Frank AK, Tran DC, Qu RW, Stohr BA, Segal DJ, Xu L

Frank AK, Tran DC, Qu RW, Stohr BA, Segal DJ, Xu L. provide the first evidence that the IFD can mediate enzyme processivity and telomerase recruitment to telomeres in a TPP1-dependent manner. Moreover, unlike hTERT-V791Y, hTERT-V763S, a variant with reduced activity but increased processivity, and hTERT-L805A, could both immortalize limited-life-span cells, but cells expressing these two mutant enzymes displayed growth defects, increased apoptosis, DNA damage at telomeres, and short telomeres. Our results highlight the importance of the IFD in maintaining short telomeres and in cell survival. INTRODUCTION Telomeres are the protective nucleoprotein structures that cap the ends of linear eukaryotic chromosomes, thus preventing the aberrant and fatal activation of the DNA damage repair machinery. During normal somatic cell division, the end replication problem arising from the inability of DNA polymerase to completely replicate telomeres leads to progressive telomere loss and, over time, triggers cellular senescence to prevent carcinogenesis. The renewal capacity of germ cells, stem cells, and cancer cells is limited by telomere erosion and relies on the activation of a telomere maintenance mechanism for cellular survival. In over 85% of human cancers, detectable expression of telomerase, a specialized reverse transcriptase, is a requirement for cellular immortalization (1). In humans, telomerase is minimally composed of the core catalytic subunit human telomerase reverse transcriptase (hTERT) and an intrinsic RNA moiety, human telomerase RNA (hTR), to dictate the synthesis of tandem TTAGGG repeats. Telomerase has the unique ability to synthesize long stretches of telomeric sequence repeats using its short RNA template through reiterative rounds of DNA synthesis, partial dissociation, translocation, and realignment with the newly synthesized telomere end. In human cells, this unique property, termed repeat addition SFN processivity (RAP), is a determinant of telomere maintenance and cellular survival (2). The reverse transcriptase region of the TERT subunit contains seven motifs b-AP15 (NSC 687852) (1, 2, A, B, C, D, and E) that are also conserved in other nucleic acid polymerases. Importantly, TERT distinguishes itself from other conventional reverse transcriptases by the presence of a large insertion within the fingers subdomain between the conserved motifs A and B, referred to as the insertion in fingers domain (IFD). The TERT crystal structure reveals that the IFD is located on the periphery of the TERT ring (3). In hybridization (FISH) was performed as previously described (5), using HeLa cells coexpressing hTERT-WT or hTERT-variants and hTR (22), three different Cy3-conjugated hTR probes (23), and an Oregon green-conjugated telomeric probe (8). Cy3 monoreactive dye was from GE Healthcare (Piscataway, NJ), Oregon green 488 from Invitrogen, and probes from Operon (Huntsville, AL). Images were captured using an Axio Imager M1 microscope (63; Carl Zeiss, Jena, Germany). ChIP. Chromatin immunoprecipitation (ChIP) was performed using HeLa cells overexpressing 3FLAG-tagged mutant and WT hTERTs as previously described (24) with the following modification. Ten picomoles of Alu and telomeric (T2AG3)3 probes were end labeled with 10 pmol of [-32P]ATP (PerkinElmer) and purified using G-25 columns (GE Healthcare). Quantitation of telomere binding was done using the formula (telo IP/telo input)/(Alu IP/Alu input) (25), and values are expressed relative to WT telomerase binding to telomeres. Quantitative fluorescence hybridization analysis and signal free ends. Metaphase spread analysis for detection of signal free ends (SFE) was performed as described previously (2, 5). b-AP15 (NSC 687852) b-AP15 (NSC 687852) Imaging was performed using b-AP15 (NSC 687852) an Axio Imager M1 microscope (63; Carl Zeiss, Jena, Germany). Quantitative analysis of telomere length and SFE was performed with TFL-Telo (Peter Lansdorp). Apoptosis analysis by fluorescence-activated cell sorting (FACS). Retrovirally infected hTERT-HA5 cells were grown to confluence in a 10-cm dish. Cell medium was collected and combined with trypsinized cells from the plate. Cells were treated with propidium iodide (Sigma-Aldrich, St. Louis, MO) and annexin V-fluorescein isothiocyanate (BD Bioscience) using a BD LSRFortessa b-AP15 (NSC 687852) analyzer at the Lady Davis Institute Flow Cytometry Facility. Data were analyzed using BD FACSDiva dongle software. Immunofluorescence combined with FISH for TIF detection. For visualization of telomere dysfunction-induced foci (TIF), HA5 cells were grown on coverslips for 24 h, and then the previously described protocol (23) for detection of telomeres by FISH was carried out using.