title: Simultaneous measurement of passage through the restriction point and MCM loading in single cells.
abstract: Passage through the Retinoblastoma protein (RB1)-dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyper-phosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1-phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells.
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310 Oslo, Norway Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: T W
authors.initials: TW
authors.lastname: Håland
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310 Oslo, Norway Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: E
authors.initials: E
authors.lastname: Boye
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310 Oslo, Norway.
authors.forename: T
authors.initials: T
authors.lastname: Stokke
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310 Oslo, Norway Beata.Grallert@rr-research.no.
authors.forename: B
authors.initials: B
authors.lastname: Grallert
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310 Oslo, Norway.
authors.forename: R G
authors.initials: RG
authors.lastname: Syljuåsen
mesh_terms: D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005434:Flow Cytometry; D059585:G1 Phase Cell Cycle Checkpoints; D006801:Humans; D064110:Minichromosome Maintenance Proteins / Q000032:analysis*; D015739:Nocodazole / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle; D016160:Retinoblastoma Protein / Q000378:metabolism; D059010:Single-Cell Analysis
chemical_list: D016160:Retinoblastoma Protein; D064110:Minichromosome Maintenance Proteins; D015739:Nocodazole