Eukaryotic cells possess DNA repair mechanisms to protect the chromosomes from damage inflicted by ionizing radiation. The lethal effect of X-Rays primarily appears to be in the misrepair of double- stranded DNA breaks (DSBs). Interestingly, DNA recombination and DNA repair events may be interrelated in mammalian cells. because DSBs are likely to be intermediates in both of these processes. We have observed that the scid mouse mutation has a defect in V(D)J recombination in the developing immune system, and a general sensitivity to X-Ray damage. These observations have prompted a model proposing a common function for the SCID protein in both events. In this proposal we will examine the role of the scid mutation in several general DNA recombination events and characterize the repair defect in order to understand the function of scid. We will characterize on the molecular level the repair of X- Ray lesions generated in chromosomal genes in scid cells. We will analyze the specificity of the scid repair defect to DSBs in the chromosomes when these structures have been induced by several methodologies. We will investigate the potential restriction of the scid repair defect to a portion of the cell cycle. Several DNA recombination reactions involving double-stranded DNA intermediates will be studied in scid cells. DNA end joining reactions (non-homologous recombination) will be examined by DNA transfection of defined plasmid molecules: both stable plasmid integration and reclosure of plasmid circles are to be evaluated. Similarly, DNA recombination events requiring DNA homology will also be studied with transfected plasmid DNA substrates: gene targeting and homologous recombination. A third general objective is to derive a molecular clone of the murine SCID gene by complementation of the scid repair defect. We will then examine the molecular phenotype of this gene and the scid mutation. Eukaryotic chromosomes are exposed to high rates of mutagenesis in the life of an organism, with surprisingly low rates of mutation. The highly evolved defense to these insults must repair breaks in the chromosomes efficiently. However, many instances of tumor phenotypes are correlated on the molecular level with chromosomal translocations, rearrangements, and deletions. A fundamental understanding of the mechanisms by which the integrity of the chromosome content is maintained is necessary for a basic conceptualization and solution to the cancer problem.
