XCdc7 loaded onto chromatin is functional. Interphase Xenopus egg extracts were immunodepleted with antibodies against XCdc7 (XCdc7−) or with an equal quantity of nonimmune antibodies (NI−). Extract was supplemented with [α-³²P]dATP and incubated for 3 hr with different DNA templates. DNA replication was assessed by the proportion of ³²P incorporated into acid-insoluble material. (A) Replication in XCdc7-depleted and nonimmune-depleted extracts was measured using Xenopus sperm nuclei (Sperm) or various chromatin templates, prepared by incubating Xenopus sperm nuclei in interphase extract for 20 min minus (LS Chrom) or plus p21^Cip1 (LS p21 Chrom) or for 15 min in 6-DMAP-treated extract (6-DMAP Chrom), followed by isolation in low salt buffer. (B) XCdc7-depleted extract was supplemented with 0.05 volume of different fractions, and its ability to replicate chromatin previously assembled in 6-DMAP-treated extract was assessed. Added fractions: (Extract) membrane-free egg extract; (XCdc7) partially purified XCdc7; (Buffer) LFB2/50. Replication is shown as the percentage obtained with the identical chromatin template incubated in nonimmune-depleted (NI−) extract.

XCdc7 is not involved in licensing-dependent origin inactivation but is required for XCdc45 loading. (A) Sperm nuclei were incubated for 40 min in XCdc7-depleted extract (XCdc7−) or nonimmune-depleted extract (NI−) plus or minus geminin. Samples were split in two, and chromatin was isolated in low (LS) or high salt (HS) buffers and blotted for XOrc1 and XMcm3. (B) Sperm nuclei were incubated for 40 min in interphase extract (Inter.), XCdc7-depleted extract (XCdc7−) or nonimmune-depleted extract (NI−) plus or minus p21^Cip1 or geminin. Chromatin was isolated in low salt buffer and immunoblotted for XCdc6 and XMcm3. (C) Sperm nuclei were incubated for 45 min in interphase extract plus or minus added p21^Cip1 or geminin. Chromatin was isolated in low salt buffer and blotted for XCdc45, XCdc7, XMcm3, and XOrc1. (D) Sperm nuclei were incubated in XCdc7-depleted extract (XCdc7−) or nonimmune-depleted extract (NI−) plus or minus added p21^Cip1 for 150 min (the long incubation is due to the slow rate that immunodepleted extracts assemble DNA into interphase nuclei). Complete nuclear envelope formation was verified by microscopy, and chromatin was then isolated in low salt buffer and immunoblotted for XCdc45, XCdc7, XMcm3, and XOrc1.

XCdc7 can perform its essential function in the absence of CDK activity. (A) Schematic representation of the experimental procedure. Sperm nuclei were incubated for 25 min in interphase extract supplemented with p21^Cip1. Chromatin was isolated in low or high salt buffer and reincubated for 150 min in XCdc7-depleted extract (XCdc7−) or nonimmune-depleted extract (NI−), plus or minus further added p21^Cip1. DNA synthesis was assessed by incorporation of [α-³²P]dATP; alternatively, chromatin was reisolated and immunoblotted for XCdc45, XMcm3, and XCdc7. (B) Low salt chromatin was blotted for bound XCdc45, XMcm3, and XCdc7 after reincubation in XCdc7- or nonimmune-depleted extract. (C) High salt chromatin was blotted for bound XCdc45, XMcm3, and XCdc7 after reincubation in XCdc7- or nonimmune-depleted extract. (D) DNA synthesis in XCdc7- or nonimmune-depleted extract. Chromatin templates were untreated sperm nuclei (Sperm), sperm nuclei incubated in 6-DMAP-treated extract and isolated under low salt conditions (6-DMAP Chrom), sperm nuclei incubated in p21^Cip1-treated extract and isolated under low salt conditions as in B (LS p21 Chrom), or sperm nuclei incubated in p21^Cip1-treated extract and isolated under high salt conditions as in C (HS p21 Chrom).

