Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.
Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.
复制标题
作者：
大肠杆菌RecA是一种普遍存在的DNA链交换蛋白的定义成员，它对同源重组至关重要，同源重组是一种通过修复断裂的DNA来维持基因组完整性的途径。为了发挥作用，RecA的细丝必须在单链DNA （ssDNA）上成核并生长，与ssDNA结合蛋白（SSB）直接竞争，后者快速结合并持续隔离ssDNA，从而动态地阻断RecA的组装。这种动态自组装在DNA晶格上，与另一种蛋白质竞争，对于reca家族来说是独特的，相对于其他的丝形成蛋白，如肌动蛋白和微管蛋白。这个过程的复杂性阻碍了我们对RecA细丝组装的理解，因为集合测量不能可靠地区分成核和生长阶段，尽管进行了广泛和多样化的尝试。以前的单分子分析已经测量了reca及其真核同源物rad51在裸dsDNA和ssDNA上的成核和生长；然而，RecA在体内自组装的模板是ssb包被的ssDNA。利用单分子显微镜，我们直接看到了ssb包覆的ssDNA单分子上的RecA丝组装，同时测量了成核和生长。我们确定了RecA的二聚体是成核所必需的，其次是通过单体添加的长丝的生长，这与成核而不是生长受核苷酸和镁离子辅助因子调节的发现是一致的。纤维的生长是双向的，但在5 ‘→3 ’方向上生长更快。在生理条件下，成核和生长都受到抑制，这突出了重组介质在增强体内组装中的重要作用。我们定义了一个两步动力学机制，在SSB滑动和/或部分解离（即DNA解包裹）过程中，RecA在瞬时暴露的ssDNA上成核，然后生长。我们进一步证明了重组中介蛋白对RecOR加速了RecA成核和丝的生长，并且RecF的引入进一步刺激了RecA成核。
Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA-family relative to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured nucleation and growth of RecA—and its eukaryotic homolog RAD51—on naked dsDNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, we directly visualized RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′→3′ direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism where RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (i.e., DNA unwrapping) and then grows. We further demonstrate that the recombination mediator protein pair, RecOR, accelerates both RecA nucleation and filament growth, and that introduction of RecF further stimulates RecA nucleation.