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Visualization of the MCM DNA helicase at replication factories before the onset of DNA synthesis

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Abstract

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.

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Abbreviations

BrdU:

5-bromo-2′-deoxyuridine

EdU:

5-ethynyl-2′-deoxyuridine

IF:

Immunofluorescence

MCM:

Minichromosome maintenance

PCNA:

Proliferating cell nuclear antigen

RF:

Replication foci

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Acknowledgments

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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Author notes

  1. Tomás Aparicio

    Present address: Institute for Cancer Genetics, Columbia University Medical Center, 1130 Saint Nicholas Avenue, New York, NY, 10032, USA

Authors and Affiliations

  1. DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro, 3, 28029, Madrid, Spain

    Tomás Aparicio & Juan Méndez

  2. Confocal Microscopy Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro, 3, 28029, Madrid, Spain

    Diego Megías

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  1. Tomás Aparicio
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Correspondence to Juan Méndez.

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Communicated by John Diffley

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Figure S1

Incorporation of EdU to DNA does not affect cell cycle progression. a Flow cytometric detection of EdU incorporation (y-axis) vs DNA content (x-axis) in HeLa cells previously labeled for 30 min with 10 μM EdU. Cells were processed and analyzed immediately (NC, no chase), or 2 h, 4 h, 16 h after the EdU pulse. b The experimental design is similar to that of part a, but cells were treated with Triton X100 to pre-extract soluble proteins prior to flow cytometry, and chromatin-bound PCNA, which is only present in S phase cells, was immunodetected (y-axis). EdU-positive cells, labeled in blue, can be seen in S phase immediately after the pulse (NC), in G2/M and G1 (8 h) and back in S phase (24 h). (JPEG 109 kb)

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Aparicio, T., Megías, D. & Méndez, J. Visualization of the MCM DNA helicase at replication factories before the onset of DNA synthesis. Chromosoma 121, 499–507 (2012). https://doi.org/10.1007/s00412-012-0381-x

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