Geminin inhibits a late step in the formation of human pre-replicative complexes

Abstract

The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2-7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2-7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2-7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2-7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.

Keywords: DNA Replication; DNA-Protein Interaction; Geminin; Pre-RC Assembly; Protein Assembly; Protein Complex; Protein-Protein Interaction.

FIGURE 1.

Analysis of purified human pre-RC components by SDS-PAGE followed by silver staining. A, HsORC. B, Myc-HsCdc6 (the faster migrating band (*) is a degradation product of HsCdc6 as demonstrated by analysis of tryptic peptides by mass spectrometry). C, FLAG-HsCdt1. D, HsMCM467 (the asterisk indicates the 70-kDa insect heat shock protein). E, HsMCM2–7. F, wild-type HsGeminin (the asterisk indicates the 70-kDa insect heat shock protein). G, mutant HsGeminin deficient in Cdt1 binding (HsGeminin-BD) (the asterisk indicates the 70-kDa insect heat shock protein).

FIGURE 2.

Interactions of purified recombinant human ORC, Cdc6, and Cdt1 with DNA, and inhibition of the DNA-binding activity of HsCdt1 by HsGeminin. A, DNA-binding assays. Control magnetic beads or beads with plasmid DNA containing the lamin B2 origin of DNA replication were incubated with the indicated proteins in a two-step reaction (see ”Experimental Procedures“ for details). The bound proteins were detected by Western blotting. B, HsGeminin inhibits DNA binding by HsCdt1. HsCdt1 was incubated with control buffer or the indicated amount of HsGeminin, and then the protein mixture was incubated with magnetic beads containing pUC19-lamin-B2 plasmid DNA in the absence (a) or presence (b) of HsORC and HsCdc6. C, interaction between HsCdt1 and HsGeminin is necessary for inhibition of HsCdt1 DNA binding activity. a, co-immunoprecipitation (IP) of HsCdt1-HsGeminin complexes. Purified HsCdt1 was incubated with control buffer (C), purified wild-type HsGeminin (wt) or purified HsGeminin BD mutant (BD), and complexes were collected on protein A/G plus-agarose beads containing anti-HsGeminin polyclonal antibodies. b, DNA-binding assay. HsCdt1 was preincubated with wild-type (wt) or BD mutant (BD) HsGeminin, or control buffer, and then incubated with magnetic beads containing linear lamin B2 DNA fragments.

FIGURE 3.

Requirements for recruitment of purified HsMCM complexes to DNA. A, requirements for HsMCM467 recruitment to origin DNA. Magnetic beads containing pUC19-lamin-B2 plasmid DNA were incubated with HsORC or control buffer for 30 min, and then further incubated with HsMCM467 in the presence of HsCdc6 and/or HsCdt1 for 30 min. HsCdt1 was preincubated with control buffer or HsGeminin as indicated. B, requirements for recruitment of HsMCM2–7 to origin DNA. Reaction mixtures were assembled and incubated as described in A, except that HsMCM2–7 was used in place of HsMCM467.

FIGURE 4.

Differential effects of HsGeminin on the recruitment of HsMCM467 and HsMCM2–7 complexes to DNA. A, comparison of the effects of HsGeminin on recruitment of HsMCM2–7 and HsMCM467. Magnetic beads containing pUC19-lamin-B2 plasmid DNA were incubated with HsORC for 30 min, and then further incubated with HsCdc6 and HsCdt1 in the presence of HsMCM2–7 or HsMCM467 for 30 min. HsCdt1 was preincubated with control buffer or the indicated amount of HsGeminin. B, binding of HsGeminin to HsMCM2–7. Magnetic beads containing lamin B2 DNA fragments were incubated with HsORC and HsCdc6 for 30 min, and then further incubated with either HsMCM467 or HsMCM2–7 for 30 min, followed by incubation with HsGeminin for 30 min. C, recruitment of HsMCM467 to origin DNA is not reversed by HsGeminin. Magnetic beads containing pUC19-lamin-B2 plasmid DNA were preincubated with HsORC for 30 min, and then further incubated with HsCdt1 and HsMCM467 for 20 (lanes 1–3) or 40 min (lanes 4 and 5), in the presence of either control buffer or HsGeminin. HsGeminin or control buffer was added at various times as follows: lane 1: control buffer was added at t = −30 min (preincubated with HsCdt1 for 30 min prior to addition to the reaction). Lane 2, HsGeminin was added at t = −30 min. Lane 3, HsGeminin was added at t = 0 min (the start of second incubation). Lane 4, control buffer was added at t = +20 min (20 min after the second incubation started). Lane 5, HsGeminin was added at t = +20 min.

