DNA-protein kinase catalytic subunit-interacting protein KIP binds telomerase by interacting with human telomerase reverse transcriptase

Telomere homeostasis, a process that is essential for continued cell proliferation and genomic stability, is regulated by endogenous telomerase and a collection of associated proteins. In this paper, a protein called KIP (previously reported as a protein that binds specifically to DNA-dependent protein kinase), has been identified as a telomerase-regulating activity based on the following pieces of evidence. First, complexes between KIP and the catalytic subunit of telomerase (hTERT) were identified using the yeast two-hybrid technique. Second, antibodies specific to KIP immunoprecipitate human telomerase in cell-free extracts. Third, immunolocalization experiments demonstrate that KIP is a nuclear protein that co-localizes with hTERT in cells. Fourth, KIP binds to hTERT both in vitro and in vivo in the absence of human telomerase RNA or telomeric DNA, thus defining the catalytic subunit of telomerase as the site of physical interaction. Fifth, co-immunoprecipitation experiments suggest that KIP-hTERT complexes form readily in cells and that overexpression of KIP in telomerase-positive cells increases endogenous telomerase activity. Finally, continued overexpression of KIP (60 population doublings) resulted in cells with elongated telomeres; thus, KIP directly or indirectly stimulates telomerase activity through hTERT and contributes to telomere lengthening. The collective data in this paper suggest that KIP plays a positive role in telomere length maintenance and/or regulation and may represent a novel target for anti-cancer drug development.     

Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1

The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.     

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in?vitro and in?vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:     

Human cancer cells harbor T-stumps, a distinct class of extremely short telomeres

Using a modified single telomere length analysis protocol (STELA) to clone and examine the sequence composition of individual human XpYp telomeres, we discovered a distinct class of extremely short telomeres in human cancer cells with active telomerase. We name them "t-stumps," to distinguish them from the well-regulated longer bulk telomeres. T-stumps contained arrangements of telomeric repeat variants and a minimal run of seven canonical telomeric TTAGGG repeats, but all could bind at least one TRF1 or TRF2 in vitro. The abundance of t-stumps was unaffected by ATM alteration but could be changed by manipulating telomerase catalytic subunit (hTERT) levels in cancer cells. We propose that in the setting of active telomerase and compromised checkpoints characteristic of human cancer cells, t-stumps define the minimal telomeric unit that can still be protected by a TRF1/TRF2-capping complex and, further, that hTERT (or telomerase) may have a role in protecting t-stumps.     

Telomerase maintains telomere structure in normal human cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.     

A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.