Figure 8.

NPL facilitates pre-RC assembly on erythrocyte chromatin in egg extract. Permeable erythrocyte nuclei were incubated in control (CON), mock-depleted (MOC), NPL-depleted (ΔNPL), or depleted extract reconstituted with NPL (ΔNPL+NPL) for 45 min. Initiation events occurred within virtually all nuclei in both mock-depleted and NPL-depleted extracts by 60 min, as judged by the incorporation of biotin-dUTP into nascent DNA (our unpublished observation). Each sample was diluted with buffer containing 0.1% Triton X-100 and sedimented, and the chromatin proteins were separated by SDS-PAGE and transferred to nitro-cellulose. Western blots were probed with antibodies to XOrc1, XOrc2, XCdc6, XMcm3, and XMcm7, incubated with enzyme-conjugated secondary antibody, and developed with enhanced chemiluminescence using the ECL immunoblotting kit. (B) Intact G1-phase 3T3 nuclei were incubated in mock-depleted (MOC) or NPL-depleted (ΔNPL) extract for 6 h. The samples were pro-cessed as de-scribed in MATERIALS AND METHODS. DNA replication in the NPL-depleted sample represents a mean value derived from eight separate experiments in which two different extracts were used and is expressed as a percentage of the mock-depleted sample that was designated as 100%. The SEM for the depleted sample is shown.