Abstract 2336 Human shelterin complex prefers binding to displacement loop over linear telomeric DNA

Telomeres are protective DNA-protein structures capping the end of linear mammalian chromosomes. Human telomeric DNA is composed of the repetitive hexameric DNA sequence, TTAGGG, which spans several thousands of repeats at each chromosome end. These telomeric double-stranded DNA (dsDNA) regions end in a 3′ overhang which has the same repetitive sequence. This overhang can loop back and strand invade an internal region of the telomeric dsDNA to form a telomeric loop (T-loop), of which the base of the T-loop is a ssDNA displacement loop (D-loop). The telomeric D-loop is thought to be a highly reclusive structure to telomerase, the ribonucleoprotein enzyme that extends the telomeric overhang. Presumably, the annealing of the overhang into the telomeric dsDNA prevents telomerase from accessing the 3′ overhang. The regulation of T-loop formation and stability is mediated by a major telomeric protein complex known as shelterin. How shelterin engages a telomeric D-loop and whether telomerase can utilize a telomeric overhang that is assembled within a telomeric D-loop are currently unknown. To gain further insights into these questions, we studied human shelterin binding affinity to telomeric D-loops using a reconstituted biochemical system and fluorescence polarization assays. We found that the human shelterin binds tightly to a telomeric D-loop and prefers this DNA construct over telomeric ss- or dsDNA. Direct enzyme assays using reconstituted human telomerase and telomeric D-loop suggest telomeric D-loop is a poor substrate for telomerase. Collectively, our biochemical studies demonstrate that the telomeric D-loop is a natural DNA structure for tight binding by shelterin and a challenging substrate for telomerase. Thank you to the Mary Shine Peterson Biochemistry Scholarship, the Hilldale Undergraduate Research Fellowship and the NIH for their support in making this project possible.