Telomeres, the nucleoprotein structures at the ends of eukaryotic chromosomes, are maintained and protected by a suite of highly specialized proteins. These proteins are responsible for chromosome maintenance and play important roles in cancer and aging. A critical activity at telomeres is the proper capping and processing of a conserved GT-rich single-stranded overhang. Loss of the ability to discriminate this natural end from damaged DNA is catastrophic, leading to severe genomic instability. Access of the replicative enzyme telomerase to the overhang is tightly controlled, as this activity regulates the proliferative potential of the cell. One of the key hallmarks of cancer cells is their unlimited proliferative potential, which is, in turn, dependent on telomere maintenance and length regulation. Telomerase is activated in approximately 85% of tumor cells, but not in neighboring non-tumorous cells. However, premature telomere shortening can also lead to a variety of serious disease states. The goal of our work is to understand how modulation and control of the protein and DNA structure at the end of the telomeres mediates these essential activities. Our project focuses on the structures and activities of key proteins found at the telomeric single stranded overhang in the model organism budding yeast and in higher eukaryotes. In the budding yeast, the essential protein Cdc13 binds the single stranded overhang, protects the ends of chromosomes from degradation, and regulates the activity of telomerase. Each of these activities is critical for maintaining telomere integrity and function. The knowledge gained from the study of Cdc 13 will accelerate our ability to understand telomere maintenance in humans and other higher eukaryotes. This project will extend our knowledge of Cdc13 function, by examining the elements of molecular recognition involved in the interaction of Cdc13 with telomeric DNA using biochemical, biophysical, and structural methods. In addition, this project will investigate the structural and biochemical attributes of an important regulatory domain within Cdc 13 that plays a crucial role in telomere length regulation. Finally, this project will examine telomere structure in higher eukaryotes, where the recently discovered Pot1 proteins bind the single-stranded overhang and perform end protection and telomerase recruitment activities. This work will contribute to a molecular level understanding of telomere end protection and length regulation, and will provide the structural and functional knowledge needed to help design new therapeutics for cancer and aging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059414-07
Application #
6911583
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1999-05-01
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
7
Fiscal Year
2005
Total Cost
$266,318
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Glustrom, Leslie W; Lyon, Kenneth R; Paschini, Margherita et al. (2018) Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives function. Proc Natl Acad Sci U S A 115:10315-10320
Lloyd, Neil R; Wuttke, Deborah S (2018) Discrimination against RNA Backbones by a ssDNA Binding Protein. Structure 26:722-733.e2
Hom, Robert A; Wuttke, Deborah S (2017) Human CST Prefers G-Rich but Not Necessarily Telomeric Sequences. Biochemistry 56:4210-4218
Lloyd, Neil R; Dickey, Thayne H; Hom, Robert A et al. (2016) Tying up the Ends: Plasticity in the Recognition of Single-Stranded DNA at Telomeres. Biochemistry 55:5326-40
Pinzaru, Alexandra M; Hom, Robert A; Beal, Angela et al. (2016) Telomere Replication Stress Induced by POT1 Inactivation Accelerates Tumorigenesis. Cell Rep 15:2170-2184
Lewis, Karen A; Pfaff, Danielle A; Earley, Jennifer N et al. (2014) The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain. Nucleic Acids Res 42:475-84
Lloyd, Neil R; Wuttke, Deborah S (2014) Less is more: structures of difficult targets with minimal constraints. Structure 22:1223-1224
Dickey, Thayne H; Wuttke, Deborah S (2014) The telomeric protein Pot1 from Schizosaccharomyces pombe binds ssDNA in two modes with differing 3' end availability. Nucleic Acids Res 42:9656-65
Rao, Timsi; Lubin, Johnathan W; Armstrong, Geoffrey S et al. (2014) Structure of Est3 reveals a bimodal surface with differential roles in telomere replication. Proc Natl Acad Sci U S A 111:214-8
Altschuler, Sarah E; Lewis, Karen A; Wuttke, Deborah S (2013) Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions. J Nucleic Acids Investig 4:19-28

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