Rtt105 functions as a chaperone for replication protein A to preserve genome stability

Abstract Generation of single‐stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication ProteinA (RPA) is an ssDNA‐binding protein essential for protecting ssDNA at replicationforks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replicationforks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replicationforks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replicationforks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA– importininteraction 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 replicationforks.