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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.

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Affiliation: From the Program in Molecular Biology and.

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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).
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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).

Mentions: Previous studies in the Xenopus system have shown that Geminin inhibits pre-RC assembly, and therefore prevents de novo formation of the pre-RC during S, G2, and M phases, as one of the mechanisms that eukaryotic cells employ to prevent re-replication (26). To study the effect of HsGeminin on human pre-RC formation in vitro, we purified all known protein components involved in this process, and studied the requirements for pre-RC assembly on DNA coupled to magnetic beads. The purified proteins were free of significant contamination as assessed by SDS-polyacrylamide gel electrophoresis (Fig. 1). Recombinant human ORC was purified by affinity chromatography from Sf9 insect cells co-infected with baculoviruses encoding the six HsOrc subunits, as previously described (34). Consistent with our previous observations, the HsOrc1 and HsOrc6 subunits were substoichiometric in the purified complex (Fig. 1A). HsCdc6, tagged at the N terminus with Myc epitope (Fig. 1B), and HsCdt1, tagged at the N terminus with FLAG epitope (Fig. 1C), were also expressed in Sf9 cells and purified to near homogeneity by affinity chromatography. Recombinant human Mcm4 (tagged at the N terminus with His6 and FLAG epitopes), Mcm6, and Mcm7 were co-expressed in Sf9 cells, and HsMCM467 complexes were purified by affinity chromatography (Fig. 1D). Recombinant wild-type HsGeminin (Fig. 1F), and a mutant form of HsGeminin that is unable to interact with HsCdt1 (Fig. 1G, described later), both tagged at the N terminus with the FLAG epitope and at the C terminus with His6, were expressed in Sf9 cells, and purified by two steps of affinity chromatography, using Ni-NTA beads followed by FLAG antibody-conjugated agarose beads.


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)

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

License
Show All Figures
getmorefigures.php?uid=PMC4215257&req=5

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).
Mentions: Previous studies in the Xenopus system have shown that Geminin inhibits pre-RC assembly, and therefore prevents de novo formation of the pre-RC during S, G2, and M phases, as one of the mechanisms that eukaryotic cells employ to prevent re-replication (26). To study the effect of HsGeminin on human pre-RC formation in vitro, we purified all known protein components involved in this process, and studied the requirements for pre-RC assembly on DNA coupled to magnetic beads. The purified proteins were free of significant contamination as assessed by SDS-polyacrylamide gel electrophoresis (Fig. 1). Recombinant human ORC was purified by affinity chromatography from Sf9 insect cells co-infected with baculoviruses encoding the six HsOrc subunits, as previously described (34). Consistent with our previous observations, the HsOrc1 and HsOrc6 subunits were substoichiometric in the purified complex (Fig. 1A). HsCdc6, tagged at the N terminus with Myc epitope (Fig. 1B), and HsCdt1, tagged at the N terminus with FLAG epitope (Fig. 1C), were also expressed in Sf9 cells and purified to near homogeneity by affinity chromatography. Recombinant human Mcm4 (tagged at the N terminus with His6 and FLAG epitopes), Mcm6, and Mcm7 were co-expressed in Sf9 cells, and HsMCM467 complexes were purified by affinity chromatography (Fig. 1D). Recombinant wild-type HsGeminin (Fig. 1F), and a mutant form of HsGeminin that is unable to interact with HsCdt1 (Fig. 1G, described later), both tagged at the N terminus with the FLAG epitope and at the C terminus with His6, were expressed in Sf9 cells, and purified by two steps of affinity chromatography, using Ni-NTA beads followed by FLAG antibody-conjugated agarose beads.

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