Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells |
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| Authors: | Andrew Burgess Thierry Lorca Anna Castro |
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| Affiliation: | 1. The Kinghorn Cancer Center, Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.; 2. St. Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.; 3. Universités Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Montpellier, France.; University of Ottawa, Canada, |
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| Abstract: | 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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