Concentration-dependent exchange of replication protein A on single-stranded DNA revealed by single-molecule imaging.
Concentration-dependent exchange of replication protein A on single-stranded DNA revealed by single-molecule imaging.
复制标题
DOI：
10.1371/journal.pone.0087922
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Greene EC
中科院分区：
文献类型：
--
作者：
Gibb B;Ye LF;Gergoudis SC;Kwon Y;Niu H;Sung P;Greene EC
复制蛋白A（RPA）是一种普遍存在的真核单链DNA（ssDNA）结合蛋白，其对于涉及ssDNA中间体的DNA代谢的所有方面都是必需的，包括DNA复制、修复、重组、DNA损伤应答和检查点激活以及端粒维持。RPA在大多数这些反应中的作用是保护ssDNA，直到它可以被递送到下游酶。因此，RPA的一个关键特征是它必须与ssDNA非常紧密地结合，但也必须很容易从ssDNA中置换出来，以允许其他蛋白质进入底物。在这里，我们使用全内反射荧光显微镜和纳米制造的DNA窗帘，以可视化的行为酿酒酵母RPA的单链ssDNA的实时。我们的研究结果表明，RPA保持绑定到ssDNA的时间很长，当游离蛋白质是从溶液中缺席。相比之下，当游离RPA存在于溶液中时，RPA快速地从ssDNA解离，从而允许游离状态和结合状态之间的快速交换。此外，S.酿酒酵母DNA重组酶Rad51和E. coli单链结合蛋白（SSB）也能促进RPA从ssDNA上的去除。这些结果揭示了结合和游离RPA之间的意外交换，表明可以赋予异常缓慢的解离速率的结合机制，还能够通过依赖于溶液中游离ssDNA结合蛋白的存在的直接交换机制快速置换。我们的研究结果表明，RPA在所有条件下进行恒定的微观解离，但这只表现为宏观解离（即交换）时，游离蛋白质存在于溶液中，这种效果是由于质量作用。我们提出RPA从ssDNA的解离涉及部分解离的中间体，其暴露了ssDNA的一小部分，从而允许其他蛋白质进入DNA。
Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA.
登录
查看更多内容
影响因子：
16.8
作者：
Conway, AB;Lynch, TW;Rice, PA
通讯作者：
Rice, PA
影响因子：
10.5
作者：
Gasior, SL;Wong, AK;Bishop, DK
通讯作者：
Bishop, DK
影响因子：
2.9
作者：
Kunzelmann, Simone;Morris, Caroline;Webb, Martin R.
通讯作者：
Webb, Martin R.
影响因子：
64.8
作者：
Benson, FE;Baumann, P;West, SC
通讯作者：
West, SC
影响因子：
5.3
作者：
Hays, SL;Firmenich, AA;Berg, P
通讯作者：
Berg, P