Institute of Chemical Biology and Fundamental Medicine, Lavrentiev av. 8, Novosibirsk, 630090, Russia.
Institute of Chemical Biology and Fundamental Medicine, Lavrentiev av. 8, Novosibirsk, 630090, Russia; Department of Natural Sciences, Novosibirsk State University, 1 Pirogov Street, Novosibirsk, 630090, Russia.
DNA Repair (Amst). 2018 Dec;72:28-38. doi: 10.1016/j.dnarep.2018.09.010. Epub 2018 Sep 24.
Replication protein A contributes to all major pathways of DNA metabolism and is a target for post-translation modifications, including poly(ADP-ribosyl)ation catalyzed by PARP1. Here we demonstrate that the efficiency of RPA poly(ADP-ribosyl)ation strongly depends on the structure of DNA used for PARP1 activation and on the polarity of RPA binding. Moreover, RPA influences PARP1 activity, and this effect also depends on DNA structure: RPA inhibits PAR synthesis catalyzed by PARP1 in the presence of ssDNA and stimulates it in the presence of a DNA duplex, in particular that containing a nick or a gap. Using fluorescently labeled proteins, we showed their direct interaction and characterized it quantitatively. RPA can accelerate the replacement of poly(ADP-ribosyl)ated PARP1 molecules bound to DNA by the unmodified ones. Thus, our data allow us to suggest that the balance between the affinities of PARP1 and RPA for DNA and the interaction of these proteins with each other are the cornerstone of the modulating effect of RPA on PARP1 activity. This effect might contribute to the regulation of PARP1 activity in various DNA processing mechanisms including DNA replication and repair pathways, where both PARP1 and RPA participate.
复制蛋白 A 参与 DNA 代谢的所有主要途径，是翻译后修饰的靶点，包括 PARP1 催化的聚（ADP-核糖基）化。在这里，我们证明了 RPA 聚（ADP-核糖基）化的效率强烈依赖于用于 PARP1 激活的 DNA 结构和 RPA 结合的极性。此外，RPA 影响 PARP1 活性，这种效应也取决于 DNA 结构：RPA 在 ssDNA 存在下抑制 PARP1 催化的 PAR 合成，在 DNA 双链体存在下刺激其合成，特别是含有缺口或缺口的 DNA 双链体。使用荧光标记的蛋白质，我们显示了它们的直接相互作用并对其进行了定量分析。RPA 可以加速结合有 DNA 的聚（ADP-核糖基）化 PARP1 分子被未修饰的分子所取代。因此，我们的数据表明，PARP1 和 RPA 与 DNA 的亲和力之间的平衡以及这些蛋白质之间的相互作用是 RPA 对 PARP1 活性的调节作用的基石。这种效应可能有助于调节各种 DNA 处理机制中的 PARP1 活性，包括 PARP1 和 RPA 都参与的 DNA 复制和修复途径。
