Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.
Department of Chemistry-MS60, Rice University, 6100 Main Street, Houston, TX 77005-1892, USA.
Int J Mol Sci. 2023 Jun 6;24(12):9800. doi: 10.3390/ijms24129800.
The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.
突触蛋白-DNA 复合物由专门的蛋白质形成，这些蛋白质在 DNA 上的两个或多个远距离位点之间架桥，对于各种遗传过程至关重要。然而，蛋白质如何搜索这些位点以及如何将它们聚集在一起的分子机制尚不清楚。我们之前的研究直接可视化了 SfiI 所使用的搜索途径，我们确定了两种途径，DNA 穿线途径和位点结合转移途径，它们是突触 DNA-蛋白质系统位点搜索过程所特有的。为了研究这些位点搜索途径背后的分子机制，我们组装了各种 DNA 底物的 SfiI 复合物，这些 DNA 底物对应于不同的瞬时状态，并使用单分子荧光方法测量它们的稳定性。这些组装对应于特定特异性（突触）、非特异性-非特异性（非特异性）和特异性-非特异性（预突触）SfiI-DNA 状态。出乎意料的是，发现与特异性和非特异性 DNA 底物组装的预突触复合物具有更高的稳定性。为了解释这些令人惊讶的观察结果，我们开发了一种理论方法来描述这些复合物的组装，并将预测与实验进行比较。该理论通过利用熵论据来解释这种效应，根据该论据，在部分解离后，非特异性 DNA 模板具有多种重新结合的可能性，有效地增加了稳定性。与特异性和非特异性 DNA 的 SfiI 复合物稳定性的这种差异解释了在时间分辨原子力显微镜实验中发现的突触蛋白-DNA 复合物搜索过程中穿线和位点结合转移途径的利用。
