Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors Slawomir Kumala 1 , Krzysztof Fujarewicz 2 , Dheekollu Jayaraju 1 , Joanna Rzeszowska-Wolny 3 , Ronald Hancock 1 * 1 Laval University Cancer Research Centre, Ho ˆ tel-Dieu Hospital, Que ´ bec, Canada, 2 Bioinformatics Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland, 3 Biosystems Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland Abstract To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ,170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with c photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair. Citation: Kumala S, Fujarewicz K, Jayaraju D, Rzeszowska-Wolny J, Hancock R (2013) Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors. PLoS ONE 8(1): e52966. doi:10.1371/journal.pone.0052966 Editor: Marco Muzi-Falconi, Universita’ di Milano, Italy Received May 31, 2012; Accepted November 26, 2012; Published January , 2013 Copyright: ß 2013 Kumala et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported partially by the Polish Ministry of Education and Science (Grant N N518 4976 39 to K.F. and J.R-W.). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ronald.hancock@crhdq.ulaval.ca Introduction The molecular events implicated in repair of strand breaks in DNA are becoming more clear (reviewed in [1–6]), but an overall and quantitative picture of their repair in vivo which would contribute to understanding the systems biology of repair and the effects of inhibitors is not yet available. Current methods do not allow simultaneous and precise quantitation of repair of single and double strand breaks. Repair of double strand breaks, which are believed to be the crucial lesions leading to cell death [7], is commonly assayed by restoration of the normal length of genomic DNA or restriction fragments using pulsed-field gel electrophoresis (PFGE) [8–10]. Repair of single strand breaks, which may contribute to loss of viability by relaxing superhelical stress in genomic DNA loops and thus arresting transcription [11], cannot yet be quantitated specifically by methods with comparable precision. As a model system to approach this question we are studying the repair of strand breaks in vivo in a ,170 kb circular minichromosome, the Epstein-Barr virus (EBV) episome, which is maintained in the nuclei of Raji cells at 50–100 copies localised at the periphery of interphase chromosomes [12–17]. Two features of this minichromosome make it an attractive model for genomic chromatin: it can be considered as a defined region of chromatin in view of its canonical nucleosomal conformation [13] and the well-studied sequence and properties of its DNA [14], and its closed circular topology and length resemble those of the constrained loops which genomic chromatin forms in vivo [11,18,19]. After irradiating cells with 60 Co c photons we assayed the repair of single strand breaks in the minichromosome by quantitating the loss of nuclease S1- sensitive sites, and the repair of double strand breaks by PFGE assays of the reformation of supercoiled DNA from molecules which had been linearised. Circular molecules containing single strand breaks could not be quantitated directly, and instead their levels were calculated using a mathematical model developed to fit the experimental data. We exploited the possibility of quantitating repair in this system to examine the implication of particular enzymes, particularly topoisomerases I and II whose participation in repair has long been controversial [20–24], poly(ADP-ribose) polymerase-1 (PARP-1) [25–32], Rad51 [33], the catalytic subunit of DNA-protein kinase (DNA-PKcs) [2–6,34], and ATM kinase [2–6,35,36]. New features of the repair of strand breaks in vivo and of their kinetics were revealed by mathematical modeling. PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e52966 30