lanes 2–5) as in (A).
(D) Plot of the data in (C). Data are represented as mean ± standard deviation.
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4. Figure 3 Mapping of the ATR-Activating Domain from XtopBP1
(A) Domains of XtopBP1. Roman numerals indicate BRCT domains, as designated in the literature. Fragments containing the indicated amino acids of XtopBP1 (1–348, 333–646, 623–984, 972–1279, and 1197–1513) were produced as GST fusion proteins in bacteria. The Δ993–1196 mutant was produced as a His6 fusion in Sf9 cells.
(B) Xatr-XatripΔN222 complex (lanes 1–6) and control eluate (lane 7) were incubated in the absence (lane 1) or presence (lanes 2–6) of GST fusion proteins containing the indicated segments of XtopBP1 at a final concentration of 40 μg/ml in kinase buffer containing [32P]ATP and PHAS-I. Reactions were subjected to SDS-PAGE. GST fusion proteins and PHAS-I were stained with Coomassie blue (top and middle panels). Incorporation of 32P into PHAS-I was detected by phosphorimaging (bottom panel).
(C) The ATR-activating function is conserved in human TopBP1. The Xatr-XatripΔN222 complex (lanes 1–3) was incubated with buffer alone (lane 1), XtopBP1(972–1279) (lane 2), or human GST-TopBP1(978–1192) (lane 3) and assayed for kinase activity toward PHAS-I.
(D) Preparations of His6-XtopBP1 and His6-XtopBP1Δ993–1196 from Sf9 cells (stained with Coomassie blue).
(E) Control eluate (lanes 1–3) and Xatr-XatripΔN222 complex (lanes 4–6) were incubated with buffer alone (lanes 1 and 4), full-length His6-XtopBP1 (lanes 2 and 5), or His6-XtopBP1Δ993–1196 (lanes 3 and 6) and assayed for kinase activity toward PHAS-I.
(F) XtopBP1 activates Xatr but not Xatm. Egg extracts were immunoprecipitated with control (lanes 1 and 2), anti-Xatr antibodies (lanes 3 and 4), or anti-Xatm antibodies (lanes 5 and 6). The immunoprecipitates were incubated in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of GST-XtopBP1(972–1279) in kinase buffer containing [32P]ATP and PHAS-I. The samples were subjected SDS-PAGE and immunoblotted with anti-Xatm (top) and anti-Xatr antibodies (middle). Kinase activity was determined by 32P incorporation into PHAS-I (bottom).
Cell  , DOI: ( /j.cell )
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5. Figure 4
XtopBP1 Associates with Xatr in a Manner that Depends on Xatrip
(A) GST-XtopBP1(972–1279) associates with Xatr-Xatrip. Egg extracts (lane 1) were incubated with no recombinant protein (lane 2), GST-XtopBP1(972–1279) (lane 3), or GST-XtopBP1(1197–1513) (lane 4). The extracts were filtered through a G25 Sephadex column to remove endogenous glutathione and were incubated with glutathione-agarose beads. The beads were isolated and immunoblotted for Xatr (top) and Xatrip (bottom).
(B) Egg extracts (lane 1) were immunodepleted with control (lane 2) or anti-Xatrip antibodies (lanes 3 and 4). For lane 4, recombinant XatripΔN222-FLAG was added back later. Extracts were immunoblotted with anti-Xatr (top) and anti-Xatrip antibodies (bottom).
(C) Control buffer (lane 1) and GST-XtopBP1(972–1279) (lanes 2–4) were incubated with the indicated extracts from (B). Binding of Xatr to glutathione beads was determined by immunoblotting as in (A).
(D) Xatr was immunoprecipitated from mock-depleted (lanes 1 and 2) and Xatrip-depleted extracts (lanes 3 and 4). We immunoprecipitated twice as much Xatrip-depleted extract due to its reduced content of Xatr. The immunoprecipitates were incubated with control buffer (lanes 1 and 3) or GST-XtopBP1(972–1279) (lanes 2 and 4) and assayed for kinase activity toward PHAS-I (bottom). Samples were also immunoblotted for Xatr (top) and Xatrip (middle).
Cell  , DOI: ( /j.cell )
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6. Figure 5
Identification of a Point Mutant in the ATR-Activating Domain and Activation of Recombinant Human ATR
(A) Alignment of residues 993–1196 from the ATR-activating domain of XtopBP1 with corresponding segments of human, chicken, and zebrafish TopBP1. Two conserved sequences (RQLQ and WDDP) are marked with lines, and a conserved tryptophan (W1138 in XtopBP1) is denoted with an asterisk.
(B) Xatr-XatripΔN222 was incubated with GST-XtopBP1(1197–1513) (lane 1) or wild-type (lane 2), W1138R (lane 3), ΔWDDP (lane 4), and ΔRQLQ (lane 5) versions of GST-XtopBP1(972–1279) in the presence of PHAS-I and [32P]ATP. Reactions were stained with Coomassie blue to detect GST fusions (top) and PHAS-I (middle) and were analyzed with a phosphorimager (bottom).
