We developed a novel system to create DNA double-strand breaks (DSBs) at defined endogenous sites in the human genome, and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation (ChIP). The detection of human ATM protein at site-specific DSBs required functional NBS1 protein, ATM kinase activity and ATM autophosphorylation on Ser 1981. DSB formation led to the localized disruption of nucleosomes, a process that depended on both functional NBS1 and ATM. These two proteins were also required for efficient recruitment of the repair cofactor XRCC4 to DSBs, and for efficient DSB repair. These results demonstrate the functional importance of ATM kinase activity and phosphorylation in the response to DSBs, and support a model in which ordered chromatin structure changes that occur after DNA breakage depend on functional NBS1 and ATM, and facilitate DNA DSB repair.

The formation of protein aggregates (foci) at sites of DNA double-strand breaks (DSBs) is mainly studied by immunostaining and is hence limited by the low resolution of light microscopy and the availability of appropriate and selective antibodies. Here, we describe a system using enzymatic creation of site-specific DNA DSBs within the human genome combined with chromatin immunoprecipitation (ChIP) that enables molecular probing of a DSB. Following induction of the I-PpoI enzyme and generation of DSBs, cellular DNA and proteins are crosslinked and analyzed by ChIP for specific proteins at the site of the break. The system allows the direct detection of protein and chromatin dynamics at the site of the break with high resolution, as well as direct measurement of DNA repair defects in human cells. Starting with fragmented chromatin, results can be achieved in 2-3 d.

Genomic instability, a hallmark of leukemic cells, is associated with malfunctioning cellular responses to DNA damage caused by defective cell cycle checkpoints and/or DNA repair. Adult T-cell leukemia, which can result from infection with human T-cell leukemia virus type 1 (HTLV-1), is associated with extensive genomic instability that has been attributed to the viral oncoprotein Tax. How Tax influences cellular responses to DNA damage to mediate genomic instability, however, remains unclear. Therefore, we investigated the effect of Tax on cellular pathways involved in recognition and repair of DNA double-strand breaks. Premature attenuation of ATM kinase activity and reduced association of MDC1 with repair foci were observed in Tax-expressing cells. Following ionizing radiation-induced S-phase checkpoint activation, Tax-expressing cells progressed more rapidly than non-Tax-expressing cells toward DNA replication. These results demonstrate that Tax expression may allow premature DNA replication in the presence of genomic lesions. Attempts to replicate in the presence of these lesions would result in gradual accumulation of mutations, leading to genome instability and cellular transformation.Preserving genomic integrity is critical for all living systems. The integrity and survival of a cell depend on the stability of its DNA. Damaged DNA is detected by cellular sensing systems, which activate specific DNA repair pathways. Malfunction of this repair network, collectively known as the DNA damage response, leads to DNA mutations, a subset of which can promote cellular transformation. Double-stranded DNA breaks (DSBs) arise from genotoxic insults and normal physiological processes such as DNA replication (24, 38). The mechanisms by which eukaryotic cells sense DNA breaks remain to be elucidated, but one of the earliest detectable events in DNA damage sensing is the activation of the ataxia telangiectasia mutated (ATM) kinase (45).ATM is a member of the PI3K-like kinase family and is mutated in ataxia telangiectasia patients (45). Immediately after exposure of cells to DSB-inducing agents such as ionizing radiation (IR) and radiomimetic drugs, changes in chromatin structure activate the intermolecular autophosphorylation of ATM on Ser1981, resulting in the dissociation of inactive ATM dimers into active monomers that allow substrate accessibility to the ATM kinase domain (2). ATM substrates include cellular targets such as NBS1, Chk2, p53, MDC1, histone 2AX (H2AX), and BRCA1, which are key players in the maintenance of genomic integrity (5,19,44,47). Some of these substrates are phosphorylated by ATM in the nucleoplasm, while others are phosphorylated at sites of DNA damage where ATM is recruited via interaction with the Mre11/Rad50/NBS1 (MRN) complex. At sites of DNA breaks within chromatin, the phosphorylation of H2AX and MDC1 by ATM establishes a positive-feedback loop that maintains ATM autophosphorylation and amplifies the DNA damage response (31). Genetic defects in crucial parts of this network lead to a grou...

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