ABSTRACT
Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein-protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.
Subject(s)
DNA, Single-Stranded/metabolism , Mutation, Missense , Point Mutation , Polymorphism, Single Nucleotide , Telomere Homeostasis/physiology , Telomere-Binding Proteins/physiology , Telomere/metabolism , Amino Acid Substitution , CRISPR-Cas Systems , HCT116 Cells , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Protein Binding , Recombinant Proteins/metabolism , Shelterin Complex , Telomere-Binding Proteins/geneticsABSTRACT
The identification of telomerase-mediated telomeric misincorporation of 5-fluoro-2'-deoxyuridine (5-FdU) uncovered a unique approach to telomeric-based therapeutics. Additionally, identification of such a mechanism supports the utility of telomere maintenance mechanisms in guiding therapeutic decisions. Presented here is a unique perspective of 5-FdU and its clinical implications as a telomeric-based therapeutic.
ABSTRACT
Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches for inhibiting its activity have been clinically ineffective. As opposed to inhibiting telomerase, we use its activity to selectively promote cytotoxicity in cancer cells. We show that several nucleotide analogs, including 5-fluoro-2'-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by telomerase into a telomere DNA product. Administration of 5-FdU results in an increased number of telomere-induced foci, impedes binding of telomere proteins, activates the ATR-related DNA-damage response, and promotes cell death in a telomerase-dependent manner. Collectively, our data indicate that telomerase activity can be exploited as a putative anti-cancer strategy.