Abstract:Telomeres are specialized nucleoprotein complexes that comprise the ends of linear chromosomes. Human telomeres end in a short, single-stranded DNA (ssDNA) overhang that is recognized and bound by two telomere proteins, POT1 and TPP1. Whereas POT1 binds directly to telomere ssDNA, its interaction with TPP1 is essential for localization of POT1 to the telomere. TPP1 also provides enhanced binding and sequence discrimination that regulates POT1-TPP1 interactions exclusively with telomere ssDNA. Finally, TPP1 rec… Show more
“…59 The top three asparagine residues with a glycosylation prediction score of >0.71 out of 1 (i.e., N32, N34, and N94) were verified by MS/MS. The predicted glycosylation sites, N32 and N34, were confirmed by the MS/MS analysis of peptide 29 TYTDSDSTRGPGENGPNY 46 (Figure 2B). A third glycosylation site (N94) with a prediction score of 0.78 was indicated by the server but was not confirmed by mass spectrometry.…”
Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N32 and N34 are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.
“…59 The top three asparagine residues with a glycosylation prediction score of >0.71 out of 1 (i.e., N32, N34, and N94) were verified by MS/MS. The predicted glycosylation sites, N32 and N34, were confirmed by the MS/MS analysis of peptide 29 TYTDSDSTRGPGENGPNY 46 (Figure 2B). A third glycosylation site (N94) with a prediction score of 0.78 was indicated by the server but was not confirmed by mass spectrometry.…”
Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N32 and N34 are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.
“…2a ). The K d of POT1 binding to G-rich ssDNA representing telomeres is 38.2 nM 30 ; TLS/FUS to telomeric G4 is 37 nM 31 ; and nucleolin to telomeric G4 is 2.574 μM 32 . Fig.…”
BRCA2-deficient cells precipitate telomere shortening upon collapse of stalled replication forks. Here, we report that the dynamic interaction between BRCA2 and telomeric G-quadruplex (G4), the non-canonical four-stranded secondary structure, underlies telomere replication homeostasis. We find that the OB-folds of BRCA2 binds to telomeric G4, which can be an obstacle during replication. We further demonstrate that BRCA2 associates with G-triplex (G3)-derived intermediates, which are likely to form during direct interconversion between parallel and non-parallel G4. Intriguingly, BRCA2 binding to G3 intermediates promoted RAD51 recruitment to the telomere G4. Furthermore, MRE11 resected G4-telomere, which was inhibited by BRCA2. Pathogenic mutations at the OB-folds abrogated the binding with telomere G4, indicating that the way BRCA2 associates with telomere is innate to its tumor suppressor activity. Collectively, we propose that BRCA2 binding to telomeric G4 remodels it and allows RAD51-mediated restart of the G4-driven replication fork stalling, simultaneously preventing MRE11-mediated breakdown of telomere.
“…The OB domain of TPP1 has not only been implicated in telomerase binding but also in stabilizing the POT1-DNA complex (Rajavel et al, 2016). Telomerase activity and processivity measured in our primer extension assay depends not just on the TPP1-telomerase interaction but also on tethering TPP1 to POT1-bound DNA.…”
Section: Resultsmentioning
confidence: 99%
“…In both mammals and budding yeast, the NOB region borders this helix, which is capped at its N terminus by a “PWI” sequence (Figure 1B; top). In the structures of Est3 and human TPP1, the conserved tryptophan is stabilized by an essential aspartate located in a loop between the second and third beta strands of the OB domain (Lubin et al, 2013; Rao et al, 2014) that has also been identified as important for DNA binding with POT1 (Rajavel et al, 2016). Both the tryptophan and the aspartate residues are conserved in Tpz1, and an analogous interaction occurs in ciliate TEBPβ with a phenylalanine taking the place of the tryptophan (Rao et al, 2014).…”
SUMMARY
Telomerase recruitment to telomeres and enzymatic processivity are mediated by TPP1, an essential component of telomere integrity and telomerase function. A surface on the OB domain of TPP1 called the TEL patch is critical for TPP1’s telomerase-associated functions. Here, we identify a separate region in the N terminus of the OB domain (termed NOB) of TPP1 that, like the TEL patch, is essential for telomerase repeat addition processivity in vitro as well as telomerase recruitment to telomeres and telomere lengthening in cells. Although well-conserved among most mammalian TPP1 homologs, the NOB region in mice is distinct. Swapping the sequence of human NOB into mouse TPP1 allows it to stimulate human telomerase, qualifying NOB as an important determinant of species specificity for TPP1-telomerase interaction. Our studies show that TPP1 NOB is critical for telomerase function and demonstrate that the telomerase interaction surface on TPP1 is more elaborate than previously appreciated.
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