A conserved telomerase motif within the catalytic domain of telomerase reverse transcriptase is specifically required for repeat addition processivity.
Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The catalytic protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT) domain that mediates nucleotide addition. The RT domain of TERT is distinguishable from retroviral and retrotransposon RTs in having a sizable insertion between conserved motifs A and B', within the so-called fingers domain. Sequence analysis revealed the existence of conserved residues in this region, named IFD (insertion in fingers domain). Mutations of some of the conserved residues in Saccharomyces cerevisiae TERT (Est2p) abolished telomerase function in vivo, testifying to their importance. Significant effects of the mutations on telomerase activity in vitro were observed, with most of the mutants exhibiting a uniform reduction in activity regardless of primer sequence. Remarkably, one mutant manifested a primer-specific defect, being selectively impaired in extending primers that form short hybrids with telomerase RNA. This mutant also accumulated products that correspond to one complete round of repeat synthesis, implying an inability to effect the repositioning of the DNA product relative to the RNA template that is necessary for multiple repeat addition. Our results suggest that the ability to stabilize short RNA-DNA hybrids is crucial for telomerase function in vivo and that this ability is mediated in part by a more elaborate fingers domain structure.
Lue
Neal F
NF
Department of Microbiology & Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, New York, New York 10021, USA. nflue@med.cornell.edu
Lin
You-Chin
YC
Mian
I Saira
IS
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D017124:Conserved Sequence; D004268:DNA-Binding Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019098:Telomerase