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. 2006 Nov 23;25(24):5764–5774. doi: 10.1038/sj.emboj.7601436

Figure 4.

Figure 4

Free Cdt1, but not the Cdt1–geminin complex, leads to rereplication and checkpoint activation. (A) Left panel: kinetics of sperm chromatin replication supplemented with buffer (circles), 40 nM free Cdt1 (rectangles) or 40 nM Cdt1–geminin complex (triangles). Right panel: the same reactions were performed in the presence of 5 mM caffeine. (B) Western blot analysis of purified nuclei with a phospho-Chk1 antibody. Nuclei were purified after 100 min incubation of sperm chromatin in egg extract supplemented with either buffer, free Cdt1, Cdt1–geminin complex (all 40 nM), or Aphidicolin (50 μg/ml). A Western blot with ORC2 antibody as loading control is also shown. (C) Analysis of replicated DNA half-substituted with BrdUTP (‘heavy–light', HL) or double-substituted after rereplication (‘heavy–heavy', HH) on a CsCl gradient. Before sperm chromatin addition, reactions were supplemented either with buffer (circles), free Cdt1 (rectangles) or Cdt1–geminin complex (diamonds). (D) Immunofluorescence of G2 chromatin (120 min after sperm chromatin addition) and 20 min after addition of either buffer, free Cdt1, Cdt1–geminin complex, or free geminin. DNA is shown in blue stained with Hoechst, geminin in green (FITC) and MCM3 in red (Texas red). (E) Analysis of replicated DNA half-substituted with BrdUTP after one round of replication (‘heavy–light', HL) or double-substituted after rereplication (‘heavy–heavy', HH) on a CsCl gradient in reactions supplemented in G2 (120 min after sperm DNA was added) either with buffer (circles), free Cdt1 (rectangles) or Cdt1–geminin complex (diamonds).