Despite his deeds, Garadon never sought promotion, nor was it ever offered to him. Fate had cursed the young Garadon with a straightforward and stubborn nature, ill-matched to the glittering societal circles his kin frequented.Дuring his first solar decade of service, Garadon earned commendation after commendation. His wealthy family were only too glad to see him depart. Garadon was recruited to the Imperial Fists from the orbitals of Callisto, a moon of Jupiter. Tested on a thousand worlds, he is a master of the battlefield, and an unyielding bulwark against those who would oppose Mankind. He is an unstoppable warrior in whom the lessons of the Primarch Rogal Dorn have been distilled to their most punishing form. Tor Garadon is a Captain of the Imperial Fists Chapter and the longest-serving commander of the Imperial Fists 3rd Company, the 'Sentinels of Terra.' The structure of these elements and their interaction with known DNA repair and signaling factors will be examined using a combination of biochemical and electron microscopic approaches.Tor Garadon, Captain of the Imperial Fists 3rd Company, during the Era Indomitus. Key elements in this damage sensor are the t-loop, the t-loop junction, and the newly recognized telomeric R-loops. The application of combined quantitative EM analysis with biochemical assays will be applied throughout. Using our large t-loop substrates generated by transcription and ones containing R-loops, we will examine the action of these proteins together with GEN1, Mus81-EME-1 and other nucleases on these highly relevant biological substrates, heretofore not available. BLM, WRN, and RTEL1 helicases act at the telomere and RTEL1 resolves t-loops in vivo. If so, this would mimic the ALT cancer phenotype and provide a new vision for the role of t-loops. To test this, we will determine whether t-loop junctions generated by transcription can initiate replicative telomere extension. Our guiding hypothesis is that t-loops evolved in part to provide a means to self-prime telomere extension and this may have preceded the evolution of telomerase. Employing our new affinity method for purifying telomeric DNA we will isolate telomeric DNA containing R-loops generated in vivo and map the distribution and size of the R-loops. We are poised to ask whether Rad51/52 alone or together with hnRNPA1 will insert TERRA RNA in trans into telomeric DNA to form telomeric R-loops. The nature of the highly stable t-loop junction which also contains RNA will be examined using biochemical probes. Approaches will include EM analysis combined with single molecule magnetic tweezers studies. We can now produce telomeric DNA >10 kb in length and we will apply biophysical and biochemical tools to further understand the structure of large telomeric single stranded (ss) DNA and telomeric RNA (TERRA).

These discoveries provide a wealth of new tools and approaches toward understanding telomere structure and its function as a damage sensor. R-loops may be the single greatest source of DNA damage at telomeres. We observed that telomeric R-loops are unusually stable and bind p53 tightly. R-loops can lead to genomic instability, gross chromosomal rearrangements, and cancer. Transcription also leaves behind highly stable telomeric R-loops. Transcription generates t-loops at high efficiency in model templates including ones the size of human telomeres. In our past grant cycle, we made a paradigm-shifting finding linking t-loop formation to telomere transcription. In addition to the t-loop, the telomere is rich in complex structures including the t-loop junction, the single strand (ss) overhang, and the newly discovered telomeric R-loops- all bound by the shelterin proteins. In 1999 we demonstrated that mammalian telomeres are arranged into large loops (t-loops) subsequently confirmed in numerous systems. Little is known about how DNA damage triggers structural changes in the telomere to activate damage signaling pathways. This is accomplished via damage signaling pathways which respond to the presence of damage in the telomeric DNA. Containing only ~1/3000th of the total DNA in a human cell, they shelter us from oncogenic progression, monitor the number of cell divisions and respond to insult from environmental agents including UV, toxic metals, and organic pollutants. Telomeres provide a sensitive sentinel of DNA damage.

Sentinels of terra formation