突触蛋白-DNA复合物,由桥接DNA上两个或多个遥远位点的特殊蛋白质形成,严重参与各种遗传过程。然而,蛋白质搜索这些位点的分子机制以及它是如何将它们结合在一起的,目前还没有很好的理解。我们以前的研究直接可视化了SfiI使用的搜索途径,我们确定了两条途径,DNA穿线和位点结合转移途径,特定于突触DNA-蛋白质系统的位点搜索过程。为了研究这些位点搜索途径背后的分子机制,我们组装了SfiI与对应于不同瞬态的各种DNA底物的复合物,并使用单分子荧光方法测量了它们的稳定性。这些组件对应于特定的(突触),非特异性-非特异性(非特异性),和特异性-非特异性(突触前)SfiI-DNA状态。出乎意料的是,发现与特异性和非特异性DNA底物组装的突触前复合物的稳定性提高。为了解释这些令人惊讶的观察,开发了一种描述这些复合物组装并将预测与实验进行比较的理论方法。该理论通过利用熵论点来解释这种影响,据此,部分解离后,非特异性DNA模板具有多种重新结合的可能性,有效提高稳定性。具有特异性和非特异性DNA的SfiI复合物的稳定性的这种差异解释了在延时AFM实验中发现的突触蛋白-DNA复合物的搜索过程中穿线和位点结合转移途径的利用。
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.