Hereditary deficiencies in DNA damage surveillance and signaling are invariably associated with cancer predisposition, radiation sensitivity, immunodeficiency, gonadal abnormalities, and tissue degeneration. These so-called chromosomal instability disorders include ataxia telangiectasia (AT), ataxia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS). AT is caused by null mutations in the protein kinase ATM, whereas hypomorphic mutations in Mre11 and Nbs1 underlie ATLD and NBS respectively. Mre11, Rad50 and Nbs1 form a complex (MRN) that is required for the activation and recruitment of ATM to DNA breaks, which may explain the phenotypic overlap between NBS, ATLD and AT. The objective of this project is to delineate the critical regions in ATM and Nbs1 that regulate tumor suppression, immune system function and meiotic recombination. To accomplish this, we will generate humanized mouse models of AT and NBS by reconstituting ATM-/- and Nbs1-/- mice with bacterial artificial chromosomes (BACs) that carry mutations in critical domains of human ATM and Nbs1. We will explore the in vivo consequences of specific amino acid changes that individually abrogate ATM activity, ATM-dependent Nbs1 phosphorylation, and ATM recruitment to DNA breaks. The analysis of these humanized transgenic mice will provide mechanistic insight into the central question of how the DNA damage signal is transmitted to and activates ATM. We have also developed and applied comprehensive genomics tools to directly map DNA breaks and identify DNA end structures at high-resolution. This sensitive and unbiased method, called END-seq, can monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We have utilized END-seq to determine the frequency and spectrum of restriction enzyme-, zinc-finger nuclease-, and RAG-induced DSBs. Beyond sequence preference, we have noted that chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least 1 DSB per cell amongst 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class switch recombination. Thus END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice. We are currently applying END-Seq in a variety of biological contexts including the evaluation of faithful targeting as well as off-target cleavage sites of therapeutic nucleases such as ZFNs, TALENs and CRISPR/Cas9. We are also evaluating END-seq to map DSBs produced by uncharacterized genotoxic agents in drug development as well as in the interrogation of structural variation during tumor evolution.
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