Geminin inhibits a late step in the formation of human pre-replicative complexes.
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.
Affiliation: From the Program in Molecular Biology and.Show MeSH
Related in: MedlinePlus
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).