Journal
PLOS ONE
Volume 7, Issue 9, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0045726
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Funding
- Ligue Nationale Contre le Cancer (Equipe Labellisee)
- Agence Nationale de la Recherche (ANR) [2010 BLAN 120701]
- Cancer Institute NSW [10/FRL/3-02]
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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.
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