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首页> 外文期刊>Journal of Molecular Biology >Mapping of DNA binding sites in the Tetrahymena telomerase holoenzyme proteins by UV cross-linking and mass spectrometry.
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Mapping of DNA binding sites in the Tetrahymena telomerase holoenzyme proteins by UV cross-linking and mass spectrometry.

机译：通过紫外线交联和质谱分析四膜虫端粒酶全酶蛋白中的DNA结合位点。

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摘要

The Tetrahymena telomerase holoenzyme consists of a major catalytic protein [telomerase reverse transcriptase (TERT)], an RNA subunit, and accessory proteins. We used site-specific UV cross-linking and mass spectrometry to map interactions between the holoenzyme and the telomeric DNA. In one series of experiments, an oligodeoxyribonucleotide containing a 5-iododeoxyuridine residue or 4-thio-deoxythymidine residue was cross-linked to the telomerase by irradiation with UV light-emitting diodes. The DNA was extended by the cross-linked enzyme with a radioactively labeled or unlabeled nucleotide. The complexes were subsequently resolved by SDS-PAGE. Proteins were isolated from strips in the unlabeled gels corresponding to bands observed in the radioactive gels. Mass spectrometric analysis of these proteins revealed a major cross-linking site in TERT. Serendipitous cleavage of TERT near amino acid 254 indicated that this site maps within the N-terminal cleavage product, which includes primarily the telomerase essential N-terminal (TEN) domain. Moreover, the absence of this N-terminal segment in TERT was found to cause a reduction in DNA binding by the telomerase and/or its activity to undetectable levels. In other experiments, similar unresolved cross-linked complexes were digested with trypsin, two exonucleases, and alkaline phosphatase. Tandem mass spectrometry was then used to search for peptides linked to the residual deoxyribonucleoside. Using this approach, we identified the phenylalanine residue F351 in the accessory protein p45 as a minor DNA cross-linking site. Our study constitutes the first direct mapping of DNA interaction sites in telomerase holoenzyme complexes. This mapping represents a significant contribution to the understanding of the mechanism of telomere extension by telomerase.
机译：四膜虫端粒酶全酶由主要的催化蛋白[端粒酶逆转录酶（TERT）]，RNA亚基和辅助蛋白组成。我们使用了特定于位点的紫外线交联和质谱技术来绘制全酶和端粒DNA之间的相互作用图。在一系列实验中，通过用UV发光二极管照射，将包含5-碘脱氧尿苷残基或4-硫代脱氧胸苷残基的寡脱氧核糖核苷酸交联至端粒酶。通过具有放射性标记或未标记核苷酸的交联酶使DNA延伸。随后通过SDS-PAGE分离复合物。从未标记的凝胶中的条带分离蛋白质，其对应于在放射性凝胶中观察到的条带。这些蛋白质的质谱分析揭示了TERT中的主要交联位点。 TERT的氨基酸254附近的意外切割表明该位点位于N端裂解产物内，该产物主要包括端粒酶必需的N端（TEN）结构域。此外，发现TERT中该N末端片段的缺失导致端粒酶的DNA结合减少和/或其活性降低到不可检测的水平。在其他实验中，用胰蛋白酶，两个核酸外切酶和碱性磷酸酶消化类似的未解析的交联复合物。然后使用串联质谱法搜索与残留的脱氧核糖核苷连接的肽。使用这种方法，我们确定了辅助蛋白p45中的苯丙氨酸残基F351是次要的DNA交联位点。我们的研究构成端粒酶全酶复合物中DNA相互作用位点的第一个直接映射。该定位代表对端粒酶端粒延伸机制的理解的重要贡献。

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