Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California; Department of Structural Biology, Stanford University School of Medicine, Stanford, California.
Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California.
Biophys J. 2023 Sep 5;122(17):3447-3457. doi: 10.1016/j.bpj.2023.07.019. Epub 2023 Jul 27.
Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we report the development and proof-of-concept results of a single-molecule approach to monitor the catalytic activity of human telomerase in real time and with single-nucleotide resolution. Using zero-mode waveguides and multicolor FRET, we recorded the processive addition of multiple telomeric repeats to individual DNA primers. Unlike existing biophysical and biochemical tools, the novel approach enables the quantification of nucleotide-binding kinetics before nucleotide incorporation. Moreover, it provides a means to dissect the unique translocation dynamics that telomerase must undergo after synthesis of each hexameric DNA repeat. We observed an unexpectedly prolonged binding dwell time of dGTP in the enzyme active site at the start of each repeat synthesis cycle, suggesting that telomerase translocation is composed of multiple rate-contributing sub-steps that evade classical biochemical analysis.
在增殖细胞中，基因组稳定性严重依赖于端粒酶逆转录酶维持端粒。在这里，我们报告了一种单分子方法的开发和概念验证结果，该方法可实时、单核苷酸分辨率监测人端粒酶的催化活性。我们使用零模波导和多色 FRET 记录了多个端粒重复序列到单个 DNA 引物的连续添加过程。与现有的生物物理和生物化学工具不同，新方法能够在核苷酸掺入之前定量核苷酸结合动力学。此外，它还提供了一种手段来剖析端粒酶在合成每个六聚体 DNA 重复后必须经历的独特易位动力学。我们在每个重复合成循环开始时观察到 dGTP 在酶活性位点中出乎意料地延长了结合停留时间，这表明端粒酶易位由多个逃避经典生化分析的速率贡献子步骤组成。
