Huard Sylvain, Autexier Chantal
Department of Anatomy and Cell Biology, McGill University, Montréal, Québec H3A 2B4, Canada.
Nucleic Acids Res. 2004 Apr 19;32(7):2171-80. doi: 10.1093/nar/gkh546. Print 2004.
Telomerase is a reverse transcriptase that uses an integral RNA molecule to add de novo G-rich repeats onto telomeric DNA, or onto nontelomeric DNA generated during chromosome fragmentation and breakage events. A telomerase-mediated DNA substrate cleavage activity has been reported in ciliates and yeasts. Nucleolytic cleavage may serve a proofreading function, enhance processivity or ensure that nontemplate telomerase RNA sequences are not copied into DNA. We identified and characterized a human telomerase-mediated nucleolytic cleavage activity using enzyme reconstituted in a rabbit reticulocyte lysate in vitro transcription/translation system and native enzyme extracted from cells. We found that telomerase catalyzed the removal of nucleotides from DNA substrates including those that can form a mismatch with the RNA template or that contain nontelomeric sequences located 3' to a telomeric sequence. Unlike Tetrahymena telomerase, human telomerase catalyzed the removal of more than one nucleotide (up to 13) from telomeric primers. DNA substrates predicted to align at the 3'-end of the RNA template were not cleaved, consistent with cleavage being dictated by the template 5'-end. We also found some differences in the nuclease activity between RRL-reconstituted human telomerase and native enzyme.
端粒酶是一种逆转录酶，它利用一个整合的RNA分子将富含G的重复序列从头添加到端粒DNA上，或添加到染色体断裂和破碎事件中产生的非端粒DNA上。在纤毛虫和酵母中已报道了端粒酶介导的DNA底物切割活性。核酸酶切割可能起到校对功能、增强持续性或确保非模板端粒酶RNA序列不被复制到DNA中。我们使用在兔网织红细胞裂解物体外转录/翻译系统中重组的酶和从细胞中提取的天然酶，鉴定并表征了一种人端粒酶介导的核酸酶切割活性。我们发现端粒酶催化从DNA底物中去除核苷酸，这些底物包括那些可能与RNA模板形成错配的底物，或那些在端粒序列3'端含有非端粒序列的底物。与嗜热四膜虫端粒酶不同，人端粒酶催化从端粒引物中去除不止一个核苷酸（最多13个）。预计在RNA模板3'端对齐的DNA底物未被切割，这与切割由模板5'端决定一致。我们还发现了在兔网织红细胞裂解物中重组的人端粒酶和天然酶之间核酸酶活性的一些差异。
