Telomere protection against oxidative stress

Telomeres are nucleoprotein structures at the ends of linear chromosomes, being essential for the maintenance of genomic integrity. Telomeres have a unique structure which distinguishes chromosome termini from DNA damage sites. Shelterin complexes are the most abundant proteins associated with telomeric DNA. They prevent intact telomeres from being recognized as double-stranded DNA breaks, thus preventing the inappropriate activation of DNA damage signaling. The shelterin protein complex consists of 6 subunits: TRF1, TRF2, TPP1, POT1, Rap1 and TIN2. In addition to shelterin, a very large number of less well-characterized proteins have been detected at telomeres. Despite their heterochromatin features, telomeres are transcribed into the long noncoding RNA TERRA. Telomeric DNA is particularly susceptible to oxidative damage due to the repression of some DNA repair pathways at intact telomeres, triple-G-containing 5'-TTAGGG-3' repeats, which are highly prone to oxidation, and 3' overhangs, which cannot be repaired by the base excision repair pathway due to the lack of a complementary strand. Reactive oxygen species (ROS) trigger telomeric DNA strand breaks and nucleotide oxidation, causing telomerase inhibition, telomere shortening and cellular senescence. Several DNA repair factors and antioxidant enzymes have been reported to protect telomeres from oxidative damage. However, it is not well understood how telomeres counteract ROS and how telomeric oxidative lesions are repaired. In my thesis, I aimed at elucidating the molecular mechanisms underlying telomere protection against oxidative damage by studying the changes in telomeric chromatin composition and structure upon oxidative stress induction. Using menadione, which damages mitochondria and mimics endogenous oxidative stress, we successfully induced damage at telomeres indicated by the accumulation of single-stranded telomeric DNA breaks. We observed elevated levels of telomeric RNA:DNA hybrids (R-loops) upon menadione treatment, coinciding with upregulation of TERRA transcription. The elevation in telomeric R-loops was not only mediated by increased telomeric transcription but also due to an increase in TERRA recruitment to damaged telomeres. In addition, we discovered by 2D gel electrophoresis and Electron Microscopy the accumulation of internal loop structures at damaged telomeres. Interestingly, our findings show that the telomeric protein TRF1 dissociates from telomeres upon oxidative damage possibly to render the damaged telomeres accessible to damage signaling and repair. Several mechanisms for TRF1 dissociation can be envisaged. Oxidative lesions in telomeric DNA may reduce the binding affinity of TRF1. In addition, as TRF1 protein cannot bind to R-loops, the increase in R-loop levels at damaged telomeres may prevent TRF1 from binding. Also, upregulated TERRA levels may sequester nucleoplasmic TRF1 protein, thereby reducing the concentration of telomere-binding competent TRF1. We also examined the changes in the whole telomeric chromatin upon oxidative stress implementing a quantitative telomeric chromatin isolation protocol (QTIP) combined with Mass Spectrometry analysis. We detected the recruitment of DNA repair proteins, antioxidant enzymes, chromatin remodelers and PTM enzymes. Overall, our results uncover that oxidative damage induces dramatic remodeling of the telomeric chromatin composition to orchestrate protection, repair and DNA damage signaling pathways.