REPAIR AND RECOMBINATION OF BROKEN CHROMOSOMES

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Chromosome abnormalities such as translocations, deletions, and gene amplifications are frequently observed in tumor cells. In many cases, these abnormalities result in the activation of oncogenes, the loss of tumor suppressor genes, and the overexpression of oncogenes and drug resistance genes. Recent evidence indicates that a single form of DNA damage, chromosome breaks, may be involved in the formation of these abnormalities. Although mammalian cells have been shown to repair or heal broken chromosomes, the precise nature or mechanism of these processes has not been elucidated. The difficulty in determining, at the DNA sequence level, where a break has occurred or in predicting where a break will occur has been the major obstacle in studying the repair processes. The identification of a broken chromosome resulting from amplification of the DHFR gene in CHO cells, whose break site has been mapped at the DNA level, provides a system for the study of the molecular mechanisms of repair or healing of broken chromosomes. The terminus sequence will be cloned and sequenced to determine whether any sequences such as telomeric repeats, have been added to the free end of the break. In order to study the various mechanisms of repair and healing as they occur, a means of creating a double-strand break in a defined DNA sequence in a chromosome is proposed. The introduction of one FLP recognition and cleavage site into a defined location in a CHO chromosome and the induced expression of the FLP protein should create such a defined break. This system will be used to study the ability of CHO cells to heal or repair the break as it occurs. Fluorescent in situ hybridization will be used to track the events that occur at the chromosomal level. Healed or repaired ends will be cloned and sequenced to determine the precise nature of these repair events. This site-specific cleavage system will also provide a means of studying homologous recombination of free ends and its involvement in double-strand break repair. Finally the proposed role of double-strand breaks mediating deletion and gene amplifications events will also be tested directly with this system.