Telomerase recognizes G-quadruplex and linear DNA as distinct substrates.
Telomerase recognizes G-quadruplex and linear DNA as distinct substrates.
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DOI：
10.1021/bi700993q
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发表时间：
2007-09
期刊：
影响因子：
2.9
通讯作者：
L. Oganesian;M. Graham;P. Robinson;T. Bryan
中科院分区：
文献类型：
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作者：
L. Oganesian;M. Graham;P. Robinson;T. Bryan
端粒DNA可以组装成一个非线性的，更高阶的构象，称为G-四链体。在这里，我们证明了通过电喷雾电离质谱的两个重复端粒序列d（TGGGGTTGGGGT）从嗜热四膜虫在钠的存在下产生一个新的平行的四链G-四链体。G-四链体以微摩尔亲和力直接与四膜虫端粒酶（TERT）的催化亚基相互作用，并且端粒酶RNA的存在对于这种相互作用不是必需的。TERT的N-和C-末端两半独立地结合G-四链体。该G-四链体是体外重组和细胞提取物衍生的端粒酶的稳健底物。此外，G-四链体减弱了野生型端粒酶对进入的核苷酸（dTTP）的亲和力，并且可能扰乱酶的核苷酸结合口袋。与此一致，在TERT基序1内的氨基酸538（K538 A）处的赖氨酸至丙氨酸的取代显著降低端粒酶延伸G-四链体而非线性DNA的能力。K538 A突变体保留了对四链体的结合亲和力。这表明端粒酶在其活性位点经历构象变化以特异性地适应G-四链体DNA的结合和随后的延伸。我们提出端粒酶识别G-四链体DNA作为不同于线性DNA的底物。
Telomeric DNA can assemble into a nonlinear, higher-order conformation known as a G-quadruplex. Here, we demonstrate by electrospray ionization mass spectrometry that the two repeat telomeric sequence d(TGGGGTTGGGGT) from Tetrahymena thermophila gives rise to a novel parallel four-stranded G-quadruplex in the presence of sodium. The G-quadruplex directly interacts with the catalytic subunit of Tetrahymena telomerase (TERT) with micromolar affinity, and the presence of telomerase RNA is not obligatory for this interaction. Both N- and C-terminal halves of TERT bind the G-quadruplex independently. This G-quadruplex is a robust substrate for both recombinant and cell extract-derived telomerase in vitro. Furthermore, the G-quadruplex weakens the affinity of wild-type telomerase for the incoming nucleotide (dTTP) and likely perturbs the nucleotide binding pocket of the enzyme. In agreement with this, a lysine to alanine substitution at amino acid 538 (K538A) within motif 1 of TERT dramatically reduces the ability of telomerase to extend G-quadruplex but not linear DNA. The K538A mutant retains binding affinity for the quadruplex. This suggests that telomerase undergoes changes in conformation in its active site to specifically accommodate binding and subsequent extension of G-quadruplex DNA. We propose that telomerase recognizes G-quadruplex DNA as a substrate that is distinct from linear DNA.