Flexible positioning of the telomerase-associated nuclease leads to preferential elimination of nontelomeric DNA.
In addition to a reverse transcriptase activity, telomerase is associated with a DNA endonuclease that removes nucleotides from a primer 3' terminus prior to telomere repeat addition. Here we examine the DNA specificity of the primer cleavage-elongation reaction carried out by the Euplotes crassus telomerase. We show that the primer cleavage activity copurified with the E. crassus telomerase polymerase, indicating that it either is an intrinsic property of telomerase or is catalyzed by a tightly associated factor. Using chimeric primers containing stretches of telomeric DNA that could be precisely positioned on the RNA template, we found that the cleavage site is more flexible than originally proposed. Primers harboring mismatches in dT tracts that aligned opposite nucleotides 37 to 40 in the RNA template were cleaved to eliminate the mismatched residues along with the adjacent 3' sequence. The cleaved product was then elongated to generate perfect telomeric repeats. Mismatches in dG tracts were not removed, implying that the nuclease does not track coordinately with the polymerase active site. Our data indicate that the telomerase-associated nuclease could provide a rudimentary proofreading function in telomere synthesis by eliminating mismatches between the DNA primer and the 5' region of the telomerase RNA template.
Greene
E C
EC
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Bednenko
J
J
Shippen
D E
DE
D000818:Animals; D001665:Binding Sites; D017931:DNA Primers / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides; D003851:Deoxyribonucleases / Q000378:metabolism*; D018900:Dinucleotide Repeats; D016812:Euplotes / Q000201:enzymology*; D012313:RNA / Q000378:metabolism; D013379:Substrate Specificity; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere; D013698:Templates, Genetic; D013936:Thymidine
D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; D012313:RNA; D019098:Telomerase; D003851:Deoxyribonucleases; D013936:Thymidine