Human Protection of Telomeres 1 (POT1) Is a Negative Regulator of Telomerase Activity In Vitro

Abstract

The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3 overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.Telomeres, the nucleoprotein complexes at the ends of eukaryotic linear chromosomes, serve a number of vital cellular functions. Telomere capping protects the chromosome ends from nucleolytic degradation and provides a mechanism for cells to distinguish natural from broken ends, which signal DNA damage and are substrates for DNA repair processes (reviewed in references 7 and 13). The DNA component of telomeres is composed of tandem, simple repeats (TTAGGG in mammals), terminating with a 3Ј-protruding single strand of the guanosine-rich strand. Due to lack of a template to replicate the 3Ј overhang, most human somatic cells lose terminal DNA with each division; thus, telomeres also provide a buffer between the ends and the more internally located coding region of the genome (12,19). In germ line cells and some cells in highly proliferative tissues, telomere loss is counterbalanced through the activities of the ribonucleoprotein telomerase. In vitro assays reveal that catalytically active telomerase is minimally composed of an RNA (TER) and a protein (TERT) subunit (5,38,39). Telomerase adds telomeric repeats by iteratively reverse transcribing the template portion of its RNA subunit, using the 3Ј single-strand telomeric overhang as a primer (18, 42; reviewed in references 20 and 22).Proteins that specifically bind the telomere single-strand overhang have been identified in numerous organisms. The 3Ј telomeric overhang of the ciliate Oxytricha nova is bound by OnTEBP, a heterodimeric end-binding protein composed of an ␣ subunit and a ␤ subunit (14, 17). Crystal structures reveal that the ␣ subunit contains three oligonucleotide/oligosaccharide binding (OB) folds: the first two OB folds bind telomeric DNA with sequence specificity, and the third participates in protein-protein interactions with the ␤ subunit (21). A search for homologs of the OnTEBP ␣ subunit identified the Pot1 protein in Schizosaccharomyces pombe and human based on a weak sequence similarity with the first OB fold in the ␣ subunit (3). Genetic studies with S. pombe demonstrate a role for S. pombe POT1 in telomere end protection, since deletion of the pot1 gene results in the rapid loss of ...

Expression of hPOT1 in HeLa Cells and the Probability of Gene Variation of hpot1 Exon14 in Endometrial Cancer are Much Higher than in Other Cancers

“…So EMSA is an important technique underlying a wide range of qualitative and semi-quantitative analyses for the characterization of interacting systems (Hellman et al, 2007). EMSA have been applied in detecting telomere proteins contains hPOT1 (BaumannCech, 2001;Kelleher et al, 2005). Because hPOT1 binds to telomeric ssDNA TTAGGG repeats, so we designed oligo probes of (TTAGGG)4 (total 20 bp).…”

Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro

“…Both TEBP, the telomere end-binding protein from hypotrichous ciliates, and hPOT1, the corresponding protein from humans (7), can sequester the telomeric DNA 3Ј end and render it unavailable for telomerase extension (8)(9)(10). This inhibition is expected from the x-ray crystal structures of these DNA-protein complexes (11,12).…”

Replication of Telomeres and the Regulation of Telomerase

“…Whether telomerase recruitment is regulated in mammals by Atm or Atr, as in fission and budding yeast, is unknown, nor is it clear if in addition to Tpp1 other factors contribute to recruitment. The single-stranded telomere-binding protein Pot1 is an inhibitor of telomerase per se when binding to the 3 0 end of the substrate (Kelleher et al 2005;Lei et al 2005). When associated with Tpp1, however, Pot1 -Tpp1 decreases the dissociation of primer from telomerase and increases the efficiency of translocation (Wang et al 2007;Latrick and Cech 2010).…”