DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents.

Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (γH2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of γH2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.

The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a “primary” DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11–Rad50–Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.