Poster abstracts
Poster number 169 submitted by Mengyuan Xu
Investigation of multiple binding properties of POT1-TPP1 proteins in telomere ssDNA protection
Mengyuan Xu (Department of Pharmacology, Case Western Reserve University), Janna Kiselar (Center for Proteomics and Bioinformatics, Case Western Reserve University), Derek Taylor (Department of Pharmacology, Case Western Reserve University)
Abstract:
Telomeres are specialized nucleoprotein complexes that compose the very ends of linear chromosomes. Telomeres are needed to prevent the illicit induction of DNA damage response and end-to- end chromosome fusions. Telomere DNA consists of thousands of bases of double-strand DNA (dsDNA) before ending with a single strand DNA (ssDNA) overhang at the 3’ end that is 50-200 nucleotides in length. Both dsDNA and ssDNA of the telomere is characterized by a repeating hexameric sequence (TTAGGG in human). POT1 (Protection of Telomeres 1) is a telomere end-binding protein that binds specifically to telomere ssDNA. Together with TPP1 (Telomere Protection Protein 1), POT1 forms a heterodimer to both positively and negatively regulate telomere length. For example, POT1-TPP1 binds with nanomolar affinity to telomere ssDNA to protect it from nucleolytic attack. However, the POT1-TPP1 complex also actively recruits telomerase, the enzyme responsible for telomere extension, to enhance its activity. Most information regarding POT1-TPP1 and telomerase has been performed using short ssDNA oligonucleotides as a substrate. However, the physiological length of G-rich telomeric ssDNA is prone to form stable intermolecular structures called G-quadruplexes, which might additionally play a role in telomere maintenance. Our lab has shown that POT1-TPP1 proteins can help to disrupt G-quadruplex structures by coating long, telomere ssDNA with multiple proteins. We have used hydroxyl radiolytic footprinting coupled with mass spectrometry to better understand the protein-protein interactions that are involved in assembling long tracts of telomere ssDNA coated with multiple POT1-TPP1 proteins. Our data highlight key residues, including H266, an amino acid that is mutated (H266L) in patients suffering from chronic lymphocytic leukemia. We are mutating the H266 in recombinant POT1 protein and will quantitatively determine the consequences it imposes in disrupting G-quadruplex structures and in TPP1- and ssDNA interactions. This information will provide important knowledge for understanding how telomere proteins regulate telomere length and how cancer mutations disrupt these important processes.
Keywords: Telomere, POT1, Footprinting