XCdc7 chromatin association is dependent on licensing. (A,B) Sperm nuclei were incubated at 10 ng/μl in interphase Xenopus egg extract. At the indicated times, (A) samples were assayed for DNA synthesis by [α-³²P]dATP incorporation, or (B) chromatin was isolated and immunoblotted with antibodies specific for XOrc1, XCdc6, XMcm3, XCdc7, and XCdc45. (C) Sperm nuclei were incubated for 50 min in membrane-free extract (lane 2) or interphase extract (lane 3). Chromatin was isolated, subjected to SDS-PAGE, and blotted for XMcm3, XCdc7, and XCdc45. Lane 1 shows a control of membrane-free extract without added sperm. (D) Sperm nuclei were incubated for 40 min in interphase extracts immunodepleted previously with antibodies against XOrc1 (XOrc1−), XCdc6 (XCdc6−), and XMcm3 (XMcm3−), or with nonimmune antibodies (NI−), or in interphase extract (Inter.) minus or plus added p21 (Inter. + p21) or geminin (Inter. + geminin), or in metaphase arrested extract plus 3 mm 6-DMAP (Meta. + 6-DMAP). Chromatin was then isolated and immunoblotted for XOrc1, XCdc6, XMcm3, and XCdc7.

XCdc7 chromatin loading is independent of XOrc1 and XCdc6. (A) Xenopus sperm nuclei were incubated for 25 min in interphase Xenopus extract plus or minus p21^Cip1 or geminin. Chromatin was isolated in low salt (LS) or high salt (HS) buffers and then immunoblotted for XOrc1, XMcm3, and XCdc7. (B,C). Sperm nuclei were incubated for 20 min in interphase extract, and chromatin was isolated in high salt buffer (HS Chromatin). An aliquot was used for immunoblot (lane 1) and the rest was reincubated in nonimmune-depleted (NI−) extracts or either XOrc1-depleted (XOrc1−) (B) or XCdc6-depleted (XCdc6−) (C) extracts for a further 50 min. Chromatin was re-isolated and blotted for XOrc1 (B) or XCdc6 (C), XMcm3, and XCdc7. Controls lacking added chromatin are also shown (lanes 2, and 4). A schematic representation of the experimental procedure for B and C is shown above. (D) Sperm nuclei were incubated for 20 min in XCdc7-depleted extract or untreated interphase extract, and chromatin was isolated in high salt buffer. Chromatin from the untreated extract (HS Chrom) or the XCdc7-depleted extract (HS XCdc7− Chrom) or untreated sperm nuclei (Sperm) were incubated for 3 hr in either XOrc1-depleted (XORC−) or in nonimmune-depleted extract (NI−) supplemented with [α-³²P]dATP for 3 hr. DNA synthesis was assessed by the proportion of ³²P incorporated into acid-insoluble material.

XCdc7-dependent XMcm2 phosphorylation is independent of CDK activity. (A,B) Sperm nuclei were incubated for 25 min in interphase extract containing [γ-³²P]ATP plus or minus added p21^Cip1 or geminin. Chromatin was then isolated in the presence of phosphatase inhibitors and 250 mm KCl. (A) Samples were subjected to SDS-PAGE and Western blotted; the filters were autoradiographed (lanes 1–3) and then probed with antibodies against XMcm2 and XMcm3 (lanes 4–6). (B) Isolated chromatin was treated with 1 m KCl to elute MCM/P1 proteins from chromatin. The supernatant was immunoprecipitated with XMcm2 and XMcm3 antibodies, and samples were run on SDS-PAGE and autoradiographed. (C) Sperm nuclei were incubated for 25 min in XCdc7-depleted extract (XCdc7−) or nonimmune-depleted extract (NI−) containing [γ-³²P]ATP. Chromatin was isolated in the presence of phosphatase inhibitors and 250 mm KCl and subjected to SDS-PAGE and Western blotted; the filters were autoradiographed (lanes 1,2) and then probed with antibodies against XMcm2 and XMcm3 (lanes 3,4).

A model for XCdc7 function. (A) The loading of the RLF-M complex of XMCM/P1 proteins onto chromatin (licensing) is dependent on XORC and XCdc6 and RLF-B. As a consequence of licensing, XORC is destabilized and XCdc6 is removed, independent of XCdc7 function. (B) XCdc7 then binds to chromatin, plausibly by physical interaction with MCM/P1 proteins, and (C) phosphorylates them. After this phosphorylation, which can occur in the absence of CDK activity, XCdc7 is no longer required for DNA replication. (D) After nuclear assembly, the XCdc7-dependent phosphorylation of XMCM/P1 proteins is required for the CDK-dependent loading of XCdc45. (E) The preinitiation complex at origins, containing XORC, MCM/P1 proteins, XCdc7, and XCdc45 (though XORC and XCdc7 are no longer essential), promotes the loading of DNA polymerase α and the initiation of replication.