Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Abstract

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.

Figure 1

Cdc6-depleted chromatin is unable to replicate in Cdt1-depleted extract. (A) A schematic representation of an experimental procedure. Xenopus sperm nuclei (100 000 nuclei) were incubated for 20 min with 20 μl of Cdt1- or Cdc6-double depleted extract (ΔCdt1 or ΔCdc6, respectively), or mock-treated extract in the absence or presence of 0.5 μM recombinant geminin (mock or mock + gem, respectively). After the incubation, 2 μl of chromatin fraction isolated from each extract was incubated for 3 h with 10 μl of Cdt1-single depleted extract supplemented with [α-³²P]dATP (20 kBq). (B) Chromatin after the incubation with mock-treated extract without or with geminin (mock or mock + gem, respectively), Cdt1-depleted extract (ΔCdt1), or Cdc6-depleted extract (ΔCdc6) was isolated and further incubated with the Cdt1-single depleted extract. DNA synthesis during the second incubation is represented as a percentage of the radioactivity incorporated into DNA in the Cdt1-depleted extract to that in a mock-treated extract.

Figure 2

Cdc6-depleted chromatin is incapable of being licensed for DNA replication. (A) A schematic representation of experimental procedures. Xenopus sperm nuclei (100 000 nuclei) were incubated for 20 min with 20 μl of Cdt1- (ΔCdt1) or Cdc6-double depleted (ΔCdc6) extract (1st incubation), resulting in Cdt1-depleted chromatin (ΔCdt1-chromatin) or Cdc6-depleted chromatin (ΔCdc6-chromatin), respectively, after isolation of the chromatin fraction. The Cdt1-depleted or the Cdc6-depleted chromatin was further incubated for 20 min with 20 μl of Cdc6- (ΔCdc6) or Cdt1-double depleted (ΔCdt1) extract, respectively (2nd incubation). For a biochemical assay detecting the licensing activity, a fresh egg extract supplemented with 0.5 μM of recombinant geminin and [α-³²P]dATP (20 kBq) was added to the reaction mixture without an isolation of the chromatin and incubated for 90 min (3rd incubation). For an analysis of chromatin-bound proteins, the chromatin fraction was isolated from each reaction mixture of the 2nd incubation and subjected to immunoblotting. (B) The result of the biochemical assay for the licensing activity was indicated. Reaction mixtures after the 1st and 2nd incubation were added to a fresh egg extract in which the licensing activity was inhibited by geminin. DNA synthesis during a further 90 min incubation (‘3rd incubation’ in A) was measured and plotted as a percentage of the radioactivity incorporated into DNA during the incubation in the geminin-containing extract to that in a geminin-free extract. Open and shaded bars indicate the DNA synthesis in the geminin-containing extract after the 1st and 2nd incubation, respectively. (C) Isolated chromatin prepared after the sequential incubation in mock-treated (mock), Cdt1-double depleted (ΔCdt1) and/or Cdc6-double depleted (ΔCdc6) extract was immunoblotted using indicated antibodies. The chromatin fractions after the 1st and 2nd incubation were applied to lanes 1 and 2, respectively.

Figure 3

Cdc6-depleted chromatin associated with an amount of Cdt1 sufficient for RLF-B activity. (A) Sperm nuclei (80 000 nuclei) were incubated in 16 μl of Cdc6-depleted extract for 20 min and chromatin was isolated. Cdt1 on the isolated chromatin (ΔCdc6-chromatin) was detected by immunoblotting using anti-Xenopus Cdt1 antibody together with 0.7, 1.05 and 1.4 pmol of His-Cdt1. (B) GS T-Cdt1 was electrophoresed and stained with Coomassie Brilliant Blue R-250. A molecular weight marker (lane 1) and 1.2 μg of GST-Cdt1 (lane 2) were applied to a SDS–10% polyacrylamide gel for electrophoresis. An arrowhead indicates the migrated position of GST-Cdt1. (C) Licensing activity was assayed with a Cdt1-depl eted extract supplemented with GST-Cdt1. Sperm nuclei (10 000 nuclei) were incubated for 20 min at 23°C with 2 μl of Cdt1-single depleted extract after a 2-fold dilution with 0.01% Triton X-100 and 50 mM KCl in LFB2 containing increasing amounts of GST-Cdt1. The extent of the licensing reaction during the incubation was measured as the amount of DNA synthesized in a 3 h incubation with a geminin-containing extract supplemented with [α-³²P]dATP (20 kBq). The amount of DNA synthesized is represented as a percentage of the radioactivity incorporated into DNA during the incubation in the geminin-containing extract to that in a geminin-free extract.

