3912–3923 Nucleic Acids Research, 2009, Vol. 37, No. 12 Published online 28 April 2009 doi:10.1093/nar/gkp237 Cellular responses to DNA double-strand breaks after low-dose c-irradiation Aroumougame Asaithamby and David J. Chen* Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA Received February 10, 2009; Revised March 25, 2009; Accepted March 29, 2009 ABSTRACT DNA double-strand breaks (DSBs) are a serious threat to genome stability and cell viability. Although biological effects of low levels of radiation are not clear, the risks of low-dose radiation are of societal importance. Here, we directly monitored induction and repair of single DSBs and quantita- tively analyzed the dynamics of interaction of DNA repair proteins at individual DSB sites in living cells using 53BP1 fused to yellow fluorescent protein (YFP-53BP1) as a surrogate marker. The number of DSBs formed was linear with dose from 5 mGy to 1 Gy. The DSBs induced by very low radiation doses (5 mGy) were repaired with efficiency similar to repair of DSBs induced at higher doses. The YFP- 53BP1 foci are dynamic structures: 53BP1 rapidly and reversibly interacted at these DSB sites. The time frame of recruitment and affinity of 53BP1 for DSB sites were indistinguishable between low and high doses, providing mechanistic evidence for the similar DSB repair after low- and high-dose radiation. These findings have important implica- tions for estimating the risk associated with low- dose radiation exposure on human health. INTRODUCTION Cell lethality, mutations, chromosomal translocations, apoptosis and cancer induced by ionizing radiation (IR) result principally from an inefficient or inaccurate repair of DNA double-strand breaks (DSBs) (1). Humans are exposed to low doses of radiation during air travel, from radon in homes, during space travel or in areas of low- level contamination (including former sites of nuclear weapon production) and can encounter much higher radi- ation doses in contaminated areas such as Chernobyl or during radiotherapy (2). Radiobiologists have struggled to estimate the biological consequences of low levels of radiation exposure in humans for decades. The current risk estimates for low-dose radiation are based on assump- tion that there is a linear, nonthreshold, dose–response relationship with detrimental health effects of low-dose IR extrapolated from comparatively high doses (3). However, the biological effects associated with occupa- tional and environmental low-dose radiation are consider- ably more complex than predicted by the linear nonthreshold model due to the fact that the radiation- induced biological effects in humans depend on several factors, including the influence of cellular responses to DNA repair, sensitivity of bystander cells and delayed genomic instability (4). Currently, insufficient data are available to determine the impact of low levels of radiation exposure on human health (5). As risks associated with low-dose radiation are of societal importance (6), it is crit- ical to further understand the cellular responses to low quantities of radiation exposure. In response to DNA DSBs, histone 2A family member X is phosphorylated at serine 139 (gH2AX) and forms discrete foci at the DSB sites (7,8). Earlier studies have revealed a close correlation between the number of gH2AX foci and the estimated number of DSBs (7,9). Recently, a dose-dependent induction of gH2AX foci was observed in the range between 1.2 mGy and 2 Gy (10). These studies clearly indicate that gH2AX foci are a reliable marker for the quantification of DSBs. However, evidence indicates that the number of gH2AX may not represent true DSBs induced by different dama- ging agents (11) and that the number of background gH2AX foci is relatively greater in exponentially growing cells than in confluent cells (12). Therefore, for an accurate measurement of the biological effects of low-dose radia- tion exposure, an alternative in vitro assay sensitive enough to measure a single DSB per cell is needed. In vitro studies suggest that the tumor suppressor p53-binding protein 1 (53BP1) participates in the cellular response to DNA damage. 53BP1 relocates to multiple nuclear foci within minutes after exposure of cells to IR (13–15). Evidence suggests that, similar to gH2AX, the number of 53BP1 foci is closely correlated with the number of DNA DSBs. Further, the kinetics of resolution of 53BP1 foci is very similar to the kinetics of DNA DSBs *To whom correspondence should be addressed. Tel: +1 214 648 5597; Fax: +1 214 648 5995; Email: David.Chen@UTsouthwestern.edu ß 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/nar/article/37/12/3912/1042652 by guest on 14 June 2022