Title: Single-molecule Study of TRF2 Mediated DNA Compaction using Physiologically Relevant Long Telomeric DNA

Abstract: Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes by shielding them from activating DNA damage response (DDR) and DNA double-strand break (DSB) repair. Human telomeres contain ~2 to 20 kb of TTAGGG repeats and a G-rich 3’ overhang. A specialized six-protein shelterin complex, including TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, binds and prevents telomeres from being falsely recognized as double-strand breaks, and regulates telomerase and DNA repair protein access. Extensive telomere shortening or dramatic telomere loss due to DNA damage causes chromosome ends to be recognized as DNA breaks, which triggers cell senescence and aging-related pathologies. Human telomeric DNA is arranged into T-loops, in which the 3’ single-stranded overhang invades the upstream double-stranded region. A recent study suggests that T-loops at telomeres function as conformational switches that regulate ATM activation. Further, it was also shown that TRF2 mediated DNA compaction drives T-loop formation. While it has been shown that TRF2 protein is required for the T-loop formation, essential information regarding the dynamics of its formation still remains unknown. Moreover, most of the previous studies were done using short telomeric DNA sequences ~ 20 kb. To elucidate the dynamics of TRF2 mediated DNA compaction and T-loop formation, we used single-molecule methods which frequently require long DNA substrates. We established a new method for extracting and purifying long telomeric DNA from mouse liver cells using Region-specific Extraction (RSE).