Date of Award
Doctor of Philosophy (PhD)
de Lange Laboratory
Phosphatidyl inositol 3-kinase-like kinases (PIKKs) have a well documented function at yeast telomeres. Although several lines of evidence suggest that members of the PIKK family also play a role in vertebrate telomere biology, little is known about their specific functions. We report that the human shelterin component, TRF2, overexpressed in 293T cells, is phosphorylated on serine 368 (S368) in a caffeine and wortmannin sensitive manner. Phosphorylation is induced by hydroxyurea (HU) and ultraviolet (UV) radiation but not Ionizing Radiation (IR). Knockdown studies indicate that ATR is the primary kinase responsible for TRF2 S368 phosphorylation, while the mTOR kinase is implicated as a negative regulator of the phosphorylation event. In order to study the function of phosphorylation of TRF2 on S368, we replaced endogenous TRF2 in MEFs with TRF2 containing mutated S368 (S366 in mouse). TRF2 S366 mutants fulfilled the major telomeric protective functions of TRF2 and prevented cell cycle arrest, formation of telomere dysfunction induced foci, overhang loss, telomere fusions, and telomere sister chromatid exchanges. TRF2 S368 mutants were able to influence telomere length homeostasis in certain settings in both human and mouse cells. In mouse cells, expression of TRF2 containing a phosphomimetic mutation at position 366 (S366E) caused dramatic telomere lengthening; in human cells, overexpression of S368 mutants did not reproduce the rapid telomere shortening caused by overexpression of wildtype TRF2, indicating that these mutants may not be able to promote t-loop HR. Telomeres exhibited an unusual sensitivity to aphidicolin: 25-28% of 53BP1 foci induced by aphidicolin treatment colocalized with telomeric DNA. Expression of TRF2 S366E improved survival of MEFs after aphidicolin treatment. Cells expressing TRF2 S366E also exhibited fewer chromatid breaks and 53BP1 foci after aphidicolin treatment, and the percentage of 53BP1 foci which colocalized with telomeres was decreased by a small but significant amount. Finally, we show that a phosphomimetic mutation at position 368 weakens the interaction between TRF2 and TIN2, suggesting that phosphorylation of TRF2 on S368 may relieve replication stress at the telomere by modifying the conformation of shelterin to allow the replication fork to more easily pass.
Hoke, Kristina, "A Novel Phosphorylation Site in the Telomeric Protein TRF2 is Regulated by the ATR Kinase and Plays a Role in Relieving Replication Stress at the Telomere" (2008). Student Theses and Dissertations. 198.