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An inactive geminin mutant that binds cdt1.

Suchyta M, Miotto B, McGarry TJ - Genes (Basel) (2015)

Bottom Line: In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication.GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase.We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University Chicago, IL 60610, USA. msuchyta@gmail.com.

ABSTRACT
The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7), the replication helicase, into the pre-replication complex (pre-RC) at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.

No MeSH data available.


GemininAWA inhibits MCM loading in vitro but not in vivo. (A) GemininAWA inhibits the MCM ←→Cdt1 interaction in vitro. Increasing concentrations of recombinant Geminin protein (5 μg/mL, 10 μg/mL, or 40 μg/mL) were mixed with translated Myc-Cdt1. After 40 min, purified MCM4/6/7 complex was added. After another 45 min, the reaction was incubated with anti-Myc antibody, and the precipitate was blotted for the proteins shown. (B, C) GemininAWA does not inhibit MCM loading in vivo. (B) Replication extracts were depleted of endogenous Geminin with anti-Geminin antiserum, then either recombinant GemininWT or GemininAWA was added back (50 μg/mL). Permeabilized sperm heads were added, and nuclei were allowed to assemble. The chromatin was then pelleted through a sucrose cushion, and the amount of MCM4 in the precipitate was determined by immunoblotting. (C) In a separate aliquot of the reaction, DNA replication was allowed to proceed to completion and the extent of replication was measured. Average of two experiments
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genes-06-00252-f003: GemininAWA inhibits MCM loading in vitro but not in vivo. (A) GemininAWA inhibits the MCM ←→Cdt1 interaction in vitro. Increasing concentrations of recombinant Geminin protein (5 μg/mL, 10 μg/mL, or 40 μg/mL) were mixed with translated Myc-Cdt1. After 40 min, purified MCM4/6/7 complex was added. After another 45 min, the reaction was incubated with anti-Myc antibody, and the precipitate was blotted for the proteins shown. (B, C) GemininAWA does not inhibit MCM loading in vivo. (B) Replication extracts were depleted of endogenous Geminin with anti-Geminin antiserum, then either recombinant GemininWT or GemininAWA was added back (50 μg/mL). Permeabilized sperm heads were added, and nuclei were allowed to assemble. The chromatin was then pelleted through a sucrose cushion, and the amount of MCM4 in the precipitate was determined by immunoblotting. (C) In a separate aliquot of the reaction, DNA replication was allowed to proceed to completion and the extent of replication was measured. Average of two experiments

Mentions: Our results argue against a model where Geminin inhibits MCM loading by direct steric interference. It has been previously shown, however, that purified Cdt1 and MCM proteins directly associate with one another in vitro and that Geminin inhibits this interaction [11,12]. To see if GemininAWA inhibits the Cdt1-MCM association in vitro, we added different concentrations of recombinant GemininWT or GemininAWA to a mixture of purified MCM4/6/7 complex and in vitro-translated Myc-Cdt1. The Cdt1 was immunoprecipitated using anti-Myc antibody, and the amount of MCM4 in the precipitate was determined by immunoblotting. We found that GemininWT and GemininAWA both inhibited the interaction between Cdt1 and MCM4/6/7 in vitro at about the same concentration (Figure 3A). This result is at odds with our observation that GemininAWA does not inhibit DNA replication in vivo.


An inactive geminin mutant that binds cdt1.

Suchyta M, Miotto B, McGarry TJ - Genes (Basel) (2015)

GemininAWA inhibits MCM loading in vitro but not in vivo. (A) GemininAWA inhibits the MCM ←→Cdt1 interaction in vitro. Increasing concentrations of recombinant Geminin protein (5 μg/mL, 10 μg/mL, or 40 μg/mL) were mixed with translated Myc-Cdt1. After 40 min, purified MCM4/6/7 complex was added. After another 45 min, the reaction was incubated with anti-Myc antibody, and the precipitate was blotted for the proteins shown. (B, C) GemininAWA does not inhibit MCM loading in vivo. (B) Replication extracts were depleted of endogenous Geminin with anti-Geminin antiserum, then either recombinant GemininWT or GemininAWA was added back (50 μg/mL). Permeabilized sperm heads were added, and nuclei were allowed to assemble. The chromatin was then pelleted through a sucrose cushion, and the amount of MCM4 in the precipitate was determined by immunoblotting. (C) In a separate aliquot of the reaction, DNA replication was allowed to proceed to completion and the extent of replication was measured. Average of two experiments
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488664&req=5

genes-06-00252-f003: GemininAWA inhibits MCM loading in vitro but not in vivo. (A) GemininAWA inhibits the MCM ←→Cdt1 interaction in vitro. Increasing concentrations of recombinant Geminin protein (5 μg/mL, 10 μg/mL, or 40 μg/mL) were mixed with translated Myc-Cdt1. After 40 min, purified MCM4/6/7 complex was added. After another 45 min, the reaction was incubated with anti-Myc antibody, and the precipitate was blotted for the proteins shown. (B, C) GemininAWA does not inhibit MCM loading in vivo. (B) Replication extracts were depleted of endogenous Geminin with anti-Geminin antiserum, then either recombinant GemininWT or GemininAWA was added back (50 μg/mL). Permeabilized sperm heads were added, and nuclei were allowed to assemble. The chromatin was then pelleted through a sucrose cushion, and the amount of MCM4 in the precipitate was determined by immunoblotting. (C) In a separate aliquot of the reaction, DNA replication was allowed to proceed to completion and the extent of replication was measured. Average of two experiments
Mentions: Our results argue against a model where Geminin inhibits MCM loading by direct steric interference. It has been previously shown, however, that purified Cdt1 and MCM proteins directly associate with one another in vitro and that Geminin inhibits this interaction [11,12]. To see if GemininAWA inhibits the Cdt1-MCM association in vitro, we added different concentrations of recombinant GemininWT or GemininAWA to a mixture of purified MCM4/6/7 complex and in vitro-translated Myc-Cdt1. The Cdt1 was immunoprecipitated using anti-Myc antibody, and the amount of MCM4 in the precipitate was determined by immunoblotting. We found that GemininWT and GemininAWA both inhibited the interaction between Cdt1 and MCM4/6/7 in vitro at about the same concentration (Figure 3A). This result is at odds with our observation that GemininAWA does not inhibit DNA replication in vivo.

Bottom Line: In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication.GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase.We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University Chicago, IL 60610, USA. msuchyta@gmail.com.

ABSTRACT
The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7), the replication helicase, into the pre-replication complex (pre-RC) at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.

No MeSH data available.