Rescue of an hTERT Mutant Defective in Telomere Elongation by Fusion with hPot1

Abstract

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.Telomeres are essential DNA-protein structures that cap and protect the ends of eukaryotic chromosome from illegitimate recombination, degradation, and detection as DNA damage (10). In humans, the telomere is composed of hundreds to thousands of tandem repeats of the G-rich sequence 5Ј-TTA GGG-3Ј (whereby the G strand overhangs the complementary C strand) and has been proposed to loop around, forming a T loop, and invade the duplex DNA to form a higher-order structure termed the D loop (25). The replication of telomeres poses a unique problem to eukaryotes, as removal of the terminal RNA primer during semiconservative replication of the leading strand leaves a gap that cannot be replicated by known DNA polymerases. In most eukaryotes, this is overcome by the de novo addition of telomeric DNA via the enzyme telomerase (9). Human telomerase is minimally composed of a reversetranscriptase subunit (hTERT) that copies a template region of the accompanying RNA subunit (hTR) onto telomeres as DNA (34). Little is known regarding the mechanism by which the enzyme recognizes telomeres in vivo in higher eukaryotes. However, mutations to either the large N-terminal or smaller C-terminal DAT domains of hTERT render the enzyme catalytically active in vitro but unable to elongate telomeres or extend the life span of telomerase-negative cells in vivo (1, 3). Targeting N-terminal DAT mutants to telomeres by fusion to the double-stranded telomeric DNA-binding protein hTRF2 can extend the life span of telomerase-negative cells, although the growth of these cells was noticeably retarded (2). These results suggest that the DAT domain is involved in telomeretelomerase associations, although this model would be significantly strengthened if it could be demonstrated that a DAT mutant ...

Regulation of Cellular Immortalization and Steady-State Levels of the Telomerase Reverse Transcriptase through Its Carboxy-Terminal Domain

“…Furthermore, these results show that the N-DAT domain in mTERT, which is 82% similar to the human DAT region, functions properly in human cells (2). The N-DAT domain has been proposed to mediate telomerase access to telomeric chromatin (3,4) or to contribute to interactions between telomerase and telomere DNA repeats (40,52). Instead, our data identify the C terminus as a critical regulatory domain controlling the ability of TERT to stabilize telomeres in cells and to extend replicative lifespan.…”

“…These DAT (dissociates activities of telomerase) domains were proposed to be required for the recruitment of telomerase to telomeric chromatin (3,4). Other analyses of these TERT mutants found that the DAT mutants showed reduced processivity, principally on oligonucleotide primers matching the natural telomere sequence, indicating that the DAT regions contribute to interactions between TERT and its telomere substrate (40,52).…”

“…Two micrograms of digested DNA was fractionated on a 0.8% agarose gel and transferred overnight to Hybond-N membrane (Amersham) in 10ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The ultraviolet light-cross-linked membrane was blocked by prehybridization and then hybridized with a 32 P-end-labeled (TTAGGG) 4 oligonucleotide by using Rapid Hyb (Amersham Biosciences) at 42°C for 1 h. Membranes were washed once in 5ϫ SSC and 0.1% SDS at room temperature for 20 min and twice in 1ϫ SSC and 0.1% SDS at 42°C for 15 min and then exposed to film. Telomere signal intensities were quantitated by scanning the autoradiographs and using ImageJ software and the following equation: mean telomere length ϭ ⌺(intensity i )/⌺(intensity i /kb i ) (28).…”

Telomerase promotes efficient cell cycle kinetics and confers growth advantage to telomerase-negative transformed human cells

“…This last observation is likely reflective of a gain in the rate of cell cycle progression (see below as well as Figures 7a and b), cumulative over the 2-week period of CFU growth. (DN710), which did not have reverse transcriptase activity, we included the amino-terminal (NDAT92 and NDAT122) and carboxy-terminal (CDAT1127) DAT TERT mutants, which are catalytically active but cannot support telomere maintenance, due to telomere localization defects (Armbruster et al, 2001(Armbruster et al, , 2004Banik et al, 2002), as well as primer-template positioning dysfunctions (Lee et al, 2003). The DAT mutants were previously reported to retain the capacity to promote the non-canonical functions of TERT in chromatin remodeling and are therefore competent to promote DNA-damage repair, despite defects in their canonical TERT functions .…”

Relevance and safety of telomerase for human tissue engineering

“…However, the short duration of gene expression achieved by these methods may not allow for sufficient telomere elongation to ensure adequate cell replicative potential for tissue engineering applications. In light of this consideration, one additional potential approach is the transient delivery of a highly processive mutant of telomerase that rapidly elongates telomeres in vitro (44).…”