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.
Highlights
The replication of genomic DNA must be completed with absolute accuracy, and is one of the most critical steps of cell division
Cell Culture and Synchrony Experiments were performed on the HeLa Chromobody cell line (Chromotek), which stably expresses a Proliferating Cell Nuclear Antigen (PCNA) targeting Chromobody fused to GFP [12]
Significant co-localization between sites of EdU incorporation and GFP signal confirmed the specificity of the PCNA Chromobody to accurately identify sites of active DNA replication (Figure 1A)
Summary
The replication of genomic DNA must be completed with absolute accuracy, and is one of the most critical steps of cell division. Errors in replication can lead to cell death and or genomic instability, a hallmark of cancer, highlighting its importance. Experiments have primarily been limited to the use of static time-points, which provide only a snapshot of the replication process, thereby limiting our understanding of this biological step. The ability to visualize cells in real-time has enabled rapid and numerous advances in our understanding of a wide range of biological processes, such as identifying novel regulators of cell division [1,2], and uncovering the dynamics of specific protein-protein interactions and modifications [3,4]. The absence of simple methods for the quantitative live cell imaging of DNA replication has remained a notable stumbling block
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