Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
EMBO J. 2018 Sep 3;37(17). doi: 10.15252/embj.201899154. Epub 2018 Jul 31.
Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly , but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.
单链 DNA（ssDNA）的产生是 DNA 复制过程中模板链形成所必需的。复制蛋白 A（RPA）是一种 ssDNA 结合蛋白，对于真核细胞中复制叉处的 ssDNA 保护至关重要。尽管在表征 RPA-ssDNA 复合物的作用方面已经取得了重大进展，但 RPA 如何加载到复制叉上仍未得到充分探索。在这里，我们表明，Ty1 转座蛋白 105（Rtt105）蛋白调节剂与 RPA 结合并帮助其加载到复制叉上。缺乏 Rtt105 的细胞在复制叉处的 RPA 加载显著减少，复制应激下的 DNA 合成受损，基因组不稳定性增加。在机制上，我们表明 Rtt105 介导 RPA-importin 相互作用，并直接促进 RPA 与 ssDNA 结合，但不在最终的 RPA-ssDNA 复合物中。单分子研究表明，Rtt105 影响 RPA 与 ssDNA 的结合模式。这些结果支持了这样一种模型，即 Rtt105 作为 RPA 伴侣发挥作用，将 RPA 引导到细胞核，并促进其在复制叉处加载到 ssDNA 上。
