College of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 351-8511, Japan.
J Biochem. 2010 Nov;148(5):539-47. doi: 10.1093/jb/mvq085. Epub 2010 Aug 2.
RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.
RPA 由三个亚基（RPA1、2 和 3）组成，在 DNA 代谢中发挥重要作用。在 DNA 复制叉处，RPA 结合到单链 DNA 区域以稳定结构并组装其他复制蛋白。已经报道了 RPA 与几种复制蛋白之间的相互作用，但分析并不全面。我们系统地进行了定性分析以鉴定 RPA 相互作用伙伴，以了解复制叉处的蛋白质-蛋白质相互作用。我们在昆虫细胞中表达了人 RPA 的三个亚基，以及在正常条件下和/或在复制应激条件下存在于复制叉处的一种复制蛋白，以检查相互作用。在总共检查的 30 种蛋白质中，发现至少有 14 种蛋白质与 RPA 相互作用。RPA 与在 DNA 复制起始和延伸中发挥作用的蛋白质（如 MCM3-7、MCM-BP 和 CDC45 蛋白）相互作用。RPA 还与参与 DNA 复制检查点功能的 TIPIN、CLASPIN 和 RAD17 蛋白结合。RPA 还与细胞周期蛋白依赖性激酶和负调控 DNA 复制的 Rb 蛋白氨基末端片段结合。这些结果表明，RPA 与在调节复制叉进展中发挥作用的特定蛋白相互作用。
