Rtt105 promotes DNA replication by functioning as an RPA chaperone

Single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) is essential for protecting ssDNA at replication forks. However, how RPA is loaded to replication forks remains unexplored. Here, we show that Regulator of Ty1 transposition protein 105 (Rtt105) binds RPA and is required for the association of RPA with replication forks. Cells lacking Rtt105 exhibit dramatic genome instability and severe defects in DNA replication. Intriguingly, both the RPA nuclear import and its ability to bind DNA replication forks were greatly compromised in rtt105 mutant cells, however, targeting RPA to the nucleus cannot rescue the defect of RPA binding to replication forks. Importantly, Rtt105 promotes the binding of RPA to ssDNA but does not associate with the final RPA-ssDNA complex in vitro. Moreover, single-molecule studies revealed 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 nucleus and assemble RPA onto ssDNA at replication forks.单链DNA（single-stranded DNA，ssDNA）结合蛋白复制蛋白A（Replication Protein A，RPA）对于保护复制叉处的ssDNA至关重要。然而，RPA如何被招募至复制叉这一问题仍未得到探明。本研究发现，Ty1转座调控蛋白105（Regulator of Ty1 transposition protein 105，Rtt105）可结合RPA，并对RPA与复制叉的结合不可或缺。缺失Rtt105的细胞会表现出显著的基因组不稳定性与严重的DNA复制缺陷。有趣的是，在rtt105Δ突变体细胞中，RPA的核输入过程及其结合DNA复制叉的能力均大幅受损，但将RPA靶向导入细胞核并无法挽救其结合复制叉的缺陷。值得注意的是，Rtt105可促进RPA与ssDNA的结合，但在体外并不与最终形成的RPA-ssDNA复合物结合。此外，单分子研究表明，Rtt105会改变RPA与ssDNA的结合模式。上述结果支持如下模型：Rtt105作为RPA分子伴侣，负责将RPA护送至细胞核，并将RPA组装至复制叉处的ssDNA上。