Effects of nucleotide analogues on Euplotes aediculatus telomerase processivity: evidence for product-assisted translocation.
Telomerase is a unique ribonucleoprotein that reverse transcribes a defined region of its RNA subunit onto the ends of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is typically a G-rich repeated sequence, (TTTTGGGG)(n) in the ciliate Euplotes aediculatus and (TTAGGG)(n) in humans. Telomerase can extend oligonucleotide primers in vitro in a processive fashion. We used dNTP analogues to study the structure-activity relationship between substrate nucleotides and processivity of telomerase from E. aediculatus. Several analogues, including 2'-deoxyuridine triphosphate (dUTP), 2'-deoxyinosine triphosphate (dITP), and 7-deaza-2'-deoxyguanosine triphosphate (7-deaza-dGTP), were good substrates for telomerase with K(m) and V(max) values near those of the natural substrates, dTTP and dGTP. However, telomerase processivity was affected with these substrates, decreasing in the order dUTP > 7-deaza-dGTP > dITP. Telomerase did not completely reverse transcribe the template when dITP was the substrate, and it efficiently extended a primer by the addition of two repeats when 7-deaza-dGTP and dUTP were utilized. When the same nucleotide analogues were incorporated into the primers, no effects were observed except in the case of a 3'-terminal deoxyinosine. The data support a model that includes the formation of an intramolecular secondary structure within the product DNA to facilitate translocation. The most likely structure is a G-G hairpin.
Jarstfer
Michael B
MB
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA. jarstfer@unc.edu
Cech
Thomas R
TR
D000818:Animals; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D016812:Euplotes / Q000187:drug effects* / Q000201:enzymology; D006358:Hot Temperature; D020128:Inhibitory Concentration 50; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016053:RNA, Protozoan / Q000378:metabolism; D013329:Structure-Activity Relationship; D019098:Telomerase / Q000378:metabolism*
D017931:DNA Primers; D016054:DNA, Protozoan; D003854:Deoxyribonucleotides; D016053:RNA, Protozoan; D019098:Telomerase