Melek M, Greene E C, Shippen D E
Department of Biochemistry and Biophysics, Texas A&M University, College Station, 77843-2128, USA.
Mol Cell Biol. 1996 Jul;16(7):3437-45. doi: 10.1128/MCB.16.7.3437.
Telomerase is a specialized reverse transcriptase that maintains telomeres at chromosome ends by extending preexisting tracts of telomeric DNA and forming telomeres de novo on broken chromosomes. Whereas the interaction of telomerase with telomeric DNA has been studied in some detail, relatively little is known about how this enzyme processes nontelomeric DNA. In this study we recruited the Euplotes telomerase to nontelomeric 3' termini in vitro using chimeric DNA primers that carried one repeat of a telomeric sequence at various positions upstream of a nontelomeric 3' end. Such primers were processed in two distinct pathways. First, nontelomeric 3' ends could be elongated directly by positioning a primer terminus at a specific site on the RNA template. Delivery to this default site was precise, always resulting in the addition of 4 dG residues to the non-telomeric 3' ends. These same residues initiate new telomeres formed in vivo. Alternatively, 3' nontelomeric nucleotides were removed from primers prior to initiating the first elongation cycle. As with default positioning of nontelomeric 3' ends, the cleavage event was extremely precise and was followed by the addition of dG residues to the primer 3' ends. The specificity of the cleavage reaction was mediated by primer interaction with the RNA template and, remarkably, proceeded by an endonucleolytic mechanism. These observations suggest a mechanism for the precision of developmentally regulated de novo telomere formation and expand our understanding of the enzymatic properties of telomerase.
端粒酶是一种特殊的逆转录酶，它通过延长预先存在的端粒DNA片段并在断裂的染色体上重新形成端粒，从而维持染色体末端的端粒。虽然已经对端粒酶与端粒DNA的相互作用进行了一些详细研究，但对于这种酶如何处理非端粒DNA却知之甚少。在本研究中，我们使用嵌合DNA引物在体外将游仆虫端粒酶招募到非端粒3'末端，这些引物在非端粒3'末端上游的不同位置携带一个端粒序列重复。此类引物通过两种不同途径进行处理。首先，非端粒3'末端可以通过将引物末端定位在RNA模板上的特定位点而直接延长。输送到这个默认位点是精确的，总是导致在非端粒3'末端添加4个dG残基。这些相同的残基启动体内形成的新端粒。或者，在启动第一个延伸循环之前，从引物中去除3'非端粒核苷酸。与非端粒3'末端的默认定位一样，切割事件极其精确，随后在引物3'末端添加dG残基。切割反应的特异性由引物与RNA模板的相互作用介导，并且值得注意的是，通过内切核酸酶机制进行。这些观察结果提示了一种发育调控的端粒从头形成精确性的机制，并扩展了我们对端粒酶酶学性质的理解。