FIGURE 5.

Interactions between individual human MCM subunits and HsCdt1 or HsCdc6, and effects of HsGeminin on the interactions between MCM subunits and HsCdt1. A–D, extracts of Sf9 cells expressing untagged human Mcm4 (A), Mcm5 (B), Mcm6 (C), or Mcm7 (D) were incubated at 4 °C for 3 h with extracts from control cells or extract from cells expressing FLAG-HsCdt1 or Myc-HsCdc6. For each Mcm subunit, an equal amount of protein was added in each reaction. The proteins precipitated by anti-FLAG- or anti-Myc-agarose beads were analyzed by Western blotting. L indicates 8.3% of the load. E–H, extracts of Sf9 cells expressing untagged human Mcm3 (G), Mcm5 (H), Mcm6 (E), or Mcm7 (F) were incubated at 4 °C for 3 h with extracts from control cells or extracts from cells expressing FLAG-HsCdt1 and/or FLAG-HsGeminin-His₆. The proteins precipitated by anti-FLAG-agarose beads were analyzed by Western blotting. IP, immunoprecipitated.

FIGURE 6.

HsGeminin inhibits the ATP-dependent salt-stable binding of HsMCM2–7 to DNA. A, requirements for formation of salt-stable association of HsMCM2–7 with DNA. Reaction mixtures were assembled and incubated as described in the legend to Fig. 3B. The beads were then washed with buffer containing 0.3 m NaCl and bound proteins were analyzed by Western blotting. B, HsGeminin inhibits formation, but not the maintenance of salt-stable HsMCM2–7·DNA complexes. Reaction mixtures were assembled and incubated as described in the legend to Fig. 4C, except that HsMCM2–7 was used instead of HsMCM467, and HsCdc6 was also present. The beads were then washed with buffer containing 0.5 m NaCl. Lane 1: control buffer was added at t = −30 min. Lane 2: HsGeminin was added at t = −30 min. Lane 3: HsGeminin was added at t = 0 min. Lane 4: control buffer was added at t = +20 min. Lane 5: HsGeminin was added at t = +20 min. C, ATP-dependent formation of salt-stable HsMCM2–7·DNA complexes. Reactions were performed in the presence of ATP, ATPγS, ADP, or control buffer as indicated. The beads were then washed with buffer containing 0.3 m NaCl. D, HsGeminin inhibition of salt-stable complex formation. Reaction mixtures were incubated in the presence or absence of ATP as indicated. HsCdt1 was preincubated with control buffer or HsGeminin. The beads were then washed with buffer containing 0.3 m NaCl. E, ATP requirement in two-step DNA binding assays. Magnetic beads containing pUC19-lamin-B2 plasmid DNA were incubated with HsORC for 30 min, and then further incubated with HsMCM2–7, HsCdt1, and HsCdc6 for 30 min. ATP or ATPγS was added in each step as indicated. The beads were then washed with buffer containing 0.1 m potassium glutamate or 0.3 m NaCl and analyzed by Western blotting. Mcm6 l, long exposure of Mcm6 signal. Mcm6 s, short exposure of Mcm6 signal. Gem, Geminin.