Tethering Telomeric Double- and Single-stranded DNA-binding Proteins Inhibits Telomere Elongation

Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3' single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening

Signs & Wonders, Research and Public Art Project

Using the notion of the pedlar as a carrier of multiple viewpoints, Katie Etheridge, Simon Persighetti & Phil Smith peddled ideas through trade, exchange and conversations. Through located performances and walks along city streets, in the marketplace and down country tracks they distributed specially commissioned tokens and wondrous pamphlets. Through extensive research the works responded to the story and surroundings of the infamous Lancashire Witch Trials (1612) by examining the meanings we invest in objects and reacting to the evocative local landscape. Commissioned by Green Close and LICA, funded by Arts Council England., Following this public project, we reworked the performances and outcomes for a gallery setting in a process that accorded with Live at LICA interest in artists working across disciplines and contexts. Originally manifesting as performance walks and site-specific live art installations, the reconfigured work created new experiences, questions and exchanges in TRANSORMED: Double Bill, Exhibition October 2013., This project extends my research by increased involvement in generating outputs that lead to muti-media and legacy based records of the practice. Different modes of dissemination and participation have been explored through muti-modal approaches to investigation of landscape and history in a particular context

Distinct Functions of POT1 at Telomeres ▿ †

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions—ssDNA, TPP1, or TRF2 binding—was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres