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

Significance Telomere length homeostasis is an important mechanism for maintaining genomic stability. Telomere length is regulated by numerous events that include protein–DNA interactions, the length and structure of telomere DNA, and recruitment of telomerase. Here we used hydroxyl radical footprinting to identify environmental changes in the telomere end-binding heterodimer, POT1-TPP1, as a function of telomere length. Our data identified a specific residue (histidine 266) of the POT1 protein that reports differences in solvent accessibility as a function of telomere DNA length. We further show that the chronic lymphocytic leukemia-related H266L POT1 mutation disrupts the ability of POT1-TPP1 to negatively regulate telomerase activity in vitro and in cancer cells.
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.