This project will investigate the role that telomeric repeat sequences play in the cellular response to ionizing radiation. Telomeric repeat sequences are added by the enzyme telomerase to protect the ends of chromosomes and prevent chromosome fusion, although occasionally telomeric repeat sequences are also found at interstitial sites. Normal somatic cells do not express telomerase activity and telomeres are gradually lost during their life span; however, cancer cells must maintain their telomeres to become immortal. Cancer cells vary in their ability to maintain telomeres, which appears to explain why many have high levels of telomere fusion. The ability of cancer cells to maintain telomeres may also influence their response to ionizing radiation, because restoration of telomeres can heal broken chromosomes. This proposal will analyze the mechanisms of chromosome healing to test the hypothesis that this process plays an important role in determining radiation-induced cytotoxicity and chromosome instability. To achieve this end, specialized telomeres containing selectable marker genes will be introduced into various cancer cell lines to allow for selection and analysis of chromosomes with terminal deletions. Telomeric repeat sequences found within chromosomes have been reported to be hotspots for spontaneous and radiation-induced chromosome rearrangements. These interstitial sites can occur naturally or can be created by the addition of telomeric repeat sequences at double- strand breaks prior to chromosome repair. This proposal will therefore also study the mechanisms of formation and rearrangement of interstitial telomeric repeat sequences and test the hypothesis that they also play an important role in chromosome instability. This will be performed by introducing interstitial telomeric repeat sequences into various locations in the genome and determining the frequency and types of rearrangements that result. Cloning and nucleotide sequence analysis of the integrated sequences and the spontaneous or radiation-induced recombination junctions will provide information on the structural requirements for telomeric repeat sequence-induced chromosome rearrangements.
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