Date of Award
Doctor of Philosophy (PhD)
de Lange Laboratory
Telomere function is carried out by protein factors that either bind directly to telomeric DNA or that are recruited there by DNA binding activities. Thus, telomeric DNA binding activities are central to telomere function. Two types of vertebrate DNA binding activities are characterized in this thesis: proteins that can potentially bind to the 3' overhang of TTAGGG repeats and proteins that bind to duplex telomeric DNA. We have identified, characterized and partially purified an activity from chicken erythrocytes (ATEF) that has the biochemical properties expected for a telomere terminus factor. In addition, we have analyzed the DNA binding properties of the nonspecific DNA-end binding factor, human Ku, which is involved in DNA end joining. We show that long G-G folded overhangs do not prevent Ku binding to telomeric DNA in vitro, indicating that, similar to the situation in yeast, Ku could potentially bind to chromosome ends. Most of the mammalian telomeric DNA is in double-stranded form and is bound in vivo by two related factors, TRF1 and TRF2, which both contain one Myb-type repeat. We have conducted a detailed analysis of the DNA binding mode of TRF1 and shown that it binds DNA as a dimer by contacting a bipartite DNA site with each Myb domain. The relative orientation and spacing of the two sites does not affect binding. We propose a model for TRF1 binding that predicts the existence of a flexible region, located between the highly conserved dimerization and DNA binding domains, that would allow free relative rotation of the Myb domains. This is a novel binding mechanism that, although different from the one displayed by the yeast telomeric protein Rap1 p, could be of relevance to other telomeric proteins, such as the human TRF2 and the fission yeast Taz1 p, that share a similar domain organization. Finally, we have shown that TRF1 or TRF2 bound on telomerase substrates in vitro do not affect the activity of the enzyme.
Bianchi, Alessandro, "Characterization of DNA Binding Activities at Vertebrate Telomeres" (1999). Student Theses and Dissertations. 440.