Abstract
DNA fiber spreading assay is an invaluable technique to visualize and follow the spatial and temporal progress of individual DNA replication forks. It provides information on the DNA replication progress and its regulation under normal conditions as well as on replication stress induced by environmental genotoxic agents or cancer drugs. The method relies on the detection of incorporated thymidine analogues during DNA synthesis in the S phase of the cell cycle by indirect immunofluorescence. Here, we describe the procedure established in our laboratories for sequential pulse labeling of human cells with 5-chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU), cell lysis, and DNA fiber spreading on slides and sequential immunodetection of the incorporated thymidine analogues by primary antibodies recognizing specifically CldU or IdU alone. We describe also the laser scanning imaging, classification, and measurement of the detected DNA fiber tracks. The obtained quantitative data can be evaluated statistically to reveal the immediate or long-term effects of DNA-damaging agents, DNA repair inhibitors, and epigenetic modulators like HDAC inhibitors on DNA replication in normal and tumor cells.
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Acknowledgements
This work was supported by DFG (German Research Foundation), grant No. Ni1319/1-1, and by Deutsche Krebshilfe, grant No. 110909, for Anja Göder. We thank Alexandra Zielinski for excellent technical assistance.
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Nikolova, T., Göder, A., Parplys, A., Borgmann, K. (2017). DNA Fiber Spreading Assay to Test HDACi Effects on DNA and Its Replication. In: Krämer, O. (eds) HDAC/HAT Function Assessment and Inhibitor Development. Methods in Molecular Biology, vol 1510. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6527-4_8
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DOI: https://doi.org/10.1007/978-1-4939-6527-4_8
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