title: Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload.
abstract: The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Marjorie A
authors.initials: MA
authors.lastname: Kuipers
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stasevich
authors.forename: Takayo
authors.initials: T
authors.lastname: Sasaki
authors.forename: Korey A
authors.initials: KA
authors.lastname: Wilson
authors.forename: Kristin L
authors.initials: KL
authors.lastname: Hazelwood
authors.forename: James G
authors.initials: JG
authors.lastname: McNally
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.forename: David M
authors.initials: DM
authors.lastname: Gilbert
mesh_terms: D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D002843:Chromatin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000096:biosynthesis; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D036681:Fluorescence Recovery After Photobleaching; D016193:G1 Phase; D051379:Mice; D064148:Minichromosome Maintenance Complex Component 4; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D021381:Protein Transport
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D004265:DNA Helicases; C516793:Mcm4 protein, mouse; D064148:Minichromosome Maintenance Complex Component 4