Abstract
Several proteins involved in the response to DNA double strand breaks (DSB) form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF) are believed to be located where DNA damage occurs. To test this assumption, we analyzed the spatial distribution of 53BP1, phosphorylated ATM, and γH2AX RIF in cells irradiated with high linear energy transfer (LET) radiation and low LET. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. As expected, simulations produced DNA-weighted random (Poisson) distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) were non-random. This deviation from the expected DNA-weighted random pattern can be further characterized by “relative DNA image measurements.” This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density. The same preferential nuclear location was also measured for RIF induced by 1 Gy of low-LET radiation. This deviation from random behavior was evident only 5 min after irradiation for phosphorylated ATM RIF, while γH2AX and 53BP1 RIF showed pronounced deviations up to 30 min after exposure. These data suggest that DNA damage–induced foci are restricted to certain regions of the nucleus of human epithelial cells. It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair.
Highlights
DNA damage induced by ionizing radiation (IR) elicits microscopically visible nuclear domains marked by recruitment of certain proteins (e.g., 53BP1) or by particular modifications such as histone phosphorylation or as a result of both [1,2,3,4,5,6,7,8,9,10]
We introduce computer modeling and image analysis tools in order to relate the position of DNA damage markers to morphologically distinct regions of the nucleus
DNA double strand breaks (DSB) were simulated by Monte Carlo simulations for single traversal of 1 GeV/amu Fe ions or for exposure to 1 Gy of low-linear energy transfer (LET) radiation [24]
Summary
DNA damage induced by ionizing radiation (IR) elicits microscopically visible nuclear domains (i.e., foci) marked by recruitment of certain proteins (e.g., 53BP1) or by particular modifications such as histone phosphorylation (e.g., cH2AX) or as a result of both (e.g., phosphorylated ATM, ATMp) [1,2,3,4,5,6,7,8,9,10]. Radiation-induced foci (RIF) are believed to form at or adjacent to sites of DNA damage. Some reports suggest that there are nuclear regions that are excluded from forming RIF. In studies using densely ionizing particles that would lead to continuous DSB along their trajectories, nuclei showed discontinuous MRE11 RIF, with large gaps (.1 lm) in regions where DNA was present [15]. Others have shown that some types of RIF are not necessarily associated with DSB [12]
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