Greene E C, Bednenko J, Shippen D E
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Mol Cell Biol. 1998 Mar;18(3):1544-52. doi: 10.1128/MCB.18.3.1544.
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.
除了具有逆转录酶活性外，端粒酶还与一种DNA内切核酸酶相关，该酶在端粒重复序列添加之前从引物3'末端去除核苷酸。在这里，我们研究了嗜热四膜虫端粒酶进行的引物切割-延伸反应的DNA特异性。我们发现，与嗜热四膜虫端粒酶聚合酶共纯化的引物切割活性，这表明它要么是端粒酶的固有特性，要么是由紧密相关的因子催化的。使用含有可精确位于RNA模板上的端粒DNA片段的嵌合引物，我们发现切割位点比最初提出的更具灵活性。在RNA模板中与核苷酸37至40相对的dT片段中含有错配的引物被切割，以消除错配残基以及相邻的3'序列。然后将切割产物延伸以产生完美的端粒重复序列。dG片段中的错配没有被去除，这意味着核酸酶不会与聚合酶活性位点协同追踪。我们的数据表明，端粒酶相关核酸酶可以通过消除DNA引物与端粒酶RNA模板5'区域之间的错配，在端粒合成中提供基本的校对功能。
