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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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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.
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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.

Mentions: In a typical binding reaction, there were ∼16 nm linear lamin B2 DNA molecules or 6 nm pUC19-lamin-B2 molecules. HsORC, HsCdc6, HsCdt1, HsMCM467, and HsMCM2–7 were used at the concentration of 13 nm, and HsGeminin at 200 nm unless indicated otherwise. In Fig. 4B, 26 nm HsMCM467, 13 nm HsMCM2–7, and 60 nm HsGeminin were used. When HsGeminin was used, it was preincubated with HsCdt1 at 4 °C for 30 min, unless indicated otherwise. The reactions were carried out in Thermomixer R (Eppendorf) under constant agitation at 1,100 rpm, and the beads were collected with the 3 in 1 MPS magnetic particle separator (PureBiotech, LLC).


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)

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

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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.
Mentions: In a typical binding reaction, there were ∼16 nm linear lamin B2 DNA molecules or 6 nm pUC19-lamin-B2 molecules. HsORC, HsCdc6, HsCdt1, HsMCM467, and HsMCM2–7 were used at the concentration of 13 nm, and HsGeminin at 200 nm unless indicated otherwise. In Fig. 4B, 26 nm HsMCM467, 13 nm HsMCM2–7, and 60 nm HsGeminin were used. When HsGeminin was used, it was preincubated with HsCdt1 at 4 °C for 30 min, unless indicated otherwise. The reactions were carried out in Thermomixer R (Eppendorf) under constant agitation at 1,100 rpm, and the beads were collected with the 3 in 1 MPS magnetic particle separator (PureBiotech, LLC).

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