The human telomerase holoenzyme's structure enabled visualization of key residues and nucleotides.
Major Finding: The human telomerase holoenzyme's structure enabled visualization of key residues and nucleotides.
Approach: Cryo–electron microscopy techniques were bolstered by mass spectrometry and in vitro experiments.
Impact: The intricate data provided by this study will enhance research on this cancer-associated complex.
Although the role of aberrant telomerase activity in cancer has long been known, the development of telomerase inhibitors has been hindered in part by a lack of high-resolution structural data. Using cryo–electron microscopy, Ghanim, Fountain, van Roon, and colleagues determined structures of the human telomerase holoenzyme bound to telomeric DNA at resolutions ranging from 3.4 to 3.8 Å, enabling direct visualization of mechanistically important and disease-associated amino acid residues and nucleotides that could not be resolved in previously obtained, lower-resolution structures of this ribonucleoprotein complex. Examination of the structure of the active site of the telomerase reverse transcriptase (TERT) revealed that residue L866—mutation of which to Y can cause telomere length abnormalities and immortalization in human cells—plays a key role in stabilizing TERT's binding to substrate DNA. This suggests that the observed nucleotide and repeat-addition processivity defects underlying the telomeric disturbances in TERTL866Y cells may result from premature dissociation of substrate DNA from telomerase during telomere DNA synthesis. Analysis of the structure also elucidated roles for two catalytically essential stretches of the human telomerase RNA (hTR): the pseudoknot/template (PK/t) and conserved regions 4 and 5 (CR4/5), which are highly evolutionarily conserved among vertebrates. Specifically, CR4/5 and PK/t may enable accurate organization of the TERT domains, essential for telomerase activity. Surprisingly, analysis of an unknown portion of the structure closely associated with TERT revealed the presence of a heterodimer of histones 2A and 2B (H2A and H2B), an observation verified using mass spectrometry and in vitro assays. The proximity of H2A and H2B to CR4/5 along with the apparent contradiction between the homogeneous conformations of CR4/5 in this structure and the previous observation that CR4/5 adopts heterogeneous conformations suggests that the H2A–H2B heterodimer may be important for stabilization or correct positioning of CR4/5 during telomerase holoenzyme assembly. Collectively, these and other inferences made possible by this high-resolution structure of the human telomerase holoenzyme provide critical mechanistic insights that will enable further study of this key complex.
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