Center for Quantitative Biology, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
Department of Computational Medicine, University of California, Los Angeles, CA, USA.
Nat Commun. 2023 Jun 30;14(1):3864. doi: 10.1038/s41467-023-39579-y.
The eukaryotic single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA) plays a crucial role in various DNA metabolic pathways, including DNA replication and repair, by dynamically associating with ssDNA. While the binding of a single RPA molecule to ssDNA has been thoroughly studied, the accessibility of ssDNA is largely governed by the bimolecular behavior of RPA, the biophysical nature of which remains unclear. In this study, we develop a three-step low-complexity ssDNA Curtains method, which, when combined with biochemical assays and a Markov chain model in non-equilibrium physics, allow us to decipher the dynamics of multiple RPA binding to long ssDNA. Interestingly, our results suggest that Rad52, the mediator protein, can modulate the ssDNA accessibility of Rad51, which is nucleated on RPA coated ssDNA through dynamic ssDNA exposure between neighboring RPA molecules. We find that this process is controlled by the shifting between the protection mode and action mode of RPA ssDNA binding, where tighter RPA spacing and lower ssDNA accessibility are favored under RPA protection mode, which can be facilitated by the Rfa2 WH domain and inhibited by Rad52 RPA interaction.
真核生物单链 DNA（ssDNA）结合蛋白复制蛋白 A（RPA）通过与 ssDNA 动态结合，在包括 DNA 复制和修复在内的各种 DNA 代谢途径中发挥着关键作用。虽然单个 RPA 分子与 ssDNA 的结合已被深入研究，但 ssDNA 的可及性在很大程度上取决于 RPA 的双分子行为，其生物物理性质尚不清楚。在这项研究中，我们开发了一个三步式低复杂度 ssDNA 帷幕方法，该方法与生化测定和非平衡物理中的马尔可夫链模型相结合，使我们能够解析多个 RPA 与长 ssDNA 的结合动力学。有趣的是，我们的结果表明，中介蛋白 Rad52 可以调节 Rad51 的 ssDNA 可及性，Rad51 是通过相邻 RPA 分子之间的动态 ssDNA 暴露在 RPA 涂层 ssDNA 上引发的。我们发现，这个过程受 RPA ssDNA 结合的保护模式和作用模式之间的转换控制，在 RPA 保护模式下，RPA 间隔更紧，ssDNA 可及性更低，这可以通过 Rfa2 WH 结构域促进，也可以通过 Rad52-RPA 相互作用抑制。
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