Figure 5

Catalysis and formation of the FACT-MCM complex is regulated in a cell cycle-dependent manner. (A) FACT-associated immunocomplexes were isolated by mAbs 10D1 (left panel) and 8D2 (right panel) from HeLa cells at different cell cycle stages: exponentially growing (As), G₁/S phase, and M phase and G₂. The immunoprecipitate was subsequently subjected to DNA helicase assay as in Figure 1B. Displacement of the annealed substrate by heat denaturation is also shown ("B"). (B) Indirect immunofluorescence analysis was performed on HeLa cells to observe localization of endogenous MCM4 and SSRP1 using the respective antibodies. Nuclear DNA was counter-stained by DAPI. Cells in indicated phases of the cell cycle were examined: early G₁, G₁/S, mid- and late- S phase (cells at 2 and 4 hours after G₁/S release, respectively). Images were generated by laser scanning confocal microscopy.