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

Wu M, Lu W, Santos RE, Frattini MG, Kelly TJ - J. Biol. Chem. (2014)

Bottom Line: However, HsGeminin does not inhibit recruitment of HsMCM2-7 to DNA to form complexes containing all of the pre-RC proteins.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.

View Article: PubMed Central - PubMed

Affiliation: From the Program in Molecular Biology and.

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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.
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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.

Mentions: DNA binding assays were performed with circular plasmid DNA containing a 2-kb fragment from the human lamin B2 origin of DNA replication (36). The DNA was coupled to magnetic beads and incubated with proteins in a buffer containing 0.1 m NaCl and 1 mm ATP at 37 °C for 30 min. DNA-bound proteins were analyzed by SDS-PAGE and Western blotting. As shown in Fig. 2A, none of the purified proteins showed detectable binding to control beads without DNA. As expected, HsORC efficiently associated with DNA-coupled beads (>25% of input) in the absence of the other factors, and the recruitment of HsORC was enhanced by the presence of HsCdc6. Both HsCdc6 and HsCdt1 were also capable of binding to DNA beads in the absence of other factors, albeit with relatively low efficiency. Interestingly, the association of both proteins increased modestly when they were incubated together, suggesting that HsCdc6 and HsCdt1 can form a complex on DNA in the absence of HsORC. The binding of both HsCdc6 and HsCdt1 was also enhanced significantly in the presence of HsORC, and maximal association was achieved when all three proteins were present (Fig. 2A). Similar results were obtained when the beads contained only the linear 2-kb fragment of the lamin B2 origin DNA (data not shown), consistent with our previous observation that the binding of HsORC to DNA is sequence-independent (34). These observations indicate that the formation of the ternary complex containing HsORC, HsCdc6, and HsCdt1 is cooperative with all three proteins interacting with each other and with origin DNA.


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

Wu M, Lu W, Santos RE, Frattini MG, Kelly TJ - J. Biol. Chem. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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.
Mentions: DNA binding assays were performed with circular plasmid DNA containing a 2-kb fragment from the human lamin B2 origin of DNA replication (36). The DNA was coupled to magnetic beads and incubated with proteins in a buffer containing 0.1 m NaCl and 1 mm ATP at 37 °C for 30 min. DNA-bound proteins were analyzed by SDS-PAGE and Western blotting. As shown in Fig. 2A, none of the purified proteins showed detectable binding to control beads without DNA. As expected, HsORC efficiently associated with DNA-coupled beads (>25% of input) in the absence of the other factors, and the recruitment of HsORC was enhanced by the presence of HsCdc6. Both HsCdc6 and HsCdt1 were also capable of binding to DNA beads in the absence of other factors, albeit with relatively low efficiency. Interestingly, the association of both proteins increased modestly when they were incubated together, suggesting that HsCdc6 and HsCdt1 can form a complex on DNA in the absence of HsORC. The binding of both HsCdc6 and HsCdt1 was also enhanced significantly in the presence of HsORC, and maximal association was achieved when all three proteins were present (Fig. 2A). Similar results were obtained when the beads contained only the linear 2-kb fragment of the lamin B2 origin DNA (data not shown), consistent with our previous observation that the binding of HsORC to DNA is sequence-independent (34). These observations indicate that the formation of the ternary complex containing HsORC, HsCdc6, and HsCdt1 is cooperative with all three proteins interacting with each other and with origin DNA.

Bottom Line: However, HsGeminin does not inhibit recruitment of HsMCM2-7 to DNA to form complexes containing all of the pre-RC proteins.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.

View Article: PubMed Central - PubMed

Affiliation: From the Program in Molecular Biology and.

Show MeSH
Related in: MedlinePlus