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Fig. 4. RPA assembles to pre-replication foci. (A) Experimental scheme. Sperm chromatin was incubated for 60 minutes at 22°C in a low-speed egg extract (LSE) in the presence of aphidicolin (5 µg/ml) to slow down DNA replication (95% inhibition of total nucleotide incorporation, data not shown) and to label replication initiation foci with biotinylated dUTP. After a first wash, fresh LSE was added and elongation allowed to proceed for 60 minutes without biotin dUTP. Then purified recombinant cyclin B
90 was added to induce mitosis. After a second wash, calcium was added to promote the exit from mitosis and entry in a new S phase cycle. (B) Nuclei were analyzed by immunofluorescence at each step of the reaction to detect DNA (DAPI staining), RPA (monoclonal antibody) and dUTP incorporation. The merged images of RPA and dUTP are also shown. DNA damage was analyzed using an 
-H2AX antibody, a known marker of DNA repair (Furuta et al., 2003; Kobayashi et al., 2002) (Fig. S2, see supplementary material). In row 2, elongation was without biotin-dUTP, but it was also followed by adding biotin-dUTP in a sample resulting in a homogenous dUTP staining (Fig. S3, see supplementary material). In row 4, the reassembly of RPA was monitored at 5 minute intervals and occurred 10 to 15 minutes after adding fresh LSE.
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