Department of Biological Sciences, Marquette University, Milwaukee, WI, USA.
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
Nat Struct Mol Biol. 2019 Feb;26(2):129-136. doi: 10.1038/s41594-018-0181-y. Epub 2019 Feb 4.
Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-stranded DNA (ssDNA) intermediates, activating the DNA-damage response and handing off ssDNA to the appropriate downstream players. Six DNA-binding domains (DBDs) in RPA promote high-affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA-interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.
复制蛋白 A（RPA）通过稳定单链 DNA（ssDNA）中间体、激活 DNA 损伤反应并将 ssDNA 传递给适当的下游因子，协调重要的 DNA 代谢事件。RPA 中的六个 DNA 结合结构域（DBD）促进与 ssDNA 的高亲和力结合，但也允许 RPA 被低亲和力蛋白取代。我们生成了酿酒酵母 RPA 的荧光版本，并在全长蛋白的背景下可视化了单个 DBD 的构象动力学。我们表明，DBD-A 和 DBD-D 都能快速结合和解离 ssDNA，而 RPA 仍与 ssDNA 结合。重组介质蛋白 Rad52 选择性调节 DBD-D 的动力学。这些发现揭示了具有较低 ssDNA 结合亲和力的 RPA 相互作用蛋白如何能够进入被封锁的 ssDNA 并重塑单个 DBD 以取代 RPA。
