Molecular basis of telomere syndrome caused by CTC1 mutations

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase alpha-primase (pol alpha-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA pol alpha-primase to promote faithful telomere DNA replication

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions