Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells
Identifieur interne : 005280 ( Main/Exploration ); précédent : 005279; suivant : 005281Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells
Auteurs : Andrew Burgess [Australie] ; Thierry Lorca [France] ; Anna Castro [France]Source :
- PLoS ONE [ 1932-6203 ] ; 2012.
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.
Url:
DOI: 10.1371/journal.pone.0045726
PubMed: 23029203
PubMed Central: 3447815
Affiliations:
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type="main">Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells</title><author><name sortKey="Burgess, Andrew" sort="Burgess, Andrew" uniqKey="Burgess A" first="Andrew" last="Burgess">Andrew Burgess</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>The Kinghorn Cancer Center, Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>The Kinghorn Cancer Center, Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales</wicri:regionArea><wicri:noRegion>New South Wales</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>St. Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>St. Vincent’s Clinical School, Faculty of Medicine, 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Castro</name><affiliation wicri:level="3"><nlm:aff id="aff3"><addr-line>Universités Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Montpellier, France</addr-line></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Universités Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Montpellier</wicri:regionArea><placeName><region type="region">Occitanie (région administrative)</region><region type="old region">Languedoc-Roussillon</region><settlement type="city">Montpellier</settlement></placeName></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>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.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Burgess, A" uniqKey="Burgess A">A Burgess</name></author><author><name sortKey="Vigneron, S" uniqKey="Vigneron S">S Vigneron</name></author><author><name sortKey="Brioudes, E" uniqKey="Brioudes E">E Brioudes</name></author><author><name sortKey="Labbe, J C" uniqKey="Labbe J">J-C Labbé</name></author><author><name sortKey="Lorca, T" uniqKey="Lorca T">T Lorca</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schmitz, Mha" uniqKey="Schmitz M">MHA Schmitz</name></author><author><name sortKey="Held, M" uniqKey="Held M">M Held</name></author><author><name sortKey="Janssens, V" uniqKey="Janssens V">V Janssens</name></author><author><name sortKey="Hutchins, Jra" 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