The hTERT N-terminal DNA binding is necessary but not sufficient for maintaining telomere and cellular immortalization

Abstract

Telomerase is a reverse transcriptase that adds telomeric DNA repeats at the end of chromosomes and prevents senescence, apoptosis and genome instability. The core components of TERT are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). The N-terminus of TERT has been implicated in processive DNA synthesis that is facilitated by telomere-binding domains in TERT called anchor sites. In this study, we characterized three evolutionarily conserved residues in human TERT, G145, Q169 and G172. We performed site-directed mutagenesis and created hTERT G145A, Q169A and G172A mutants. All three hTERT mutants were not able to immortalize human foreskin fibroblasts (HFFs) and maintain telomere length in cells. The G145A mutant did not abrogate hTERT enzymatic activity, while Q169 and G172 mutants exhibited severely reduced levels and enzymatic activity. When compared to wild type hTERT, the G145 mutant still could interact with telomeric DNA in cells. In contrast, substitutions of Q169 and G172 impaired the interaction between hTERT and telomeres in cells. Thus, characterization of these three conserved residue mutants in GQ motif suggested that the hTERT N-terminal DNA binding is necessary for telomerase function, but not sufficient for maintaining telomere length and immortalizing cells.