Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

Abstract

To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion.

Figure 1.

Xenopus Scc2–Scc4 is required for pre-RC-dependent cohesin recruitment to chromatin. (A) IP was performed with control (lanes 1,4), anti-Scc2 (lanes 2,5), or anti-Scc4 antibody (lanes 3,6) from LSS. Supernatants (IP-sup; lanes 1–3) or immunoprecipitates (IP-ppt; lanes 4–6) were probed with Scc2 (top panel) or Scc4 (bottom panel) antibody. (*) Cross-reacting band. (B) Sperm chromatin was incubated in mock-depleted (lanes 1,2), Scc2-depleted (lanes 3,4), or Scc4-depleted LSS (lanes 5,6), and isolated at the indicated times. Chromatin-bound proteins were probed with the indicated antibodies. (*) Cross-reacting band. (C) Sperm chromatin was incubated in either mock-depleted (lane 1) or Cdt1-depleted LSS (lane 2) and isolated after 90 min. Chromatin-bound proteins were probed with the indicated antibodies. (*) Cross-reacting band. (D) IVT-expressed Xenopus Scc2(1–1024) (lanes 3–5), IVT-expressed Scc4 (lanes 2,4,5), or unprogrammed IVT lysate (2.5 μL of each) was added to 10 μL of HSS. Sperm chromatin was added to the HSS-IVT mixture at 10,000/μL concentration relative to HSS (lanes 1–4) and isolated after 30 min. Chromatin-bound proteins were probed with the indicated antibodies.

Figure 2.

Cdc7-depletion impairs chromatin association of the Scc2–Scc4 complex. (A) Sperm chromatin was incubated with mock-depleted (lanes 1,2) or Cdc7-depleted LSS (lanes 3–6) supplemented with buffer (lanes 1–4) or 40 nM rCdc7–Drf1 (lanes 5,6) and isolated at the indicated time points. Chromatin-bound proteins were probed with the indicated antibodies. (*) Cross-reacting band. (B) The reactions described in A were separately incubated with [α-³²P]dATP. The replication efficiency was calculated based on incorporation of ³²P-α-dATP (see the Materials and Merthods) and the results were graphed.

Figure 3.

Cdc7-kinase complexes physically associate with Scc2–Scc4. (A) IP was performed with control (lanes 1,5), anti-Cdc7 (lanes 2,6), anti-Dbf4 (lanes 3,7), or anti-Drf1 antibody (lanes 4,8) from LSS. Supernatants (IP-sup; lanes 1–4) and immunoprecipitates (IP-ppt; lanes 5–8) were probed with the indicated antibodies. (*) Cross-reacting band. (B) IP was performed with control (lanes 1,4), anti-Scc2 (lanes 2,5), or anti-Scc4 antibody (lanes 3,6) from LSS. Supernatants (IP-sup; lanes 1–3) or immunoprecipitates (IP-ppt; lanes 4–6) were probed with the indicated antibodies. (*) Cross-reacting band.

Figure 4.

Cdc7 and cohesin associate via Scc2–Scc4. LSS was depleted with either control (lanes 1,3,4,6,7) or a mixture of Scc2 and Scc4 antibodies (lanes 2,5,8). IP from the resulting LSS was performed with control (lanes 3,6) or Cdc7 antibody (lanes 4,5,7,8). Input (lanes 1,2), supernatants (IP-sup; lanes 3–5), and immunoprecipitates (IP-ppt; lanes 6–8) were probed with the indicated antibodies. (*) Cross-reacting band.

Figure 5.

The Cdc7–kinase complex, but not free Cdc7, associates with Scc2. (A) LSS was depleted with either control (lanes 1,4,5,8,9) or a mixture of Drf1 and Dbf4 antibodies (lanes 2,3,6,7,10,11), and then supplemented with either buffer (lanes 1,2,4–6,8–10), or recombinant Drf1 (100 nM final concentration; lanes 3,7,11). IP from the resulting LSS extracts was performed with control (lanes 4,8) or Cdc7 antibody (lanes 5–7,9–11). Input (lanes 1–3), supernatants (IP-sup; lanes 4–7), immunoprecipitates (IP-ppt; lanes 8–11), and a serial dilution of the sample in lane 9 were probed with the indicated antibodies. (*) Cross-reacting band. (B) LSS was depleted with control antibody (lanes 1,6,7,12,13), a mixture of Drf1 and Dbf4 antibodies (lanes 2,3,8,9,14,15), or Cdc7-antibody (lanes 4,5,10,11,16,17), and then supplemented with either buffer (lanes 1,2,4,6–8,10,12–14,16) or 20 nM rDrf1 (lanes 3,5,9,11,15,17). IP from the resulting LSS was performed with control (lanes 6,12) or anti-Drf1 antibody (lanes 7–11,13–17). Input (lanes 1–5), supernatants (IP-sup; lanes 6–11), and immunoprecipitates (IP-ppt; lanes 12–17) were probed with the indicated antibodies. (*) Cross-reacting band.

Figure 6.

Binding of Scc2N–Scc4 complex to chromatin requires the pre-RC and DDK. (A) HSS was depleted with control antibody (lane 1) or Cdt1 antibody (lane 2), and then supplemented with Scc2N and Scc4 expressed in IVT lysates. Sperm chromatin was incubated in the HSS, and after 30 min chromatin-bound proteins were isolated and probed with the indicated antibodies. (B) HSS was depleted with control antibody (lanes 1,2) or Cdc7 antibody (lanes 3,4), and then supplemented with Scc2 and Scc4-expressed IVT lysates (lanes 1–4), buffer (lanes 1,3), or 50 nM of rCdc7–Drf1 (His-Strep purified, lanes 2,4). Sperm chromatin was incubated in the HSS and isolated at 30 min. Chromatin-bound proteins were probed with the indicated antibodies. (C) HSS was depleted with control antibody (lanes 1,2) or Cdc7 antibody (lanes 3–5), and then supplemented with unprogrammed IVT lysate (lane 1), or Scc2 and Scc4-expressed IVT lysates (lanes 2–5), buffer (lanes 1–3), 180 nM of wild-type rCdc7–Drf1 (WT; His-Flag-purified, lane 4), or 180 nM of catalytically inactive rCdc7^K59E–Drf1 (KE; His-Flag-purified, lane 5). Sperm chromatin was incubated in the HSS and isolated at 30 min. Chromatinbound proteins were probed with the indicated antibodies. (D) A model for the chromatin loading mechanism of Scc2–Scc4 and cohesin. DDK physically associates with Scc2–Scc4, which in turn binds cohesin. The DDK component of this ternary complex docks onto Mcm2-7 and brings Scc2–Scc4 and cohesin close to the chromatin, thereby allowing cohesin deposition on DNA.