(C) Isolation of recombinant human ATR proteins. Protein G magnetic beads containing no antibody (lane 1) or anti-FLAG antibodies (lanes 2 and 3) were incubated in nuclear extracts of the GW33 and GK41 U2OS cell lines that were induced to express either wild-type (WT) (lanes 1 and 2) or kinase-deficient (KD) FLAG-ATR (lane 3), respectively. The beads were reisolated and eluted with 3X-FLAG peptide. The whole preparations (beads plus eluates) were immunoblotted with anti-FLAG antibodies.
(D) Activation of recombinant human ATR. Mock preparations (lanes 1–3) and preparations containing either WT (lanes 4–6) or KD FLAG-ATR (lanes 7–9) from (C) were incubated with control buffer (lanes 1, 4, and 7), GST-XtopBP1(972–1279) (lanes 2, 5, and 8), or GST-XtopBP1(972–1279)-W1138R (lanes 3, 6, and 9) in the presence of GST-Xmcm2(62–122). Samples were subjected to SDS-PAGE and processed for staining with Coomassie blue (top) and immunoblotting with anti-phospho-S92 of Xmcm2 antibodies (bottom).
Cell  , DOI: ( /j.cell )
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7. Figure 6
The Isolated ATR-Activating Domain of TopB1 Induces Ectopic Phosphorylation of Downstream Targets of ATR
(A) Control buffer (lane 1), GST-XtopBP1(972–1279) at a final concentration of either 50 μg/ml (lane 2) or 100 μg/ml (lane 3), and 100 μg/ml GST-XtopBP1(1197–1513) (lane 4) were added to interphase egg extracts. Extracts were incubated for 90 min in the presence of 35S-Xchk1 and 3 μM tautomycin. Reactions were subjected to SDS-PAGE and phosphorimaging.
(B) Phosphorylation of [35S]Xchk1 was determined in egg extracts that were incubated with control buffer (lane 1), GST-XtopBP1(972–1279) (lane 2), or the indicated mutants of the 972–1279 fragment (lanes 3–5) as described in (A). All GST fusion proteins were added at a final concentration of 100 μg/ml.
(C) Human 293T cells were transfected with plasmids encoding EGFP (lane 1), wild-type EGFP-NLS-TopBP1(978–1286) (lane 2), and EGFP-NLS-TopBP1(978–1286)-W1145R (lane 3). In addition, untransfected cells were incubated in the absence (lane 4) or presence of 2 mM hydroxyurea for 18 hr (lane 5). Lysates from the cells were immunoblotted with antibodies against phospho-S108 of human Mcm2 (top) or the Mcm2 protein (bottom).
(D) 293T cells were transfected with the indicated plasmids. Cells were processed for indirect immunofluorescence with anti-phospho-S108 antibodies and Alexa 568-conjugated goat anti-rabbit antibodies. In addition, cells were examined for fluorescence of EGFP and stained for DNA with Hoechst
(E) Quantitation (with the ImageJ program) of the immunofluorescence signal of phospho-S108 from the cells in (D) as well as from cells transfected with EGFP alone. Signals were compared between expressing and nonexpressing cells. Data are represented as mean ± standard deviation.
Cell  , DOI: ( /j.cell )
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8. Figure 7
The W1138R Mutant of XtopBP1 Is Defective in Checkpoint Regulation in Xenopus Egg Extracts
(A) Preparations of wild-type (lane 1) and W1138R mutant (lane 2) versions of full-length His6-XtopBP1 were stained with Coomassie blue.
(B) Kinase reactions with control buffer (lane 1), wild-type His6-XtopBP1 (lanes 2 and 4), and His6-XtopBP1-W1138R (lanes 3 and 5) were conducted with PHAS-I and [32P]ATP in the presence (lanes 1-3) or absence (lanes 4 and 5) of the Xatr-XatripΔN222 complex. Samples were stained with Coomassie blue (top) or analyzed with a phosphorimager (bottom).
(C) Immunodepletion of XtopBP1. Egg extracts were left untreated (lane 1) or immunodepleted with control (lane 2) or anti-XtopBP1 antibodies (lanes 3–5). In some cases, either wild-type (lane 4) or W1138R His6-XtopBP1 (lane 5) was added back to the extracts. Extracts were immunoblotted for XtopBP1.
(D) Untreated (lanes 1 and 2), mock-depleted (lane 3), and XtopBP1-depleted extracts containing control buffer (lane 4), wild-type His6-XtopBP1 (lane 5), or W1138R His6-XtopBP1 (lane 6) were incubated with [35S]Xchk1 in the absence (lane 1) or presence (lanes 2–6) of aphidicolin. Nuclear fractions were subjected to SDS-PAGE and immunoblotted with anti-phospho-S344 of Xchk1 antibodies (top). Samples were also analyzed by phosphorimaging (bottom).
(E) DNA replication was measured in a 2 hr incubation by incorporation of 32P into chromosomal DNA in XtopBP1-depleted extracts to which either wild-type (lane 1) or W1138R His6-XtopBP1 (lane 2) had been added back.
Cell  , DOI: ( /j.cell )
Copyright © 2006 Elsevier Inc. Terms and Conditions