Salazar M, Thompson B D, Kerwin S M, Hurley L H
College of Pharmacy, Department of Molecular Biology, University of Texas, Austin 78712, USA.
Biochemistry. 1996 Dec 17;35(50):16110-5. doi: 10.1021/bi961442j.
Telomerase is a specialized reverse transcriptase that contains its own RNA template for synthesis of telomeric DNA [Greider, C. W., & Blackburn, E. H. (1989) Nature 337, 331-337; Shippen-Lentz, D., & Blackburn, E. H. (1990) Science 247, 546-552]. The activity of this ribonucleoprotein enzyme has been associated with cancer cells [Kim et al. (1994) Science 266, 2011-2015] and is thus a potential target for anticancer chemotherapy. Telomeric DNA.RNA hybrids are important intermediates in telomerase function and form after extension of the growing telomere on the telomerase RNA template. Translocation is a critical step in telomerase function and consists of unwinding of the telomeric DNA.telomerase RNA hybrid followed by repositioning of the 3'-end of the extended telomere. A central question in telomerase function is how translocation of the extended telomere occurs in the absence of ATP or GTP. It has been hypothesized that unwinding of the telomeric hybrid may be facilitated by the formation of stable hairpins or G-quadruplexes by the telomere product (i.e., a hybrid to G-quadruplex transition) and that this may provide at least part of the driving force for translocation [Shippen-Lentz & Blackburn, 1990; Zahler et al. (1991) Nature 350, 718-720]. However, so far there has been no effort aimed at examining the possibility that a hybrid/G-quadruplex equilibrium can occur and to what extent this equilibrium depends on buffer and concentration conditions. Examination of these transitions may provide insight into telomerase function and may also provide clues for the development of anti-telomerase agents. Using a model system consisting of the DNA.RNA hybrid d(GGTTAGGGTTAG).r(cuaacccuaacc), we present evidence that a thermally induced transition of telomeric DNA.RNA hybrid to G-quadruplex can occur under certain conditions. These results provide support for the hypothesis that G-quadruplex formation by the telomere product may in fact regulate telomerase function at the translocation step (Zahler et al., 1991) and suggest an Achilles' heel for indirectly targeting telomerase. Thus, on the basis of the insight gained from the present studies and the results of Zahler et al. (1991), we propose that ligands that selectively bind or cleave G-quadruplex structures may modulate telomerase processivity.
端粒酶是一种特殊的逆转录酶，它含有自身用于合成端粒DNA的RNA模板[Greider, C. W., & Blackburn, E. H. (1989)《自然》337, 331 - 337；Shippen-Lentz, D., & Blackburn, E. H. (1990)《科学》247, 546 - 552]。这种核糖核蛋白酶的活性与癌细胞相关[Kim等人(1994)《科学》266, 2011 - 2015]，因此是抗癌化疗的一个潜在靶点。端粒DNA.RNA杂交体是端粒酶功能中的重要中间体，在端粒酶RNA模板上生长的端粒延伸后形成。转位是端粒酶功能中的关键步骤，包括解开端粒DNA.端粒酶RNA杂交体，随后重新定位延伸端粒的3'端。端粒酶功能中的一个核心问题是，在没有ATP或GTP的情况下，延伸端粒的转位是如何发生的。据推测，端粒杂交体的解旋可能通过端粒产物形成稳定的发夹结构或G-四链体（即杂交体向G-四链体的转变）来促进，并且这可能为转位提供至少部分驱动力[Shippen-Lentz & Blackburn, 1990；Zahler等人(1991)《自然》350, 718 - 720]。然而，到目前为止，尚未有人致力于研究杂交体/G-四链体平衡是否能够发生以及这种平衡在多大程度上取决于缓冲液和浓度条件。对这些转变的研究可能有助于深入了解端粒酶的功能，也可能为开发抗端粒酶药物提供线索。使用由DNA.RNA杂交体d(GGTTAGGGTTAG).r(cuaacccuaacc)组成的模型系统，我们提供证据表明，在某些条件下，端粒DNA.RNA杂交体可以发生热诱导向G-四链体的转变。这些结果为以下假设提供了支持，即端粒产物形成G-四链体实际上可能在转位步骤调节端粒酶功能(Zahler等人，1991)，并提示了间接靶向端粒酶的一个薄弱环节。因此，基于本研究获得的见解以及Zahler等人(1991)的结果，我们提出，选择性结合或切割G-四链体结构的配体可能调节端粒酶的持续合成能力。
