Complex interaction network revealed by mutation of human telomerase 'insertion in fingers' and essential N-terminal domains and the telomere protein TPP1.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here, we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres, and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence.

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.

N-terminal residues of human dyskerin are required for interactions with telomerase RNA that prevent RNA degradation.

The telomerase holoenzyme responsible for maintaining telomeres in vertebrates requires many components in vivo, including dyskerin. Dyskerin binds and regulates the accumulation of the human telomerase RNA, hTR, as well as other non-coding RNAs that share the conserved H/ACA box motif. The precise mechanism by which dyskerin controls hTR levels is unknown, but is evidenced by defective hTR accumulation caused by substitutions in dyskerin, that are observed in the X-linked telomere biology disorder dyskeratosis congenita (X-DC). To understand the role of dyskerin in hTR accumulation, we analyzed X-DC substitutions K39E and K43E in the poorly characterized dyskerin N-terminus, and A353V within the canonical RNA binding domain (the PUA). These variants exhibited impaired binding to hTR and polyadenylated hTR species, while interactions with other H/ACA RNAs appear largely unperturbed by the N-terminal substitutions. hTR accumulation and telomerase activity defects of dyskerin-deficient cells were rescued by wildtype dyskerin but not the variants. hTR 3' extended or polyadenylated species did not accumulate, suggesting hTR precursor degradation occurs upstream of mature complex assembly in the absence of dyskerin binding. Our findings demonstrate that the dyskerin-hTR interaction mediated by PUA and N-terminal residues of dyskerin is crucial to prevent unchecked hTR degradation.

Modified peptide monolayer binding His-tagged biomolecules for small ligand screening with SPR biosensors.

A peptide self-assembled monolayer (SAM) was designed to bind His-tagged biomolecules for surface plasmon resonance (SPR) bioanalysis, which was applied for the determination of K(d) for small ligand screening against CD36. Nonspecific adsorption could be minimized using penta- and hexa-peptide monolayers. In particular, monolayers consisting of 3-mercaptopropionyl-leucinyl-histidinyl-aspartyl-leucinyl-histidinyl-aspartic acid (3-Mpa-LHDLHD) exhibited little (12 ng cm(-2)) nonspecific adsorption in crude serum. Modification of this peptide monolayer with Nα,Nα-bis(carboxymethyl)-L-lysine gave a surface competent for binding His-tagged proteins, as demonstrated using enzyme (human dihydrofolate reductase), protein/antibody and receptor (CD36) examples. Immobilization featured chelation of copper and the His-tagged protein by the peptide monolayer, which could be recycled by removing the copper using imidazole washes prior to reuse.