'American Society for Biochemistry & Molecular Biology (ASBMB)'
Abstract
Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.Fil: Symeonidou, Ioanna Eleni. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Kotsantis, Panagiotis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Roukos, Vassilis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Rapsomaniki, Maria Anna. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Grecco, Hernan Edgardo. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina;Fil: Bastiaens, Philippe. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania;Fil: Taraviras, Stavros. University of Patras. School of Medicine. Laboratory of Physiology; Grecia;Fil: Lygerou, Zoi. University of Patras. School of Medicine. Laboratory of General Biology; Grecia
