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Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract

¹ Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan
² Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (25K): [in a new window]   Fig. 1. Characterization of anti-RPA p32 antibody by immunoblotting. 1 µl of interphase Xenopus egg extract was subjected to SDS-electrophoresis on a 10% polyacrylamide gel. The electrophoresed proteins were transferred onto PVDF-membrane and the protein bands that reacted with the antibody were visualized as described in Materials and Methods.

View larger version (42K): [in a new window]   Fig. 2. Focus-formation of RPA in the nuclei incubated with Xenopus egg extract. (A) Demembranated Xenopus sperm nuclei were incubated at 23°C for 30 minutes (a-c), 60 minutes (d-f) or 90 minutes (g-i). RPA resistant to detergent treatment is shown in panels a, c, d, f, g and i. DNA was visualized using propidium iodide (b,e,h). Panels c, f and i are enlarged images of the nuclei indicated by arrowheads in panels a, d and g, respectively. The same nuclei in the `DNA' panels are also indicated by arrowheads. Bars, 50 µm (a,b,d,e,g,h); 20 µm (c,f,i). (B) A detergent-insoluble nuclear fraction was prepared from the nuclei incubated in Xenopus egg extract for 30 minutes, 60 minutes or 90 minutes. The p32 subunit of RPA in the nuclear fraction was visualized by immunoblotting after SDS-polyacrylamide gel electrophoresis. (C) The detergent-insoluble nuclear fraction prepared from 30 minute incubated nuclei was incubated for 10 minutes at 37°C with (lanes 1,2) or without (lanes 3,4) MNase, and the reaction mixture was centrifuged. The resultant supernatant (lanes 1,3) and precipitated (lanes 2,4) fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting.

View larger version (30K): [in a new window]   Fig. 3. Focus-formation of RPA and DNA replication in the nuclei incubated with Xenopus egg extract containing CPT. (A) Demembranated Xenopus sperm nuclei were incubated at 23°C, for the periods indicated, in the presence of CPT. RPA localization (a,c,d,f,g,i) and DNA (b,e,h) were visualized. Panels c, f and i are enlarged images of the nuclei indicated by arrowheads in panels a, d and g, respectively. The same nuclei in the `DNA' panels are also indicated by arrowheads. Bars, 50 µm (a,b,d,e,g,h); 20 µm (c,f,i). Numbers of RPA-accumulated nuclei/observed nuclei from two independent experiments are shown below the panels. The percentages of RPA-accumulated nuclei are also presented in parentheses. (B) A detergent-insoluble nuclear fraction was prepared from the nuclei incubated at 23°C for 60 minutes in the absence (lane 1) or the presence (lanes 2-6) of 25 µM CPT. The nuclear fraction was subjected to SDS-polyacrylamide gel electrophoresis directly (lanes 1,2), or after incubation with (lanes 3,4) or without (lanes 5,6) MNase followed by centrifugal separation of supernatant (lanes 3,5) and precipitated (lanes 4,6) fractions. The p32 subunit of RPA was visualized by immunoblotting. (C) The sperm nuclei were incubated in the extract at 23°C for the periods indicated in the presence of [-32P]dCTP. Circles, squares and triangles represent DNA synthesis in the absence, and presence of CPT and CPT+caffeine, respectively. The extent of DNA synthesis is represented as a percentage of accumulative DNA synthesis after a 90 minute incubation under damage-noninducing conditions. 100% DNA synthesis is estimated to be an amount equivalent to the duplication of about 90% of input DNA. Open symbols indicate accumulative DNA synthesis during a 60 minute incubation in the presence of geminin. (D) The sperm nuclei were incubated in the extract containing CPT for 60 minutes at 23°C in the presence of geminin. Localization of RPA (a,c) and DNA (b) is shown. Panel c is an enlarged image of the nucleus indicated by an arrowhead in panel a. The same nucleus in panel b is also indicated by an arrowhead. Bars, 50 µm (a,b); 20 µm (c).

