Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.
Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.
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DOI：
10.1093/nar/gkw1125
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发表时间：
2017-01-25
影响因子：
14.9
通讯作者：
Greene EC
中科院分区：
文献类型：
--
作者：
Ma CJ;Gibb B;Kwon Y;Sung P;Greene EC
同源重组 (HR) 是双链 DNA 断裂 (DSB) 修复的重要途径。在 HR 的早期阶段，新生成的 DSB 末端经过处理，产生长单链 DNA (ssDNA) 突出端，这些突出端可快速与复制蛋白 A (RPA) 结合。然后 RPA 被 DNA 重组酶 Rad51 取代，在 ssDNA 上形成延伸的螺旋丝。由此产生的核蛋白丝，称为突触前复合物，负责将 ssDNA 与同源双链 DNA (dsDNA) 配对，作为指导 DSB 修复的模板。在这里，我们使用单分子成像来可视化突触前复合物组装和拆卸过程中人类 RPA (hRPA) 和人类 RAD51 之间的相互作用。我们证明，ssDNA 结合的 hRPA 可以进行促进交换，只有当溶液中存在游离 hRPA 时，hRPA 才能在游离状态和 ssDNA 结合状态之间进行快速交换。我们的结果还表明，游离 hRPA 的存在抑制 RAD51 细丝成核，但对细丝伸长的影响较小。这一发现表明 hRPA 对 RAD51 发挥重要的调节影响，并可能反过来影响组装的 RAD51 丝的特性。这些实验为进一步研究人类突触前复合体组装的调控提供了重要基础。
Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly.