Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis

Authors

  • K. Berniak,

    1. Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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    • K. Berniak and P. Rybak contributed equally to this work.

  • P. Rybak,

    1. Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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  • T. Bernas,

    1. Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
    2. Nencki Institute of Experimental Biology, Laboratory of Functional and Structural Tissue Imaging, Polish Academy of Sciences, Warsaw, Poland
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  • M. Zarębski,

    1. Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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  • E. Biela,

    1. Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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  • H. Zhao,

    1. Brander Cancer Research Institute, Department of Pathology, New York Medical College, Valhalla, New York
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  • Z. Darzynkiewicz,

    1. Brander Cancer Research Institute, Department of Pathology, New York Medical College, Valhalla, New York
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  • J. W. Dobrucki

    Corresponding author
    • Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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  • This article is an expanded version of the communication, which was delivered during 2012 ISAC Congress in Leipzig.

Correspondence to: J. W. Dobrucki, Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. E-mail: jerzy.dobrucki@uj.edu.pl

Abstract

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA “cleavable complexes” stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry

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