Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80309.
BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309.
Proc Natl Acad Sci U S A. 2023 Jun 27;120(26):e2220537120. doi: 10.1073/pnas.2220537120. Epub 2023 Jun 20.
We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands.
我们之前已经证明，多梳抑制复合物 2 染色质修饰酶可以在没有游离酶中间状态的情况下直接在 RNA 和 DNA 之间转移。模拟表明，这种直接转移机制可能通常是 RNA 将蛋白质募集到染色质所必需的，但直接转移能力的普遍性尚不清楚。在此，我们使用荧光偏振测定法并观察到几种经过充分表征的核酸结合蛋白的直接转移：3′端修复外切酶 1、异质性核核糖核蛋白 U、Fem-3 结合因子 2 和 MS2 噬菌体外壳蛋白。对于 TREX1，在单分子测定中还观察到直接转移机制，并且数据表明直接转移通过具有部分相关多核苷酸的不稳定三元中间产物发生。通常，直接转移可以允许许多 DNA 和 RNA 结合蛋白对其靶位点进行一维搜索。此外，结合 RNA 和 DNA 的蛋白质可能能够在这些配体之间轻松转移。
