Telomeres are nucleoprotein complexes on the ends of chromosomes that are composed of a six base pair repeat sequence maintained by the enzyme telomerase. A primary function of telomeres is to, protect the ends of chromosomes and prevent chromosome fusion. One form of chromosome repair involves the addition of telomeres to the ends of broken chromosomes, termed chromosome healing, which has been demonstrated in a variety of organisms. The importance of chromosome healing in humans has been demonstrated by the fact that all seven of the terminal deletions associated with human genetic disease that have been analyzed thus far involved the addition of telomeric repeat sequences directly onto the end of a broken chromosome. Chromosome healing has also been shown to prevent chromosome instability in some organisms. This proposal will investigate chromosome healing in human cells to test the hypothesis that chromosome healing plays an important role in the cellular response to unrepaired DNA double-strand breaks. Human cell clones have been developed that contain plasmid sequences integrated immediately adjacent to a telomere. The plasmid contains a neo gene for positive selection, an 18 base pair I-Scel endonuclease recognition sequence for introducing specific double-strand breaks, and a Herpes Simplex Virus thymidine kinase gene (HSV-tk) for selection of cells that have lost the end of the chromosome. Rescue of the integrated plasmid and adjacent cellular sequences provides an easy method for the cloning and analysis of the chromosome rearrangements involved. The analysis of spontaneous HSV- tk mutants in the EJ-30 tumor cell line have demonstrated that telomere loss can result in either terminal deletions involving chromosome healing or chromosome fusions which lead to prolonged periods of chromosome instability. In the present proposal we will investigate the mechanism and factors influencing chromosome healing by using this system to study chromosome healing in cell clones that are defective in telomerase, DNA double-strand-break repair and cell cycle regulation/apoptosis. Chromosome healing will also be studied in """"""""normal"""""""" telomerase-immortalized human clones to test the hypothesis that the high rates of chromosome fusion seen in many tumor cells results from air increase in the rate of spontaneous telomere loss and a decrease in the ability to perform chromosome healing. The ability to observe chromosome healing and fusion in the same system will also allow us to test the hypothesis proposed by McClintock that chromosome healing can prevent the chromosome instability resulting from chromosome fusion.
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