Specificity Requirements for Human Telomere Protein Interaction with Telomerase Holoenzyme*

Background: The interaction specificity of telomerase and telomeres is not well understood. Results: We establish the determinants of telomere protein TPP1 association with TERT and the catalytically active telomerase holoenzyme. Conclusion: A surface loop of one TPP1 domain mediates a holoenzyme-assembled TERT interaction with telomere protein complexes. Significance: Eukaryotes conserve an interaction interface within the telomere-bound telomerase holoenzyme. Human telomeres are maintained by the enzyme telomerase, which uses a template within its integral RNA subunit (hTR) and telomerase reverse transcriptase protein (TERT) to accomplish the synthesis of single-stranded DNA repeats. Many questions remain unresolved about the cellular regulation of telomerase subunits and the fully assembled telomerase holoenzyme, including the basis for the specificity of binding and acting on telomeres. Previous studies have revealed that the telomere protein TPP1 is necessary for stable TERT and hTR association with telomeres in vivo. Here, we expand the biochemical characterization and understanding of TPP1 interaction with TERT and the catalytically active telomerase holoenzyme. Using extracts from human cells, we show that TPP1 interacts sequence-specifically with TERT when TERT is assembled into holoenzyme context. In holoenzyme context, the TERT N-terminal domain mediates a TPP1 interaction. Assays of stable subunit complexes purified after their cellular assembly suggest that other telomere proteins do not necessarily influence TPP1 association with telomerase holoenzyme or alter its impact on elongation processivity. We show that a domain of recombinant TPP1 comprised of an oligonucleotide/oligosaccharide binding fold recapitulates the full-length protein interaction specificity for the TERT N-terminal domain assembled into telomerase holoenzyme. By global analysis of TPP1 side chain requirements for holoenzyme association, we demonstrate a selective requirement for the amino acids in one surface-exposed protein loop. Our results reveal the biochemical determinants of a sequence-specific TPP1-TERT interaction in human cells, with implications for the mechanisms of TPP1 function in recruiting telomerase subunits to telomeres and in promoting telomere elongation.

• Publication year

  2012

• Venue

  Journal of Biological Chemistry

• Publication date

  2012-08-14

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1074/jbc.M112.394767PMID 22893708PMCID PMC3464550
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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