Specificity requirements for human telomere protein interaction with telomerase holoenzyme

Abstract

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.

FIGURE 1.

Domain interactions of TERT and TPP1. A, immunoblot detection of f-TPP1 co-immunopurification of zz-tagged full-length TERT or zz-TEN domain (TERT amino acids 1–325), each with wild-type (WT), G100V, or NDAT (92–97 NAAIRS) sequence. B, immunoblot detection of zz-TERT, zz-Core (TERT lacking amino acids 1–325), or zz-TEN domain following immunopurification of full-length f-TPP1 or f-TPP1 variants lacking the domain for TIN2 binding (ΔTBD) or POT1 binding (ΔPBD).

FIGURE 2.

TEN domain dependence of TPP1 association with telomerase holoenzyme. A, telomerase catalytic activity associated with f-TPP1 from cells co-expressing hTR and the indicated zz-tagged TERT (lanes 1–5) or activity associated with the indicated f-tagged TERT co-expressed with hTR alone (lanes 6 and 7). Lanes 6 and 7 were cropped from the same exposure of the same gel. A loading control (LC) was added to products before precipitation. B, Northern blot detection of co-expressed hTR co-immunopurificationed with the indicated f-TERT in the presence or absence of co-expressed Myc-tagged (m) TPP1. A recombinant RNA recovery control (RC) was added before hTR extraction and is detected with the same end-labeled probe. All lanes were cropped from the same exposure of the same blot. C, immunoblot and Northern blot detection of f-TPP1 co-immunopurification of co-expressed zz-TERT and hTR. The bound/input ratio of hTR hybridization signal was calculated after normalization for the RNA recovery control (see “Experimental Procedures”). D, immunoblot and Northern blot detection of f-TPP1 co-immunopurification of co-expressed zz-tagged full-length or domain-truncated TERTs and hTR.

FIGURE 3.

Influence of telomere protein complexes on holoenzyme RAP. A, protein and hTR overexpression was assayed by blots of cell extracts. B, telomerase catalytic activity and hTR purified in association with complexes of the indicated f-tagged protein (TERT, TPP1, TIN2, or POT1) or f-tagged TPP1 OBD were assayed following immunopurification from the extracts assessed in A. Cells co-expressed hTR, f-tagged, or zz-tagged TERT, and also f-tagged and/or Myc (m)-tagged shelterin proteins as indicated in lanes 2–5; the f-tagged subunit directly enriched by affinity purification is boxed. Cell extract with no f-tagged protein was used in parallel to verify the reproducible absence of nonspecific activity association with the FLAG antibody resin (data not shown). The bracket at the right of the gel in B indicates the product DNAs used to quantify relative RAP. Product DNAs from the entire lane were used to quantify relative specific activity. RC, recovery control; LC, loading control; FL, full length.

FIGURE 4.

Identification of a TPP1 OBD surface required for holoenzyme interaction. A, extract from cells co-expressing hTR and f-TERT was used for holoenzyme binding to Ni-NTA-agarose with His₆-tagged TPP1 OBD or the His₆-tagged N-terminal or C-terminal OBD of Tetrahymena Teb1. Either 100 pmol or 1 nmol of each purified OBD was prebound to the resin. A negative control monitored holoenzyme binding to resin lacking prebound OBD (lane 1). B, extract from cells co-expressing hTR and either WT or G100V f-tagged TERT was used for holoenzyme purification. The holoenzymes were then allowed to interact with the TPP1 OBD immobilized on Ni-NTA-agarose. Either 20 or 200 pmol of TPP1 OBD was prebound to the resin, and activity was assayed directly on resin without elution (lanes 3–6). Activity assays of the input purified holoenzymes (lanes 1 and 2) used 10% of the amount allowed to bind to TPP1 OBD. C, telomerase holoenzyme binding to resin-immobilized WT TPP1 OBD or the TPP1 OBD variant indicated. Each panel begins with a negative control monitoring holoenzyme binding to resin lacking a TPP1 OBD. Either 20 or 200 pmol of WT or 200 pmol of sequence variant TPP1 OBD was prebound to the resin. Activity was assayed directly on resin without elution. The bracket indicates variants with reproducibly and severely reduced co-immunopurification of holoenzyme activity. LC, loading control.

FIGURE 5.

Sequence dependence of TPP1 OBD association with telomerase holoenzyme co-expressed in human cells. A and B, telomerase holoenzyme activity co-immunopurification with an f-tagged TPP1 OBD co-expressed with zz-TERT and hTR in human cells. Each panel of activity assays begins with a negative control that monitors holoenzyme binding in the absence of f-tagged TPP1 OBD. The bracket indicates TPP1 OBD variants with reproducibly and severely reduced co-immunopurification of holoenzyme activity; the two additional TPP1 OBD variants indicated with an asterisk reproducibly but less severely compromise holoenzyme co-immunopurification. The TPP1 OBD variants with compromised holoenzyme interaction were expressed at the same level as wild-type TPP1 (data not shown).

FIGURE 6.

Sequence dependence of full-length TPP1 association with telomerase holoenzyme co-expressed in human cells. A, co-immunopurification of zz-TERT with f-tagged full-length TPP1 harboring the indicated single amino acid substitution in the OBD, following co-expression in human cells. A negative control monitors holoenzyme subunit binding to resin from cell extract lacking f-tagged TPP1 (lane 1). TERT bound/input ratio was calculated after general background correction of Input and Bound immunoblot signal intensities. B, telomerase holoenzyme activity co-immunopurification by co-expressed f-tagged full-length TPP1 harboring the indicated single amino acid substitution in the OBD. The bracket in A and B indicates the sequence variants with reproducibly and severely reduced co-immunopurification of holoenzyme activity. C, ribbon and space-filled representations of human TPP1 OBD structure (20). The amino acids shown here to be important for holoenzyme binding are indicated in lighter shading against the rest of the domain and also labeled. RC, recovery control; LC, loading control.