Takahashi TS, Basu A, Bermudez V, Hurwitz J, Walter JC.

GM034559 NIGMS NIH HHS , GM077440 NIGMS NIH HHS , GM62267 NIGMS NIH HHS , R01 GM077440 NIGMS NIH HHS , R01 GM062267 NIGMS NIH HHS , R01 GM034559 NIGMS NIH HHS

	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 rCdc7K59E–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.

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