title: Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.
abstract: Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns. Such a correlation can be used to estimate a probability of inducing damage in sections of replicating DNA. The damage and replication foci are imaged in 3D with confocal microscopy and their respective positions within nuclei are determined with adaptive image segmentation. Using correlation functions spatial proximity of the resultant point patterns is quantified over the range of distances in cells in early-, mid- and late S-phase. As the numbers (and nuclear densities) of γH2AX and replication foci differ significantly in the subsequent substages of S phase, the detected association values were corrected for the expected random overlap between both classes of foci. Thus, the probability of their nonrandom association was estimated. Moreover, self association (clustering) of DNA replication sites in different stages of S-phase of the cell cycle was detected and accounted for. While the analysis revealed a strong correlation between the γH2AX foci and the sites of DNA replication in cells treated with Cpt, only a low correlation was apparent in cells exposed to H2O2. © 2013 International Society for Advancement of Cytometry.
authors.affiliation: Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Tytus
authors.initials: T
authors.lastname: Bernas
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Berniak
authors.forename: Paulina
authors.initials: P
authors.lastname: Rybak
authors.forename: Mirosław
authors.initials: M
authors.lastname: Zarębski
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
authors.forename: Jerzy W
authors.initials: JW
authors.lastname: Dobrucki
mesh_terms: D002166:Camptothecin / Q000633:toxicity; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D018613:Microscopy, Confocal; D018384:Oxidative Stress / Q000502:physiology*; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology
chemical_list: C468783:H2AX protein, human; D006657:Histones; D059004:Topoisomerase I Inhibitors; D002166:Camptothecin
keywords: DDR; DNA damage; DNA damage response; DNA replication; camptothecin; cell cycle; correlation analysis; histone H2AX phosphorylation; hydrogen peroxide; oxidative stress; γH2AX
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