Nijmegen Breakage Syndrome (NBS) is an autosomal recessive disorder characterized by marked cancer predisposition, sensitivity to ionizing radiation, and defects in DNA damage dependent cell cycle checkpoints. Because the cellular phenotypes of NBS strongly resemble those of cells established from ataxia telangiectasia (AT) patients, this syndrome has been described as an AT variant syndrome although it is genetically distinct. We showed that NBS is attributable to deficiency in an intrinsic member of the hMre11/hRad50 protein complex, referred to herein as p95. We have established that this complex, which is uniformly distributed in the nucleus prior to irradiation, relocalizes to sites of DNA damage in the nuclei of cells treated with ionizing radiation, and is restored to uniform distribution upon completion of DNA repair. The association of the complex with DNA damage, in conjunction with the finding that cellular responses to ionizing radiation are abrogated by p95 deficiency lead to the hypothesis that the complex acts to sense and signal the presence of DNA damage in irradiated cells. In this proposal, we will examine the function of p95 to address this hypothesis, and to elucidate its potential role the repair of ionizing radiation-induced DNA damage as part of the Mre11/Rad50 complex. We propose to carry out phenotypic analyses of p95 deficient cells expressing variants of p95 to define functionally important domains of the protein. To examine phenotypes that are not assayable in cultured cells and to assess genetic interactions of p95, p95 deficient mice will be constructed. Of particular interest in this regard are the genetic and functional interactions between p95 and ATM which will be assessed by breeding of p95 and ATM deficient mice. These studies will provide important information regarding the relative contributions of p95 and ATM in the cellular and organismal responses to ionizing radiation. In this proposal, p95 is examined from three complementary perspectives. First, at the cellular level, second, at the molecular level, and third, in vivo through the derivation of a munbs1 mutant mouse.
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