View larger version (32K): [in a new window]   Fig. 4. Formation of -H2AX foci in the nuclei incubated in Xenopus egg extract containing CPT. Demembranated sperm nuclei were incubated in Xenopus egg extract with (B) or without (A) CPT. -H2AX foci formation (a,c,e,g) and DNA (b,d,f,h) in the absence (a,b,c,d) or the presence (e,f,g,h) of geminin were visualized. Panels c, d, g and h are enlarged images of the nuclei indicated by arrowheads in panels a, b, e and f, respectively. Bars, 50 µm (a,b,e,f); 20 µm (c,d,g,h).

View larger version (27K): [in a new window]   Fig. 5. Focus-formation of RPA in nuclei incubated with Xenopus egg extract containing EcoRI. (A) Demembranated Xenopus sperm nuclei were incubated at 23°C for the periods indicated in the presence of EcoRI. Localization of RPA (a,c,d,f,g,i) and DNA (b,e,h) is shown. Panels c, f and i are enlarged images of the nuclei indicated by arrowheads in panels a, d and g, respectively. The same nuclei in the `DNA' panels are also indicated by arrowheads. Bars, 50 µm (a,b,d,e,g,h) or 20 µm (c,f,i). Numbers of RPA-accumulated nuclei/observed nuclei from two independent experiments are shown below the panels. The percentages of RPA-accumulated nuclei are also presented in parentheses. (B) A detergent-insoluble nuclear fraction was prepared from the nuclei incubated at 23°C for 60 minutes in the absence (lane 1) or the presence (lanes 2-6) of 0.05 units/µl EcoRI. The nuclear fraction was subjected to SDS-polyacrylamide gel electrophoresis directly (lanes 1,2), or after incubation with (lanes 3,4) or without (lanes 5,6) MNase followed by centrifugal separation of supernatant (lanes 3,5) and precipitated (lanes 4,6) fractions. The p32 subunit of RPA was visualized by immunoblotting. (C) The sperm nuclei were incubated in the extract containing EcoRI for 60 minutes at 23°C in the presence of geminin. Localization of RPA (a,c) and DNA (b) is shown. Panel c is an enlarged image of the nucleus indicated by an arrowhead in panel a. The same nucleus is also indicated by an arrowhead in panel b. Bars, 50 µm (a,b) or 20 µm (c).

View larger version (17K): [in a new window]   Fig. 6. Suppression of DNA replication by EcoRI-treatment. Demembranated Xenopus sperm nuclei were incubated for 90 minutes at 23°C in the extract with or without EcoRI, caffeine and geminin in the presence of [-32P]dCTP. The extent of DNA synthesis is represented as a percentage of accumulative DNA synthesis after a 90 minute incubation in the absence of DNA damaging agents. 100% DNA synthesis is estimated to be an amount equivalent to the duplication of about 90% of input DNA.

View larger version (31K): [in a new window]   Fig. 7. Caffeine did not affect focus-formation of RPA. Demembranated Xenopus sperm nuclei were incubated in the extract containing 5 mM caffeine for 60 minutes at 23°C in the presence of EcoRI (A) or CPT (B). Localization of RPA (a,c) and DNA (b) is shown. Panel c is an enlarged image of the nucleus indicated by an arrowhead in panel a. The same nucleus is also indicated by an arrowhead in panel b. Bars, 50 µm (a,b) or 20 µm (c).

View larger version (34K): [in a new window]   Fig. 8. EcoRI-induced DNA synthesis was co-localized with RPA foci and insensitive to caffeine treatment. Demembranated Xenopus sperm nuclei were incubated in the extract containing geminin and biotin-dUTP for 30 minutes at 23°C in the absence (a-c), or presence of EcoRI (d-i) or EcoRI+caffeine (j-o). Localization of biotin-dUTP (a,d,g,j,m) and RPA (b,e,h,k,n) is shown in green and red, respectively. Merged images are also shown in panels c, f, i, l and o. Panels g, h, i, m, n and o are enlarged images of the nuclei indicated by arrowheads in panels d, e, f, j, k and l, respectively. Bars, 50 µm (a-f,j-l); 20 µm (g-i,m-o).