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首页> 外文期刊>Biochemistry >Effects of Nucleotide Analogues on Euplotes aediculatus Telomerase Processivity: Evidence for Product-Assisted Translocation
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Effects of Nucleotide Analogues on Euplotes aediculatus Telomerase Processivity: Evidence for Product-Assisted Translocation

机译：核苷酸类似物对Eupplotes aediculatus端粒酶生产力的影响：产品辅助易位的证据。

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摘要

Telomerase is a unique ribonucleoprotein that reverse transcribes a defined regionof its RNA sbuit onto the ends of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is typicaly a G-rich repeated sequence, (TTTTGGGG)_n in the ciliate Euplotes aediculatus and (TTAGGG)_n in humans. Telomerase can extend oligonucleotide primers n 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-dezaz-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, dTTP > 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 anintramolecular secondary structure within the product DNA to facilitate translocation. The most likely structure is a G-G hairpin.
机译：端粒酶是一种独特的核糖核蛋白，可将其RNA片段的定义区域逆转录到真核染色体的末端。端粒酶的产物，端粒DNA，通常是纤毛的Euplotes aediculatus中的（TTTTGGGG）_n和人类中的（TTAGGG）_n，是富含G的重复序列。端粒酶可以以渐进方式在体外延伸寡核苷酸引物。我们使用dNTP类似物来研究底物核苷酸与来自埃德埃希氏菌的端粒酶之间的构效关系。几个类似物，包括2'-脱氧尿苷三磷酸（dUTP），2'-脱氧肌苷三磷酸（dITP）和7-deaza-2'-脱氧鸟苷三磷酸（7-dezaz-dGTP），是端粒酶K_m和V_max值的良好底物接近天然底物dTTP和dGTP。然而，端粒酶的合成能力受这些底物的影响，dTTP> 7-脱氮-dGTP> dITP。当以dITP为底物时，端粒酶不能完全逆转录模板，当使用7-脱氮-dGTP和dUTP时，它通过添加两个重复序列有效地延伸了引物。当将相同的核苷酸类似物掺入引物中时，除了在3′-末端脱氧肌苷的情况外，没有观察到作用。数据支持一种模型，该模型包括在产物DNA中形成分子内二级结构以促进易位。最可能的结构是G-G发夹。

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