title: Quantitative live imaging of endogenous DNA replication in mammalian cells.
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.
authors.affiliation: The Kinghorn Cancer Center, Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
authors.forename: Andrew
authors.initials: A
authors.lastname: Burgess
authors.forename: Thierry
authors.initials: T
authors.lastname: Lorca
authors.forename: Anna
authors.initials: A
authors.lastname: Castro
mesh_terms: D002453:Cell Cycle / Q000187:drug effects; D004261:DNA Replication; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D018613:Microscopy, Confocal; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D049452:Green Fluorescent Proteins; D006918:Hydroxyurea