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Rif1, DNA damage response, Rap1, human telomeres


Rifl, the ortholog of the yeast Rapl interacting factor 1, a telomere length regulator was identified. Immunofluorescence did not reveal an association of human Rifl with telomeres. Preliminary co-immunoprecipitation with known telomeric proteins and chromatin immunoprecipitation failed to show an interaction of human Rifl with telomeric protein complexes or with telomeric DNA. Unexpectedly, Rifl responded to ionizing radiation (IR), UV light, and clastogens, forming foci that co-localized with other DNA damage response factors such as 53BP1, ATM, BRCA1, Chkl, Nbsl, and Rad 17. Furthermore, Rifl localized to uncapped telomeres, as do other DNA damage response factors. Among DNA damage response proteins, Rifl showed a unique dependence on the ATM kinase. Whereas inhibition of ATR signaling did not suppress the Rifl response, ATM deficient cells treated with IR or UV lacked Rifl foci even after prolonged incubation or high radiation dose. Therefore, IR-induced Rifl foci constitute an assay for ATM status. The Rifl response also depended on the presence of 53BP1 but was not affected by reduced function of BRCA1, Chk2, Nbsl, and Mrell. RNAimediated Rifl inhibition resulted in increased radiosensitivity, indicating that Rifl contributes to ATM-mediated protection against exposure to ionizing radiation. In order to more fully understand telomere length regulation in mammalian cells and the evolutionary history of telomere-binding complexes, a search for novel hRapl -binding proteins and studies of the Rapl telomere complex at human telomeres were performed. Both genetic and biochemical methods were used in order to better understand the role of the Rapl complex, consisting of hRapl, TRF2, and the Mrell complex, in human telomere biology. A yeast two-hybrid screen was carried out using hRapl as a bait and two genes were identified. The first gene, called candidate 144 (C144), encodes a novel zinc-finger containing protein. Immunofluorescence and immunoprecipitation failed to reveal an association of C144 with telomeres. Deletion of the open-reading frame YDL175c in S. cerevisiae, the ortholog of C144, did not result in detectable changes in telomere length. The second gene, called FLASH, was previously identified to play a role as an adaptor in ligand-mediated apoptosis. In immunofluorescence experiments, FLASH did not co-localize with known telomeric proteins.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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