Tracking telomerase recruitment to model telomere DNA ends using real-time single-molecule FRET.

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Telomere homeostasis is critical to human health as demonstrated by telomerase dysfunction diseases such as dyskeratosis congenita, aplastic anemia and pulmonary fibrosis. One essential step for the function of telomerase, the enzyme that maintains telomere length, is its recruitment to the telomere and subsequent engagement of the 3’ single-stranded chromosome end. Human telomeric DNA consists of tandemly arrayed hexameric repeats bound by telomere-specific proteins and nucleosomes. Previous studies have shown that telomerase interaction with the telomeric heterodimeric protein complex, POT1-TPP1, is essential for telomerase activity in vivo. However, the precise molecular mechanism of telomerase recruitment, including the role of POT1-TPP1 in facilitating successful engagement of the 3’ single-stranded DNA, remains elusive. Furthermore, the single-stranded telomere binding protein POT1 sequesters the 3’ ends of telomeres to prevent recognition by DNA repair machinery, but how the 3’ end is handed off from POT1 to telomerase is unknown. Here, we present an in vitro biophysical assay that directly tracks telomerase dynamics with individual telomeric DNA molecules during the recruitment and 3’ end binding process. This assay permits analysis of telomerase recruitment and 3’ end binding as a function of DNA length as well as in the presence of wild-type or mutant forms of POT1 and/or TPP1 proteins. Ultimately, we aim to critically evaluate specific physical models for how telomerase solves the ‘telomere end search problem’.