Endogenous DNA breaks: γH2AX and the role of telomeres

cells but can be deadly. A single unrepaired DSB will kill a yeast cell deficient in recombination [1]. Unrepaired DSBs lead to chromosome damage and are associated with aging and cancer. Until a few years ago, methods used for their detection relied exclusively on physical techniques such as pulsed field gel electrophoresis that measure changes in the size of DNA molecules. Unfortunately, these methods are insensitive, typically recognizing 50 or more breaks per mammalian cell and necessitating the use of lethal exposures to X-rays or radiomimetic drugs. In 1998, Bonner and colleagues reported that phosphorylation of H2AX, a minor nucleosomal histone protein, occurred at sites of DSBs [2]. This process is unique in that hundreds of molecules surrounding each break become phosphorylated as the signal propagates away from the break site; development of antibodies against the serine-139 phosphorylated form (called γH2AX) allowed microscopic detection of individual DSBs [3]. This discovery revolutionized the ability to detect DSBs and provided a unique tool to examine processes involved in DNA damage signalling. Applications of γH2AX as an indicator of response to radiation and drugs soon followed [4]. Sensitive detection of drug-and radiation-induced DSBs using γH2AX requires a low endogenous expression of γH2AX. Similarly, applications of γH2AX in DNA damage signalling are dependent on low endogenous levels of the phosphorylated form. In most normal primary human cells, γH2AX foci are relatively rare so that DSBs can be detected by non-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 lethal radiation doses in the mGy range [5]. However, γH2AX foci are observed in cells undergoing meiosis or V(D)J recombination, as well as senescent cells and apoptotic cells. In each of these cases, a convincing argument can be made that DSBs underlie the formation of γH2AX foci. It is more difficult to explain large numbers of endogenous γH2AX foci seen in many tumors cells, or the variability in foci numbers between different tumor cell lines [6]. As physical methods lack the sensitivity to confirm that these foci signify true breaks, the possibility remains that either some tumor cells contain large numbers of DSBs or there are other explanations for endogenous foci. In either case, endogenous foci are a problem because they reduce the sensitivity and specificity for detecting exogenously produced breaks. In …