POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length

Proc Natl Acad Sci U S A. 2019 Nov 19;116(47):23527-23533. doi: 10.1073/pnas.1905381116. Epub 2019 Nov 4.

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.

Keywords: chronic lymphocytic leukemia; footprinting; hydroxyl radical; shelterin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • CRISPR-Cas Systems
  • DNA, Single-Stranded / metabolism*
  • HCT116 Cells
  • Humans
  • Leukemia, Lymphocytic, Chronic, B-Cell / genetics
  • Leukemia, Lymphocytic, Chronic, B-Cell / metabolism
  • Mutation, Missense*
  • Point Mutation*
  • Polymorphism, Single Nucleotide*
  • Protein Binding
  • Recombinant Proteins / metabolism
  • Telomere / metabolism*
  • Telomere Homeostasis / physiology*
  • Telomere-Binding Proteins / genetics
  • Telomere-Binding Proteins / physiology*

Substances

  • ACD protein, human
  • DNA, Single-Stranded
  • POT1 protein, human
  • Recombinant Proteins
  • Telomere-Binding Proteins
  • shelterin, human