Figure 6.

Immunodepletion of NPL from egg extract inhibits initiation but not elongation in erythrocyte nuclei. (A) Permeable erythrocyte nuclei were incubated in control (CON), mock-depleted (MOC), NPL-depleted (ΔNPL), or depleted extract reconstituted with NPL (ΔNPL+NPL) containing [α-³²P]-dATP for 8 h. The samples were processed as described in MATERIALS AND METHODS. DNA replication is expressed as a percentage of the control sample that was designated as 100%. The data shown are mean values ± SE from three separate experiments in which three different extracts were used. (B) Permeable erythrocyte nuclei were incubated for 8 h in NPL-depleted extract supplemented with BrdUTP and [α-³²P]dATP. DNA was purified from each sample and centrifuged to equilibrium in a cesium chloride gradient. The refractive index of every fifth fraction was determined. The radioactivity in each fraction was measured by liquid scintillation, and the counts per minute (cpm) are shown. The expected densities of heavy/light DNA (HL, 1.75 g/ml) and heavy/heavy DNA (HH, 1.79 g/ml) are indicated. (C) Permeable erythrocyte nuclei were incubated for 8 h in mock-depleted extract (MOC), NPL-depleted extract (ΔNPL), or mock-depleted extract supplemented with aphidicolin at 20 μg/ml (MOC+ APH), each containing [α-³²P]dATP. DNA was precipitated and separated on a 1% agarose gel under alkaline denaturing conditions. An autoradiogram of the nascent DNA is shown. (D) Permeable erythrocyte nuclei were incubated in control (CON), mock-depleted (MOC), NPL-depleted (ΔNPL), or depleted extract reconstituted with NPL (ΔNPL+NPL) containing 20 μM biotinylated dUTP for 8 h. Bulk DNA was stained with Hoechst 33258 (DNA), and nascent DNA was labeled with fluorescein-conjugated streptavidin (Biotin). An unincubated nucleus is also shown (XEN). The samples were processed as described in MATERIALS AND METHODS.