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).

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