Abstract
Telomeres are specialized nucleoprotein structures found at the end of eukaryotic chromosomes. The telomere DNA in humans is composed of the sequence "5'-TTAGGG-3'" tandemly repeated in a stretch of 5-30kb of double stranded DNA. TTAGGG Repeat Factor 2 (TRF2) is a telomere DNA binding protein that has a critical role in telomere end protection. The current model for telomere protection by TRF2 is through its ability to remodel telomeres into looped higher-order structures, called the t-loop, which sequesters the end from DNA damage sensors. Since telomeres are known to be comprised of nucleosomal chromatin, it is important to determine how TRF2 binds to and affects the structure of nucleosomal arrays. The ability of TRF2 to bind to unusual DNA structures such as the t-loop and the single stranded/double (ss/ds) stranded telomere DNA junction may facilitate its binding to DNA in the form of nucleosomal arrays and promote higher-order chromatin structures. In this study, we have reconstituted a 2kb DNA fragment containing 550bp of telomere DNA into nucleosomal arrays and tested the binding of full-lengthTRF2 and four truncation mutants to telomeric nucleosomal arrays. Our data indicates that TRF2 and its truncation mutants bind to telomere nucleosomal arrays as well as it binds to telomere DNA. We used a novel electrophoretic technique, Analytical Agarose Gel Electrophoresis (AAGE), to measure changes in surface charge density, hydrodynamic radius, and conformational flexibility of DNA and nucleosomal arrays upon protein binding. Our results indicate that the C-terminal DNA binding Myb/SANT domain of TRF2 might be rearranging nucleosomal structure through either nucleosome sliding, unwrapping, or changing the arrangement of the linker DNA, while the N-terminal basic DNA binding region is causing nucleosomal arrays compaction. Instead of significant compaction, histone-free DNA undergoes DNA condensation and self-association. This activity is observed with the full-length protein and all regions of the protein, with the exception of TRF2-DBD, participate in the process. We speculate that the ability of TRF2-DBD to rearrange nucleosomal structure and N-terminal basic region to cause nucleosomal fiber compaction may allow TRF2 to promote t-loop formation in the context of chromatin. We propose that TRF2, possessing all the features, has a new role at telomeres as a chromatin architectural protein.