Figure 4

Cdt1 released from the licensed chromatin is active for the licensing. (A) The chromatin fraction was isolated after incubation of sperm nuclei (1 000 000 nuclei) with 200 μl of Xenopus egg extract for 20 min. Proteins bound on the chromatin were eluted by incubation of the isolated chromatin with a buffer containing 300 mM KCl and 0.1% BSA. Sperm nuclei (10 000 nuclei) were incubated for 20 min with 2 μl of Cdt1-single depleted extract after a 2-fold dilution with a buffer containing the elution fraction. After the incubation, the reaction mixture was further incubated for 90 min with 10 μl of a fresh egg extract supplemented with geminin (0.5 μM) and [α-³²P]dATP (20 kBq). The extent of DNA synthesis is represented as a percentage of the radioactivity incorporated into DNA during the incubation in the geminin-containing extract to that in a geminin-free extract. (B) Xenopus sperm nuclei were incubated for indicated periods in the interphase extract. After the incubation, the chromatin fraction was isolated from the extract and subjected to SDS–10% polyacrylamide gel electrophoresis. Cdt1 in the chromatin fractions was detected by immunoblotting. Two microliters of the interphase extract was applied onto the lane indicated by ‘Ex’. (C) Proteins were eluted from chromatin after an incubation of sperm nuclei with egg extract for 20 min. Cdt1-single depleted extract was diluted 2-fold with a buffer containing the elution fraction corresponding to 0, 7.5, 15 or 30 nM Cdt1 and assayed for the licensing activity. (D) Proteins were eluted from chromatin after an incubation of sperm nuclei with egg extract for 20 or 60 min. Cdt1-depleted extract was diluted 2-fold with a buffer containing no eluate (none), an eluate from 20 min chromatin after a single depletion of Cdt1 (20' ΔCdt1) or after a mock treatment (20' mock), or an eluate from 60 min chromatin after mock treatment (60' mock).

Figure 5

The stepwise association of Cdc6, Cdt1 and Mcm2-7 onto sperm nuclei. (A) A schematic representation of experimental procedures. Sperm nuclei (100 000 nuclei) were incubated in 20 μl of Cdt1- or Cdc6-double depleted extract for 20 min and then chromatin was isolated to prepare Cdt1-depleted chromatin (ΔCdt1 chromatin) or Cdc6-depleted chromatin (ΔCdc6 chromatin), respectively. The isolated chromatin was incubated for 20 min with GST-Cdt1, the Cdc6 fraction, a mixture of GST-Cdt1 and the Mcm fraction, or a mixture of Cdc6 and Mcm fractions (1st incubation). Chromatin was isolated again from the reaction mixture of the 1st incubation and incubated for 20 min with the Mcm fraction, a mixture of the Mcm fraction and GST-Cdt1, or a mixture of Mcm and Cdc6 fractions (2nd incubation). For a biochemical assay detecting the licensing activity, a fresh egg extract supplemented with 0.5 μM of recombinant geminin and [α-³²P]dATP (20 kBq) was added to the reaction mixture without an isolation of the chromatin fraction and incubated for 3 h (3rd incubation). For an analysis of chromatin-bound proteins, the chromatin fraction was isolated from each reaction mixture after the 2nd incubation and subjected to immunoblotting. (B) ΔCdt1- or ΔCdc6-chromatin was sequentially incubated with buffer (−), GST-Cdt1 (c1), Cdc6 fraction (c6) and/or Mcm fraction (m) as indicated. The samples were subjected to a biochemical assay to detect licensing activity. Isolated chromatin after an incubation with the Cdt1-depleted extracts supplemented with GST-Cdt1 (ΔCdt1 + Cdt1) and that after an incubation with the Cdc6-depleted extract supplemented with Cdc6 (ΔCdc6 + Cdc6) were used for controls of licensed chromatin (column 1 or 8, respectively). (C) The chromatin after the 2nd incubation was isolated and subjected to immunoblotting to detect Mcm4, Cdc6 and Cdt1. The bands of Cdt1 represent GST-Cdt1 in the left panel and native Cdt1 in the right panel. (D) Chromatin sequentially bound with Cdc6 and GST-Cdt1 was incubated with the Mcm fraction as described above. After the incubation with Mcm, the sample was separated into supernatant (sup) and chromatin (ppt) fractions by centrifugation. Both samples were immunoblotted with anti-Cdt1 antibody.

Figure 6

Cdc6 bound onto chromatin after Cdt1 functions in the licensing reaction. Sperm nuclei (200 000 nuclei) were incubated with 40 μl of Cdc6-depleted extract to isolate Cdc6-depleted chromatin. The Cdc6-depleted chromatin was incubated with the Cdc6 fraction for 20 min to allow the loading of Cdc6 onto the chromatin. The chromatin fraction was isolated and incubated with the Mcm fraction (column 2) or a mixture of the Mcm fraction and GST-Cdt1 (column 3) for 20 min. The chromatin isolated after the incubation with the Cdc6-depleted extract supplemented with Cdc6 (ΔCdc6 + Cdc6) was used as a control for licensed chromatin (column 1). A fresh egg extract (10 μl) supplemented with 0.5 μM of recombinant geminin and [α-³²P]dATP (20 kBq) was added to the reaction mixture of the 2nd incubation and incubated for 3 h to assess the licensing activity. The extent of DNA synthesis is represented as a percentage of the radioactivity incorporated into DNA during the incubation in the geminin-containing extract to that in a geminin-free extract (upper panel). The chromatin fractions after the second incubation were isolated and subjected to immunoblotting to detect Mcm4 (lower panel).