The goals of the proposed research are to explore the effects of ionizing radiation-induced interchromosomal recombination in normal human cells and in mutant cells with altered radiosensitivity and recombination phenotypes. Few studies have focused on the recombinogenic effects of ionizing radiation in mammalian cells, and thus far none have examined such effects as a function of dose rate, linear energy transfer (LET), or cellular repair capacity. It has been difficult to study the mechanisms of ionizing radiation-induced recombination in mammalian cells because of difficulties in analyzing recombinant products. Furthermore, although ionizing radiation is known to induce homologous recombination between alleles present at homologous autosomal loci in mammalian cells, it is not known whether this is due to direct effects of damage in recombining genes (i.e., strand breaks) or indirect effects (i.e., by inducing enzymes involved in recombinational repair) or both. In the yeast Saccharomyces cerevisiae, ionizing radiation-induced recombination is thought to involve both direct and indirect processes. This proposal describes model systems that will provide information about the induction of recombination in human cells by radiations of various types. The proposed recombination substrates will consist of noncomplementing (heteroallelic) neomycin (neo) genes targeted to both copies of an autosomal locus, and will include several features. Recombination substrates will be flanked by convenient restriction sites and neo alleles will be heteroallelic at eleven positions consisting of phenotypically silent restriction fragment length polymorphisms (RFLPs). The flanking restriction sites and RFLPs will facilitate the rescue and fine-resolution mapping of recombinant products. The neo alleles will be regulated by the inducible mouse mammary tumor virus (MMTV) promoter, allowing experimental control of transcription rates in recombination substrates. Complementary approaches are proposed to address questions about how recombination induces recombination. One approach involves the detailed examination of spontaneous and radiation- induced recombination products of defined heteroallelic genes, including studies of the influence of transcription on induced recombination frequencies and mechanisms. Such analyses will provide increased sensitivity for detecting potential mechanistic differences among spontaneous, low LET-induced and high LET-induced recombination events. In another approach we will investigate whether induced recombination is a consequence of direct effects of damage in recombining regions and/or an indirect induction of recombinational repair enzymes. Lastly, comparisons will be made between spontaneous and radiation-induced recombination frequencies and product spectra in DNA repair-proficient normal human cells, and DNA repair-deficient cells from patients suffering from ataxia- telangiectasia and Bloom's syndrome. These experiments will help to establish the roles of the gene products altered in these mutant cells. Together these studies will further our understanding of the initiation, mechanisms, and genetic consequences of spontaneous and ionizing radiation-induced recombination in mammalian cells.
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