Tomlinson Christopher G, Moye Aaron L, Holien Jessica K, Parker Michael W, Cohen Scott B, Bryan Tracy M
*Children's Medical Research Institute, Westmead, NSW 2145, Australia.
‡ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.
Biochem J. 2015 Jan 15;465(2):347-57. doi: 10.1042/BJ20140922.
The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight.
核糖核蛋白酶端粒酶维持端粒长度，对大多数癌症细胞的永生至关重要。对端粒酶机制的深入了解可从诸如先天性角化不良等综合征中获得，在这些综合征中，端粒酶成分的突变表现为端粒功能障碍。我们对野生型端粒酶以及逆转录酶结构域中的两个与疾病相关的突变进行了详细的动力学和热力学分析。不同3'端引物解离速率的差异与DNA亲和力无关，这表明端粒酶与端粒DNA的初始结合是通过一种先前未描述的两步机制发生的，该机制涉及酶的构象变化。两种突变均影响DNA结合，但通过不同机制：P704S特异性影响DNA结合过程中的蛋白质构象变化，而R865H在与DNA的3'区域结合时表现出缺陷。为了在结构水平上进一步深入了解，我们构建了人类端粒酶逆转录酶结构域的首个同源模型；P704S和R865H的位置证实了它们观察到的机制缺陷，为结构模型提供了验证。我们的数据揭示了蛋白质与端粒DNA 3'端相互作用的重要性以及蛋白质构象变化在端粒酶DNA结合中的作用，并强调自然发生的疾病突变是深入了解机制的丰富来源。
