ArticleNucleic acids research2015
Simultaneous measurement of passage through the restriction point and MCM loading in single cells.
Article in Nucleic acids research, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed, 48 citations in OpenAlex.
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- The consequences of differential origin licensing dynamics in distinct chromatin environments.Nucleic acids research · 2022Article
- A novel, rapid and sensitive flow cytometry method reveals degradation of promoter proximal paused RNAPII in the presence and absence of UV.Nucleic acids research · 2022Article
- Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry.Nucleic acids research · 2021Article
- The CRL4DTL E3 ligase induces degradation of the DNA replication initiation factor TICRR/TRESLIN specifically during S phase.Nucleic acids research · 2021Article
- Altered G1 signaling order and commitment point in cells proliferating without CDK4/6 activity.Nature communications · 2020Article
- Distinct and sequential re-replication barriers ensure precise genome duplication.PLoS genetics · 2020Article
- Transient Hysteresis in CDK4/6 Activity Underlies Passage of the Restriction Point in G1.Molecular cell · 2019Article
- Preparation for DNA replication: the key to a successful S phase.FEBS letters · 2019Review
- 5-hydroxymethylcytosine Marks Mammalian Origins Acting as a Barrier to Replication.Scientific reports · 2019Article
- Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence.The Journal of cell biology · 2019Article
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.