Student Theses and Dissertations

Date of Award

2001

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

de Lange Laboratory

Abstract

TTAGGG Repeat Binding Factor 2 (TRF2) is a ubiquitous human telomeric protein present at all telomeres throughout the cell cycle. TRF2 has been proposed remodel telomeres into large lasso-like structures named t-loops. Removal of TRF2 from telomeres by expression of a dominant negative allele of TRF2 (TRF2ABAM) led dissolution of these structures and appearance of end-to-end fusions visible in metaphase and anaphase cells. Fusion formation was accompanied by the loss of Goverhang, but not by the loss of double stranded telomeric DNA, which can be documented to persist at the sites of fusions. Fusions were covalent and their formation was dependent on the nonhomologous end-joining (NHEJ) pathway as evidenced by the failure to form fusions in cells lacking Ligase IV. Furthermore, the fusions initiated genomic instability. Expression of TRF2ABAM in primary human cells resulted in induction of senescence. The arrested cells exhibited elevated levels of p53, stabilization of was accompanied by induction of its downstream effectors p21 and Bax. The Rb pathway was also affected, with pRb becoming hypophosphorylated. The importance of both the p53 and the Rb pathways was further evident from the fact that the expression of SV40 Tag alone bypassed the growth arrest. Separate elimination of p53 or Rb function could not do so, and the activity of the ATM PI3 kinase was not necessary the arrest. Expression of a different truncation allele of TRF2, TRF2AB also resulted in senescence but the growth arrest was accompanied by rapid loss of telomeric DNA. In addition, this allele induced an unanticipated chromosome breakage phenotype. Long-term overexpression of full length T R F 2 resulted in the gradual shortening of telomeres, suggesting that TRF2 is a regulator of telomere maintenance. Although it is clear that TRF2 acts through positively regulating the shortening activities at telomeres, it is unknown at this time whether it influences the telomerase pathway. Besides being present at telomeres, TRF2 also appears to be localized to centrosomes. The functional consequences of such localization are not known at this time; however, it is shown that telomere dysfunction can result in genome reduplication.

Comments

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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