A major function of telomeres is to protect the ends of chromosomes and prevent chromosome fusion. Chromosome fusion can result in chromosome instability through breakage/fusion/bridge (B/F/B) cycles, which occur when chromosomes repeatedly break and fuse with each cell division. B/F/B cycles can be prevented or terminated by the addition of telomeres to the ends of broken chromosomes, which results in the formation of terminal deletions. In Tetrahymena and yeast, telomeres are added on to the ends of broken chromosomes by telomerase, termed chromosome healing. Chromosome rearrangements associated with telomere loss and B/F/B cycles have been found in a variety of human genetic diseases, and are thought to play an important role in the chromosome instability associated with cancer. We have established an assay that utilizes selectable marker genes located adjacent to a telomere to monitor the consequences of telomere loss in mammalian cells. Telomere loss is induced through the introduction of a double-strand break at an I-Scel site adjacent to the telomere following the expression of the I-Scel endonuclease. Most mouse embryonic stem cells that lose a telomere have telomeres added directly on at the I-Scel site; however, sister chromatid fusion and chromosome instability involving B/F/B cycles is observed in cells that do not add a telomere on to the end of the broken chromosome. The present proposal will use this system to address the consequences of telomere loss in mammalian cells. Specific Aim 1 will investigate the role of telomerase and the Pif1 helicase in chromosome healing. Pif1 mutants in yeast have a 600-fold higher incidence of chromosome healing, and as a result, Pif1 has been proposed to negatively regulate chromosome healing to prevent terminal deletions. These experiments will test the hypotheses that telomerase and Pifl are important in chromosome healing in mammalian cells and that chromosome healing prevents B/F/B cycles. Specific Aim 2 will utilize cell lines containing mutations in DNA-PKcs or NBS1, to address the role of nonhomologous end joining and the Mre11/Rad50/Nbs1 complex in telomere maintenance, chromosome healing and chromosome fusion. Specific Aim 3 will test the hypothesis that chromosome instability due to telomere loss promotes carcinogenesis by expressing I-Scel in vivo and monitoring preneoplastic changes resulting from the amplification/rearrangement of the c-Myc gene on the chromosome 15 containing a telomeric I-Scel site.