Processing of Alternative DNA Structures in the Human Telomere

Telomeres help maintain the overall genomic stability of an organism, and telomeric homeostasis is critical to navigating between aging and cancer. Telomeric dysfunction is implicated as a contributing factor in numerous aging-related diseases, such as diabetes, impaired hematopoeisis, and atherosclerosis. Telomeric homeostasis is maintained by a shelterin complex of six proteins and an array of telomere-associated proteins that interact with the central shelterin complex, such as the Werner syndrome helicase/exonuclease protein (WRN) or p53. Telomeres also have non-canonical DNA structures that are critical towards their function, especially G-quadruplex DNA (G4 DNA) and Holliday Junctions. The former are pseudoknots that form on the G-rich 3' single-stranded tail of the telomere and may block telomere replication and lengthening, when the 3' telomeric tail is exposed in the "open" conformation. We found that the shelterin protein protection of telomeres 1 (POT1) competes with and destabilizes G4 DNA on a physiologically realistic telomeric tail substrate, leading to an equilibrium population of diminished G4 DNA coexisting with POT1. While POT1 is a passive binder of DNA, the destabilizing effect of bound POT1 on pre-existing G4 DNA leads to an emergent, de facto cooperativity in G4 DNA unfolding by POT1.;Holliday Junctions (HJ) form when the telomere is in a "closed" conformation, in which the 3' telomeric tail invades the duplex telomeric DNA, creating a displacement loop (D-loop) and sequestering the end of the chromosome from unwanted DNA damage responses. The D-loop is a homologous recombination intermediate, and we demonstrate that telomere repeat binding factor 2 (TRF2) is necessary to protect HJ DNA from unwanted WRN helicase activity, which has been thought to branch migrate the D-loop into a target for Holliday Junction cleaving enzymes, causing sudden telomere shortening. TRF2 also protects HJ DNA from Holliday Junction cleaving enzymes, and the cleavage protection is due largely to the HJ-binding B-domain on TRF2. In contrast, we found that TRF2-mediated protection against WRN depends on both the B domain and the telomeric-repeat binding Myb domain. We have therefore discovered an overlapping but distinct role for TRF2 in maintaining telomeric stability.;Our work has elucidated novel structural and functional data on the modulation of non-canonical DNA structures by shelterin and telomere-associated proteins. These data help us elucidate the mechanisms underlying cellular and animal models for telomere instability and aging.