Date of Award
de Lange Laboratory
telomere function, human Tel2, DNA damage proteins, TRF2
Tel2 influences telomere length in S. cerevisiae and DNA-damage signaling in C. elegans. We found that endogenous human Tel2 (hTel2) localized in a diffuse, granular patter to the nuclei of human cells where it was enriched in PML nuclear bodies, but telomeres. Surprisingly, hTel2 also localized to centrosomes. Although hTe!2 did not detectably interact with telomeric proteins or telomeric chromatin, the overexpression untagged hTel2 resulted in the slow elongation of telomeres in HTC75 and SK-HEP-1 cells. Furthermore, hTel2 overexpression suppressed the accumulation of cells in G2 after ionizing radiation. The depletion of hTel2 in HeLa cells by RNA interference resulted in apoptotic cell death. Furthermore, hTel2 depletion resulted in the accumulation of aberrant mitotic cells that possessed disorganized metaphase chromosomes. The metaphase chromosomes of hTel2 depleted cells were curly suggesting that hTel2 played a role in chromosome compaction. A role for hTel2 in establishing chromatin structure during S-phase could explain its multiple localizations and phenotypes. TRF2 protects telomeres from non-homologous end joining (NHEJ), possibly through the formation of a t-loop structure. A mutant allele of TRF2, TRF2AB, retained the ability to suppress NHEJ but induced dramatic deletions of telomeric DNA. This catastrophic telomere shortening was accompanied by a DNA damage response and senescence. Using FISH and CO-FISH, TRF2AB was found to induce rapid, stochastic deletions that preferentially affected leading strand telomeres (parental C-strand) DNA replication. Genetic analyses demonstrated that TRF2AB-induced deletions required Nbsl, a component of the Mrell complex, and XRCC3, a component of the RAD51C/XRCC3 Holliday junction (HJ) resolvase, suggesting the involvement of homologous recombation (HR) in the deletions. Consistent with a role for HR, TRF2AB induced the formation of t-loop-sized telomeric circles. These telomeric circles were detected in unperturbed cells suggesting that t-loop deletion by H R could explain the stochastic nature of telomere shortening and senescence in somatic cells. Furthermore, telomerase-negative ALT cells had abundant telomeric circles, suggesting that recombination-mediated elongation of ALT telomeres could involve telomeric circles. These findings show that TRF2 regulates both NHEJ and HR at mammalian telomeres and that HR at telomeres influences the integrity and dynamics of mammalian telomeres.
Wang, Richard Chih-Chien, "Functional analyses of hTel2 and TRF2 deltaB: Insights Into the Role of a New DNA Damage Pathway and Homologous Recombination in Mammalian Telomere Function" (2004). Student Theses and Dissertations. 48.