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 Saccharomyces cerevisiae 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 in vitro, 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.
Bibliographical noteFunding Information:
We thank Marc Wold for providing the yeast RPA expression constructs, Steven Brill for providing yeast Rfa1 and Rfa2 antibodies, Oscar Aparcio for the BrdU incorporation strains and plasmids, Chao Tang and Xiaojing Yang for providing GFP and mCherry plasmids, and Wenfeng Qian for providing various genetic background yeast strains. We thank Yun Zhang for assistance with the Illumina sequencing. We thank Hongxia Lv and Xiaochen Li for assistance with live-cell imaging. We thank Zhiguo Zhang, Lilin Du, and Hengyao Niu for critical reading of the manuscript. We thank Huiqiang Lou and Lee Zou for early discussion. This work was supported by grants from NSFC [31725015 (QL) and 31671332 (JF)]. Work in AC's laboratory was funded by the Swedish Research Council, the Swedish Cancer Society, and the Knut and Alice Wallenberg Foundation.
- RPA chaperone
- replication fork
- replication stress