The chromosome instability syndromes are a group of autosomal recessive diseases associated with higher cancer risks. For one of these diseases, ataxia-telangiectasia (A-T), heterozygotes carriers are also at increased relative risk, particularly for breast carcinoma. As a result, it has been estimated that 7-8% of women suffering from breast cancer are A-T heterozygotes. Cells from A-T homozygotes exhibit abnormalities consistent with a defect in DNA metabolism and/or maintenance of genomic integrity. A-T represents an important model system for the study of carcinogenesis, DNA repair and genetic recombination. Despite extensive investigation, no A-T gene has been isolated and the underlying defect is unknown. The overall goal of this proposal is to gain a better understanding of both A-T cellular responses to DNA damage by cloning and characterization A-T genes as well as studying the etiology of two aspects to the A-T phenotype: radiation sensitivity and hyper-recombination. Our approach is summarized in two specific aims: A. Identify and characterize genes that cause A-T as well as other human cDNAs that complement the phenotypic defects of A-T fibroblasts by: i) transfecting A-T fibroblasts with cDNA libraries contained in an EBV- based episomal expression vector and selecting for colonies of transfected cells that have become mutagen-resistant; ii) screening cells derived from these surviving colonies form complementation of the A-T phenotype and rescuing cDNAs from complemented fibroblasts by transfection of nuclear lysates into E. coli; iii) transfecting the recovered cDNAs into fresh A-T fibroblasts and identifying candidate cDNAs by their ability to complement multiple aspects of the A-T phenotypes upon repeated transfection; iv) identifying disease genes by mapping candidate cDNAs and analyzing the structure and expression of candidate genes in normal and affected individuals; v) characterizing A-T and related genes by isolating and analyzing full- length cDNA and genomic sequences and determining patterns of tissues expression as well as changes in expression during the cell cycle and after exposure to mutagens. B. Investigate the etiology of recombination abnormalities and radiosensitivity in A-T by"""""""" i) determining the effects of functional loss of p53 and the G1/S cell cycle checkpoint on recombination in normal mammalian cells; and ii) studying the role of apoptosis and p53 function in radiation- and mutagen-induced killing of A-T fibroblasts. This work may further define the etiology of A-T, identify new genes involved in cellular responses to DNA damage and further our knowledge of genetic recombination and the genetics of cancer.
