Molecular characterization of the interplay between DNA-PK and TRF2 in telomere protection

Abstract: The genetic material of mammalian cells is organized as linear chromosomes inside their nucleus. In order to preserve genomic stability, mammalian cells have developed several mechanisms to detect and repair DNA lesions that can occasionally occur in the genome. Nevertheless, in certain circumstances, these pathways can promote aberrant repair that can result in genomic instability and tumorigenesis. One such case are the ends of linear chromosomes, which are particularly susceptible since they can be mistakenly recognized as DNA lesions by the DNA repair machinery, which seeks to repair the damage by joining the two DNA ends together and thus causing rearrangements and genome instability. Therefore, the linear ends of chromosomes need to be protected from being sensed as sites of damage, and this is promoted by the presence of specialized structures known as telomeres, with the aim of preventing determinantal consequences of having exposed DNA ends. Telomeres are formed by a repetitive DNA sequence that is bound by a protein complex named shelterin, which forms a protective structure at the end of chromosomes. Among the shelterin subunits, TRF2 plays a crucial role. Other factors that have been found essential for the protection of telomeres are Apollo and DNA-PK proteins which, strikingly, are also involved in DNA repair mechanisms.Understanding the underlying mechanism of telomere protection and the interplay between factors involved is of relevance since defects in telomere protection activate aberrant repair, which is associated with genome instability, a prime mechanism shaping cancer.In Paper I, we focused on how Apollo and the catalytic subunit of DNA-PK (DNA-PKcs) cooperate in preserving telomere homeostasis, and we shed light on the mechanistic function of DNA-PKcs in granting access to Apollo to the telomeric ends. We found that this function requires the kinase activity of DNA-PKcs to promote autophosphorylation, and the binding of Apollo to DNA-PKcs for optimal positioning at the DNA ends. This study shows an analogous mechanism of function of DNA-PK at sites of DNA lesions and at telomeres.In Paper II, we found that the localization of DNA-PK at telomeres post-replication is essential to block the nucleolytic erosion of the telomeric ends by any other nuclease than Apollo. This protective function is also independently fulfilled by the iDDR domain of TRF2, which specifically inhibits the endonucleolytic activity of MRN protein complex. This study reveals the high pressure for cells to keep tight control of resection at telomeres.In Paper III, we expanded our studies by investigating the role of Apollo and DNA-PKcs in ALT cancerous cells, which utilize a recombination-mediated mechanism to elongate telomeres. Here, we show that DNA-PKcs and Apollo have a conserved role in promoting G-overhang formation, but in the absence of Apollo and/or DNA-PKcs kinase activity, telomeres are insensitive to fusion. Moreover, we found that Apollo promotes telomere recombination events at ALT telomeres. This study opens the possibility for different mechanisms of telomere maintenance in ALT cells.