MECHANISM OF FORMATION OF TERMINAL DELETIONS

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Terminal deletions are responsible for approximately 10 percent of human genetic diseases resulting from chromosome aberrations. All terminal deletions resulting in genetic diseases that have been analyzed thus far have resulted from new telomeres being added directly onto the ends of broken chromosomes. The restoration of lost telomeres, termed chromosome healing, has been studied extensively in single cell organisms, however, very little is known about this process in mammalian cells. This project involves the use of an assay system developed in mouse embryonic stem (ES) cells to study the mechanism of formation of terminal deletions and the genes involved in chromosome healing. This system utilizes plasmid sequences containing selectable marker genes and an I-SceI endonuclease recognition sequence integrated immediately adjacent to a telomere. The I-SceI recognition sequence is used to specifically introduce double-strand breaks and the selectable marker genes are used to identify cells that have terminal deletions on the end of the marked chromosome. Electroporation of an expression vector containing the gene for the I-SceI endonuclease produced terminal deletions resulting from chromosome healing. The new telomeres are added on with the loss of little or no DNA from the end of the chromosome at sites with short homologies to the telomeric repeat sequences. The new telomeres are much shorter than in the parental cell line, and increase in length with time in culture, consistent with de novo synthesis by telomerase. In the current grant proposal we will utilize this assay system to investigate the mechanism of chromosome healing, the factors that influence this process, and the importance of chromosome healing in the cellular response to DNA double-strand breaks. The formation of terminal deletions by chromosome healing will be studied in mouse cell lines with deficiencies in telomerase, DNA double-strand-break repair and cell cycle regulation to determine the role of these pathways in this process. The ES cell clones will also be used to establish transgenic mice, which will provide a method for studying chromosome healing in a variety of different cell types and for investigating chromosome healing in different genetic backgrounds. Using these approaches we will be able to test the hypothesis that chromosome healing is a cell-type specific mechanism that can influence the cellular response to DNA double-strand breaks. The ability of this system to monitor telomere length and the rate of telomere loss will also provide a means of investigating the role of different genes in maintaining telomere stability.