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FIGURE 1. xCtc1, xStn1, and xTen1 form a trimeric complex. A. predicted structures of xCtc1, xStn1, and xTen1 gene products. Black boxes represent putative OB-fold-containing regions. Percentages of identical and similar amino acids shared by Xenopus proteins and human ones are summarized in the table. B, recombinant xCST (3ÃFLAG-xCtc1, His6-xStn1, and His6-xTen1) expressed in insect cells was affinity-purified using anti-FLAG beads and Ni beads. Proteins were visualized by silver staining. FT, flow-through fraction; B, bead-bound proteins after elution; E1 and E2, eluate with 100 mm imidazole; E3 and E4, eluate with 200 mm imidazole; and E5 and E6, eluate with 500 mm imidazole. C, E1 and E2 fractions in B were combined and subjected to gel filtration. Proteins in each fraction were separated by SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue.
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FIGURE 2. xCST binds to ssDNA with moderate preference for G-rich sequences. A, preparation of recombinant xCST and xRPA used in EMSA. Proteins were visualized by SYPRO Ruby staining of SDS-polyacrylamide gel. B, sequences of 32-mer ssDNA probes used in EMSA. C, EMSA. The reaction mixtures included 20 nm 32P-labeled DNA probes and 10 nm xCST (lanes 6â10) or 10 nm xRPA (lanes 11â15). For lanes 1â5, control buffer was included instead of protein samples.
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FIGURE 3. xStn1 associates with chromatin and localizes at telomeres. A, xStn1 localization in in vitro reconstituted Xenopus sperm chromatin was analyzed by indirect immunofluorescence in combination with telomere FISH. A representative set of microscopic images is shown. DNA stained with DAPI is shown in blue. White arrowheads indicate the co-localization of xStn1 foci (green) and telomeric FISH signals (red). Enlarged images of the numbered white boxes on the merged picture are shown on the right. B, Xenopus sperm nuclei were incubated in interphase LSS for the indicated times. The time when sperm nuclei were added to the egg extract was set to 0 min. Isolated chromatin was analyzed by immunoblotting using the indicated antibodies. The reaction mixtures contained control buffer (lanes 1â4), 60 μg/ml GST-geminin H (lanes 5â8), or 50 μg/ml aphidicolin (lanes 9â12), respectively.
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FIGURE 4. xStn1 is required for priming on ssDNA template but not essential for bulk chromosomal replication. A, xStn1-immunodepleted or mock-immunodepleted extracts (HSS and NPE) were analyzed by immunoblotting against xStn1. The volume of the loaded extracts is 0.2 μl/lane. Positions of xStn1 protein are indicated. Although the anti-xStn1 antibody detected a single band in immunoblotting experiments using the cell lysates of Sf9 expressing recombinant xStn1, it produced many nonspecific bands in the immunoblotting using the egg extracts. This is probably because the concentration of endogenous xStn1 is so low (5 nm at most, see supplemental Fig. S8) that cross-reactions with proteins other than xStn1 are dominantly detected over the specific interaction between the antibody and xStn1. Unlike xStn1, most of the nonspecific bands detected in the immunoblotting experiments with anti-xStn1 antibody were not immunoprecipitated by anti-xStn1 antibody, suggesting that the antibody immunodepletes proteins with high specificity compared with the immunoblotting experiments (supplemental Fig. S3). Moreover, similar results were obtained when we immunodepleted xStn1 from HSS with two independently immunized anti-xStn1 antibodies that recognized different sets of nonspecific bands (KU003 and KU004, see supplemental Fig. S3), supporting the notion that the biological effects of the immunodepletion observed in B are caused by the depletion of xStn1 or its binding proteins. B, upper, DNA replication assay using pBluescript dsDNA as template. pBluescript was incubated in xStn1-immunodepleted or mock-immunodepleted HSS for 30 min. Then, 2 volumes of xStn1-immunodepleted or mock-immunodepleted NPE containing [α-32P]dCTP was added, and the reaction was incubated further. Aliquots were withdrawn at the indicated time points, and DNA was isolated and subjected to agarose gel electrophoresis. Nucleotide incorporation was visualized by autoradiography. Lower, DNA replication assay using M13 ssDNA as template. M13 was incubated in xStn1-immunodepleted or mock-immunodepleted extracts containing [α-32P]dCTP for the indicated times. Nucleotide incorporation was analyzed as described above.
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FIGURE 5. xCST(n) enhances M13 replication and reverses the neutralizing effect of anti-xStn1 antibody. A, M13 was incubated in HSS containing [α-32P]dCTP for 1.5 h in the presence of 0, 0.05, or 0.1 pmol/μl xCST(n). Replication efficiency, which was determined by measuring radioactivity on the gel, was normalized to the standard condition (without xCST(n) addition). The results of three independent experiments are shown in the bar graph. B, same experiment as A, except that anti-xStn1 KU004 was included in the reactions at the concentration of 0.04 μg/μl. The results of three independent experiments are shown in the bar graph. Replication efficiency was normalized to the standard condition (without xCST(n) and antibody addition).
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FIGURE 6. Priming is disturbed in the absence of xStn1. Left, primed or nonprimed M13 was incubated for 1 h in xStn1-immunodepleted or mock-immunodepleted HSS containing [α-32P]dCTP. KU003 antibody was used to immunodeplete xStn1 (see âExperimental Proceduresâ). Right, radioactivity on the gel was quantified to calculate the amount of newly synthesized DNA in the reactions. Replication efficiency was defined as the ratio of the amount of synthesized DNA to that of input DNA and expressed as percentage. The graph shows the results of three independent experiments.
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FIGURE 7. xStn1 immunodepletion does not affect DNA polymerase α-primase binding to ssDNA in Xenopus egg extracts. A, M13 was incubated for 1 h in xStn1-immunodepleted or mock-immunodepleted HSS supplemented with 0.2 mm aphidicolin. The reactions were terminated by transferring the test tubes onto ice, and samples were immediately fractionated by gel filtration. Fractions were collected, and the presence of DNA was revealed by agarose gel electrophoresis followed by SYBR Gold staining. Identities of the circular/linearized forms were determined on the basis of the results shown in supplemental Figs. S4B and S7A. B, fractions 12â14 in A were analyzed by immunoblotting using the indicated antibodies. HSS that did not contain M13 DNA was also processed as described in A and used for the immunoblotting experiments (lanes 7-9). As a reference, 0.2 μl of HSS was loaded in lane 10.
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