Quantitative live imaging of endogenous DNA replication in mammalian cells

PLoS One. 2012;7(9):e45726. doi: 10.1371/journal.pone.0045726. Epub 2012 Sep 20.


Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle / drug effects
  • DNA Replication*
  • Flow Cytometry
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Hydroxyurea / pharmacology
  • Microscopy, Confocal
  • Proliferating Cell Nuclear Antigen / metabolism*


  • Proliferating Cell Nuclear Antigen
  • Green Fluorescent Proteins
  • Hydroxyurea

Grant support

This work was supported by the Ligue Nationale Contre le Cancer (Equipe Labellisée), and Agence Nationale de la Recherche (ANR) 2010 BLAN 120701, and a Cancer Institute NSW Future Research Leaders Fellowship [10/FRL/3-02][AB]. Funding for open access charge: [Cancer Institute NSW, 10/FRL/3-02]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.