Characterization of physical and functional anchor site interactions in human telomerase.
Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation.
Wyatt
Haley D M
HD
Southern Alberta Cancer Research Institute, Department of Biochemistry and Molecular Biology, Room 372B HMRB, 2220 Hospital Drive NW, Calgary, AB, Canada.
Lobb
Deirdre A
DA
Beattie
Tara L
TL
D000596:Amino Acids; D001483:Base Sequence; D019921:Biotinylation; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism
D000596:Amino Acids; D017931:DNA Primers; D009841:Oligonucleotides; C422952:telomerase RNA; D012313:RNA; D004247:DNA; C509186:TERT protein, human; D019098:Telomerase