Fig. 5. AKAP149-derived RVXF peptides introduced into G1-phase nuclei inhibit DNA replication in vivo and in vitro. (A) Nuclei of early G1-phase HeLa cells were microinjected with peptide buffer (Mock), 10 nM RAXF or 10 nM RVXF peptide, together with a 150 kDa FITC-dextran (green label). Cells were cultured for eight hours with 100 µM BrdU. Mock-injected cells were also cultured with 100 µM BrdU and 50 µM aphidicolin (Aphid). BrdU incorporation was detected with anti-BrdU antibodies. Nuclei of S-phase cells were also injected with RVXF peptides, cultured for five hours and BrdU labeling monitored. Percentages (±s.d.) of cells labeled with BrdU are shown (n=40-45/treatment). Arrows point to non-injected cells. Bar, 10 µM. (B) Isolated G1-phase HeLa nuclei were loaded with RVXF or RAXF peptides or exposed to peptide buffer (Mock). Nuclei were incubated for three hours in S-phase extract containing [³²P]dCTP, dNTPs and an ATP-regenerating system. [³²P]dCTP incorporation was analyzed by autoradiography. G1-phase nuclei were also exposed to extract from G0- or S-phase cells under conditions promoting replication. (C) G1-phase nuclei were incubated in S-phase extract containing 0 mM or 1 mM olomoucine (Olo) and DNA synthesis was assessed as in A. (D) Nuclei purified from S-phase cells were loaded with peptides, incubated in S-phase extract and [³²P]dCTP incorporation into replicated DNA was evaluated as in B.

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