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Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase. We have cloned a Xenopus homologue of Dbf4 (XDbf4), the sequence of which confirms the results of Furukhori et al. We have analysed the role of XDbf4 in DNA replication using cell-free extracts of Xenopus eggs. Our results indicate that XDbf4 is the regulatory subunit of XCdc7 required for DNA replication. We show that XDbf4 binds to chromatin during interphase, but unlike XCdc7, its chromatin association is independent of pre-RC formation, occurring in the absence of licensing, XCdc6 and XORC. Moreover, we show that the binding of XCdc7 to chromatin is dependent on the presence of XDbf4, whilst under certain circumstances XDbf4 can bind to chromatin in the absence of XCdc7. We provide evidence that the chromatin binding of XDbf4 that occurs in the absence of licensing depends on checkpoint activation. We have identified XDbf4 as a functional activator of XCdc7, and show that it is required to recruit XCdc7 to chromatin. Our results also suggest that XCdc7 and XDbf4 are differentially regulated, potentially responding to different cell cycle signals.
Figure 1. Amino acid sequence of XDbf4. Alignment of the full-length XDbf4 (GenBank accession number AY460183) and human Dbf4. The underlined, the double-underlined and the dash-underlined regions indicate the three conserved domains (N-, M- and C-domains). Arrows indicate the position of the two degenerate primers used for PCR amplification. The overall and the homology between domains are indicated.
Figure 2. Reconstitution of XCdc7/XDbf4. (A) Whole Xenopus egg extract and chromatin isolated from interphase extract were immunoblotted with XDbf4, XCdc7 and XMcm7 antibodies. (B) Interphase Xenopus egg extracts were immunodepleted with antibodies against XDbf4 (XDbf4-) or XCdc7 (XCdc7-) antibodies or with an equal quantity of non-immune antibodies (NI-). Depleted extracts were immunoblotted with XDbf4, XCdc7 or XMcm7 antibodies. (C) Interphase extracts depleted with either XCdc7 antibodies or non-immune antibodies were supplemented with [α-32P]dATP and incubated for 2 hr with Xenopus sperm nuclei or licensed interphase chromatin, in the presence or absence of recombinant XDbf4 and/or XCdc7. After incubation, DNA synthesis was measured, and expressed as a percentage of that occurring in control extract.
Figure 3. The association of XDbf4 with chromatin differs from that seen with XCdc7. (A) Xenopus sperm nuclei were incubated in metaphase or interphase extract for 30 min. Chromatin was isolated and immunoblotted with antibodies specific for XOrc1, XDbf4, XCdc7, XMcm7 and XCdc6. (B, C) Sperm nuclei were incubated at 10 ng/μl in interphase Xenopus egg extract. At the indicated times, (B) samples were assayed for DNA synthesis by [α-32P]dATP incorporation, or (C) chromatin was isolated and immunoblotted with antibodies specific for XOrc1, XCdc6, XMcm7, XDbf4 and XCdc7.
Figure 4. XDbf4 chromatin association is independent of a pre-replication complex. (A) Sperm nuclei were incubated for 30 min in interphase extracts minus or plus added Cip1 (extract+Cip1) or geminin (extract+geminin). Chromatin was isolated, subjected to SDS-PAGE and immunoblotted for XOrc1, XCdc6, XMcm7, XDbf4 and XCdc7. A control lacking added sperm is also shown. (B) Sperm nuclei were incubated at 10 ng/μl in interphase Xenopus egg extract in the presence of geminin. At the indicated times, chromatin was isolated and immunoblotted with antibodies specific for XOrc1, XCdc6, XMcm7, XDbf4 and XCdc7. Equivalent exposures to the time course of Fig 3 are shown. (C) Sperm nuclei were incubated for 40 min in interphase extracts previously immunodepleted with antibodies against XOrc1 (XOrc1-), XCdc6 (XCdc6-), and XMcm7 (XMcm7-), or with non-immune antibodies (NI-). The incubations also were performed in the presence of the phosphatase inhibitor microcystin (μC) or with a control lacking added sperm. Chromatin was then isolated and immunoblotted for XOrc1, XCdc6, XMcm7 and XCdc7. A Coomassie-stained image of the gel containing the histones is shown as loading control. (D) Sperm nuclei were incubated for 1 hr in extract supplemented with geminin and/or 5 mM caffeine. Chromatin was then isolated and immunoblotted with antibodies against XDbf4 and XCdc7. In parallel, nuclei were isolated and immunoblotted for phospho-Ser344-Chk1.
Figure 5. XCdc7 chromatin interaction requires XDbf4. (A) Sperm nuclei were incubated for 30 min in Xenopus interphase extract previously depleted with antibodies specific to XDbd4 (XDbf4-) or with non-immune serum (NI-), minus or plus added recombinant GST-XDbf4 protein, minus or plus geminin. Chromatin was then isolated and immunoblotted with antibodies specific for XCdc7, XMcm7, XOrc1 and XCdc6. (B) Sperm nuclei were incubated for 30 min in XCdc7-depleted extracts (XCdc7-) or non-immune depleted extracts (NI-) containing recombinant GST-XCdc7, recombinant XDbf4 previously treated with thrombin to remove the GST tag (XDbf4), recombinant geminin and/or microcystin. Incubation of recombinant proteins in non-immune depleted extract lacking sperm are shown as negative controls. Chromatin was then isolated and immunoblotted for XDbf4, XCdc7, XMcm7 and XOrc1.
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