Visualization of the MCM DNA helicase at replication factories before the onset of DNA synthesis
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In mammalian cells, DNA synthesis takes place at defined nuclear structures termed “replication foci” (RF) that follow the same order of activation in each cell cycle. Intriguingly, immunofluorescence studies have failed to visualize the DNA helicase minichromosome maintenance (MCM) at RF, raising doubts about its physical presence at the sites of DNA synthesis. We have revisited this paradox by pulse-labeling RF during the S phase and analyzing the localization of MCM at labeled DNA in the following cell cycle. Using high-throughput confocal microscopy, we provide direct evidence that MCM proteins concentrate in G1 at the chromosome structures bound to become RF in the S phase. Upon initiation of DNA synthesis, an active “MCM eviction” mechanism contributes to reduce the excess of DNA helicases at RF. Most MCM complexes are released from chromatin, except for a small but detectable fraction that remains at the forks during the S phase, as expected for a replicative helicase.
Proliferating cell nuclear antigen
We are grateful to all members of the CNIO DNA Replication and Chromosome Dynamics Groups for the insightful discussions and Oscar Fernández-Capetillo and Almudena R. Ramiro for the useful comments on the manuscript. This work was supported by the Spanish Ministry of Science and Innovation (BFU2010-21467 and Consolider CSD2007-00015 to J.M.).
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