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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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Related in: MedlinePlus

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.
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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.

Mentions: As shown in Fig. 3A, the purified HsMCM467 complex did not bind to DNA in the absence of the other factors. The presence of HsORC was insufficient to recruit HsMCM467 to DNA, but in the presence of either HsCdc6 or HsCdt1 recruitment of a small fraction of the added HsMCM467 was observed. Interestingly, substantial binding of HsMCM467 (>50% of input) was observed when HsCdc6 and HsCdt1 were both present, but HsORC was absent. This observation confirms that a complex of HsCdt1 and HsCdc6 has DNA-binding activity and further demonstrates that the complex is capable of recruiting HsMCM467. The presence of HsORC enhanced the recruitment of both HsCdc6 and HsCdt1 to DNA and resulted in maximal binding of HsMCM467. Taken together, these observations suggest that the recruitment of HsMCM467 to origin DNA depends largely upon direct interactions with the pre-RC proteins, HsCdc6 and HsCdt1. Our data are consistent with the possibility that HsORC plays only an indirect role in HsMCM467 recruitment via stabilization of the binding of an HsCdc6-HsCdt1 complex. Alternatively, the binding of HsCdc6 and HsCdt1 may alter the conformation of HsORC, exposing sites for direct HsORC-HsMCM interactions.


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)

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

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

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.
Mentions: As shown in Fig. 3A, the purified HsMCM467 complex did not bind to DNA in the absence of the other factors. The presence of HsORC was insufficient to recruit HsMCM467 to DNA, but in the presence of either HsCdc6 or HsCdt1 recruitment of a small fraction of the added HsMCM467 was observed. Interestingly, substantial binding of HsMCM467 (>50% of input) was observed when HsCdc6 and HsCdt1 were both present, but HsORC was absent. This observation confirms that a complex of HsCdt1 and HsCdc6 has DNA-binding activity and further demonstrates that the complex is capable of recruiting HsMCM467. The presence of HsORC enhanced the recruitment of both HsCdc6 and HsCdt1 to DNA and resulted in maximal binding of HsMCM467. Taken together, these observations suggest that the recruitment of HsMCM467 to origin DNA depends largely upon direct interactions with the pre-RC proteins, HsCdc6 and HsCdt1. Our data are consistent with the possibility that HsORC plays only an indirect role in HsMCM467 recruitment via stabilization of the binding of an HsCdc6-HsCdt1 complex. Alternatively, the binding of HsCdc6 and HsCdt1 may alter the conformation of HsORC, exposing sites for direct HsORC-HsMCM interactions.

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